Circ-HSP90A expedites cell growth, stemness, and immune evasion in non-small cell lung cancer by regulating STAT3 signaling and PD-1/PD-L1 checkpoint

Abstract

Background

Circular RNAs (circRNAs) are important participators in tumor progression for their stable structure and high tissue-specific expression. The purpose of this research was to clarify the potential and mechanism of a novel circRNA-circ-HSP90A in non-small cell lung cancer (NSCLC).

Methods

Biological potentials of circ-HSP90A in NSCLC were measured by functional assays. Molecular interaction was assessed by bioinformatics analysis and mechanical assays.

Results

Results depicted that circ-HSP90A was cyclization from its host gene heat shock protein 90 alpha (HSP90A) and was up-regulated in NSCLC cells. Circ-HSP90A depletion retarded proliferation, migration, invasion, and immune evasion. Mechanistically, circ-HSP90A recruited ubiquitin specific peptidase 30 (USP30) to stabilize HSP90A and then stimulated the signal transducer and activator of transcription 3 (STAT3) signaling. Meanwhile, circ-HSP90A sponged miR-424-5p to programmed cell death ligand 1 (PD-L1).

Conclusions

Our study firstly showed that circ-HSP90A promoted cell growth, stemness, and immune evasion in NSCLC through regulating STAT3 signaling and programmed cell death 1 (PD-1)/PD-L1 checkpoint, mirroring that targeting circ-HSP90A might become a novel target of immunotherapy in NSCLC.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-022-03235-z.

Introduction

Non-small cell lung cancer (NSCLC), a subtype of lung cancer, is a dominating cause of disease-concerned death all around the world [1]. Although tremendous advances have been made in the diagnosis and treatments of lung cancer, the 5-year survival rate of NSCLC sufferers is less than 20% [2]. In recent years, tumor immune microenvironment exerts a vital part in NSCLC development, and NSCLC cells can cooperate with immune cells to promote immune evasion [3]. Therefore, cancer immunotherapies have been adopted to treat NSCLC in clinical trials, among which, programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) blockade has been recognized to be one of a novel cancer immunotherapies for NSCLC treatment [4]. PD-L1 can bind to PD-1 to repress the activity and induce CD8⁺ T cells apoptosis, resulting in the immune evasion of tumors [5]. As a result, understanding the potential mechanism related to PD-1/PD-L1 checkpoint in NSCLC is conducive to improve the immunotherapy for NSCLC.

Recently, studies on circular RNAs (circRNAs) have emerged as a popular subject in tumors. CircRNAs are a special type of noncoding RNAs and are formed by a covalently closed continuous loops [6, 7]. Abnormal expression of circRNAs has been served as promising prognostic indicators for multiple cancers, such as gastric cancer [8], triple-negative breast cancer [9] and ovarian cancer [10]. Mounting evidence has demonstrated that circRNAs are implicated in modulating cancer progression via sponging microRNAs (miRNAs) or cooperating with RNA-binding proteins (RBPs) [11]. Additionally, circRNAs are important participants in cell proliferation, apoptosis, migration, and invasion of NSCLC. For instance, circRNA 100,146 facilitates NSCLC cell proliferation and invasion via targeting miR-361-3p and miR-615-5p [12]. Circ_0078767 restrains cell cycle progression in NSCLC by the miR-330-3p/RASSF1A axis [13]. Circ-CPA4 facilitates cell growth and immune evasion in NSCLC via increasing PD-L1 expression (14). However, the potential of circRNAs in NSCLC has not been fully elucidated yet.

Signal transducer and activator of transcription 3 (STAT3) is geared to the STAT family and is activated in various cancers, including NSCLC [15]. Upon activation, STAT3 undergoes phosphorylation-induced dimerization, nuclear translocation and combination with its target genes, therefore inducing the transcription of downstream target genes and cancer progression [16]. As a consequence, inhibitors targeting STAT3 activation have provided promise in repressing cancer development and have been extensively investigated in cancers, NSCLC included [17]. Accumulating evidence has suggested that circRNAs are implicated in NSCLC progression via regulating STAT3. Yu Dong et al. have pointed that circ_0076305 influences cisplatin resistance of NSCLC via positively modulating STAT3 [18]. CircHIPK3 influences autophagy by STAT3 signaling in NSCLC [19].

In this study, we investigated a circRNA derived from its host gene heat shock protein 90 alpha (HSP90A) in NSCLC. We probed the function of circ-HSP90A on NSCLC progression and immune evasion. Moreover, the relation between circ-HSP90A with PD-L1 and STAT3 was also explored further. Our findings might provide potential therapeutic targets for this cancer.

Material and methods

Cell culture

NSCLC cells (H1299, A549, PC9, CAL-12T, and H1915), normal human lung epithelial cells (BEAS-2B), and human embryonic kidney cells (HEK293T) were offered from the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. H1299, PC9 as well as H1915 cells were grown in RPMI-1640 medium (Gibco, Rockville, MD, USA). A549 cells were grown in F-12K medium (Gibco). CAL-12T and HEK293T cells were cultured in DMEM. BEAS-2B cells were grown in BEGM (Lonza/Clonetics Corporation). All mediums were contained 10% fetal bovine serum with 5% CO₂ at 37 °C.

Reagents

The cycloheximide (CHX; 5 µM), MG132 (10 Μm), RNase R (40 U), and actinomycin D (4 µg/ml) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Quantitative real-time polymerase chain reaction (RT-qPCR)

Extraction of total RNA was used with a TRIzol reagent (Invitrogen) and then performed cDNA synthesis via M-MLV reverse transcriptase (Applied Biosystems, USA). qPCR was utilized to detect relative expression with SYBR Premix Ex Taq II (Takara, Tokyo, Japan) and calculated using 2^−∆∆Ct method, which was normalized for GAPDH or U6. The sequences used in the experiments are listed in Table 1.

Table 1.

The sequences of the primers have been listed

Primers	Sequences (5′ > 3′)
hsa_circ_0033393	GCATTGGACAAAATCCGGTATGAAAGCTTGACAGATCCCAGTAAATTAGACTCTGGGAAAGAGCTGCATATTAACCTTATACCGAACAAACAAGATCGAACTCTCACTATTGTGGATACTGGAATTGGAATGACCAAGGCTGACTTGATCAATAACCTTGGTACTATCGCCAAGTCTGGGACCAAAGCGTTCATGGAAGCTTTGCAGGCTGGTGCAGATATCTCTATGATTGGCCAGTTCGGTGTTGGTTTTTATTCTGCTTATTTGGTTGCTGAGAAAGTAACTGTGATCACCAAACATAACGATGATGAGCAGTACGCTTGGGAGTCCTCAGCAGGGGGATCATTCACAGTGAGGACAGACACAGGTGAACCTATGGGTCGTGGAACAAAAGTTATCCTACACCTGAAAGAAGACCAAACTGAGTACTTGGAGGAACGAAGAATAAAGGAGATTGTGAAGAAACATTCTCAGTTTATTGGATATCCCATTACTCTTTTTGTGGAGAAGGAACGTGATAAAGAAGTAAGCGATGATGAGGCTGAAGAAAAGGAAGACAAAGAAGAAGAAAAAGAAAAAGAAGAGAAAGAGTCGGAAGACAAACCTGAAATTGAAGATGTTGGTTCTGATGAGGAAGAAGAAAAGAAGGATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTCAACAAAACAAAGCCCATCTGGACCAGAAATCCCGACGATATTACTAATGAGGAGTACGGAGAATTCTATAAGAGCTTGACCAATGACTGGGAAGATCACTTGGCAGTGAAGCATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033386	GTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033387	GTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033388	CTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033389	ACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033390	CATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033391	GTGGAGAAGGAACGTGATAAAGAAGTAAGCGATGATGAGGCTGAAGAAAAGGAAGACAAAGAAGAAGAAAAAGAAAAAGAAGAGAAAGAGTCGGAAGACAAACCTGAAATTGAAGATGTTGGTTCTGATGAGGAAGAAGAAAAGAAGGATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTCAACAAAACAAAGCCCATCTGGACCAGAAATCCCGACGATATTACTAATGAGGAGTACGGAGAATTCTATAAGAGCTTGACCAATGACTGGGAAGATCACTTGGCAGTGAAGCATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033392	GTGAACCTATGGGTCGTGGAACAAAAGTTATCCTACACCTGAAAGAAGACCAAACTGAGTACTTGGAGGAACGAAGAATAAAGGAGATTGTGAAGAAACATTCTCAGTTTATTGGATATCCCATTACTCTTTTTGTGGAGAAGGAACGTGATAAAGAAGTAAGCGATGATGAGGCTGAAGAAAAGGAAGACAAAGAAGAAGAAAAAGAAAAAGAAGAGAAAGAGTCGGAAGACAAACCTGAAATTGAAGATGTTGGTTCTGATGAGGAAGAAGAAAAGAAGGATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTCAACAAAACAAAGCCCATCTGGACCAGAAATCCCGACGATATTACTAATGAGGAGTACGGAGAATTCTATAAGAGCTTGACCAATGACTGGGAAGATCACTTGGCAGTGAAGCATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033393	GCATTGGACAAAATCCGGTATGAAAGCTTGACAGATCCCAGTAAATTAGACTCTGGGAAAGAGCTGCATATTAACCTTATACCGAACAAACAAGATCGAACTCTCACTATTGTGGATACTGGAATTGGAATGACCAAGGCTGACTTGATCAATAACCTTGGTACTATCGCCAAGTCTGGGACCAAAGCGTTCATGGAAGCTTTGCAGGCTGGTGCAGATATCTCTATGATTGGCCAGTTCGGTGTTGGTTTTTATTCTGCTTATTTGGTTGCTGAGAAAGTAACTGTGATCACCAAACATAACGATGATGAGCAGTACGCTTGGGAGTCCTCAGCAGGGGGATCATTCACAGTGAGGACAGACACAGGTGAACCTATGGGTCGTGGAACAAAAGTTATCCTACACCTGAAAGAAGACCAAACTGAGTACTTGGAGGAACGAAGAATAAAGGAGATTGTGAAGAAACATTCTCAGTTTATTGGATATCCCATTACTCTTTTTGTGGAGAAGGAACGTGATAAAGAAGTAAGCGATGATGAGGCTGAAGAAAAGGAAGACAAAGAAGAAGAAAAAGAAAAAGAAGAGAAAGAGTCGGAAGACAAACCTGAAATTGAAGATGTTGGTTCTGATGAGGAAGAAGAAAAGAAGGATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTCAACAAAACAAAGCCCATCTGGACCAGAAATCCCGACGATATTACTAATGAGGAGTACGGAGAATTCTATAAGAGCTTGACCAATGACTGGGAAGATCACTTGGCAGTGAAGCATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033394	ATGCCTGAGGAAACCCAGACCCAAGACCAACCGATGGAGGAGGAGGAGGTTGAGACGTTCGCCTTTCAGGCAGAAATTGCCCAGTTGATGTCATTGATCATCAATACTTTCTACTCGAACAAAGAGATCTTTCTGAGAGAGCTCATTTCAAATTCATCAGATGCATTGGACAAAATCCGGTATGAAAGCTTGACAGATCCCAGTAAATTAGACTCTGGGAAAGAGCTGCATATTAACCTTATACCGAACAAACAAGATCGAACTCTCACTATTGTGGATACTGGAATTGGAATGACCAAGGCTGACTTGATCAATAACCTTGGTACTATCGCCAAGTCTGGGACCAAAGCGTTCATGGAAGCTTTGCAGGCTGGTGCAGATATCTCTATGATTGGCCAGTTCGGTGTTGGTTTTTATTCTGCTTATTTGGTTGCTGAGAAAGTAACTGTGATCACCAAACATAACGATGATGAGCAGTACGCTTGGGAGTCCTCAGCAGGGGGATCATTCACAGTGAGGACAGACACAGGTGAACCTATGGGTCGTGGAACAAAAGTTATCCTACACCTGAAAGAAGACCAAACTGAGTACTTGGAGGAACGAAGAATAAAGGAGATTGTGAAGAAACATTCTCAGTTTATTGGATATCCCATTACTCTTTTTGTGGAGAAGGAACGTGATAAAGAAGTAAGCGATGATGAGGCTGAAGAAAAGGAAGACAAAGAAGAAGAAAAAGAAAAAGAAGAGAAAGAGTCGGAAGACAAACCTGAAATTGAAGATGTTGGTTCTGATGAGGAAGAAGAAAAGAAGGATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTCAACAAAACAAAGCCCATCTGGACCAGAAATCCCGACGATATTACTAATGAGGAGTACGGAGAATTCTATAAGAGCTTGACCAATGACTGGGAAGATCACTTGGCAGTGAAGCATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033395	GCATGCGTAGGCGCGCGGCCGCGGCGGCGGCTGGGGAGGGTTCTTCCGGAAGGTTCGGGAGGCTTCTGGAAAAAGCGCCGCGCGCTGGGCGGGCCCGTCGCTATATAAGGCAGGCGCGGGGGTGGCGCGTCAGTTGCTTCAGCGTCCCGGTGTGGCTGTGCCGTTGGTCCTGTGCGGTCACTTAGCCAAGATGCCTGAGGAAACCCAGACCCAAGACCAACCGATGGAGGAGGAGGAGGTTGAGACGTTCGCCTTTCAGGCAGAAATTGCCCAGTTGATGTCATTGATCATCAATACTTTCTACTCGAACAAAGAGATCTTTCTGAGAGAGCTCATTTCAAATTCATCAGATGCATTGGACAAAATCCGGTATGAAAGCTTGACAGATCCCAGTAAATTAGACTCTGGGAAAGAGCTGCATATTAACCTTATACCGAACAAACAAGATCGAACTCTCACTATTGTGGATACTGGAATTGGAATGACCAAGGCTGACTTGATCAATAACCTTGGTACTATCGCCAAGTCTGGGACCAAAGCGTTCATGGAAGCTTTGCAGGCTGGTGCAGATATCTCTATGATTGGCCAGTTCGGTGTTGGTTTTTATTCTGCTTATTTGGTTGCTGAGAAAGTAACTGTGATCACCAAACATAACGATGATGAGCAGTACGCTTGGGAGTCCTCAGCAGGGGGATCATTCACAGTGAGGACAGACACAGGTGAACCTATGGGTCGTGGAACAAAAGTTATCCTACACCTGAAAGAAGACCAAACTGAGTACTTGGAGGAACGAAGAATAAAGGAGATTGTGAAGAAACATTCTCAGTTTATTGGATATCCCATTACTCTTTTTGTGGAGAAGGAACGTGATAAAGAAGTAAGCGATGATGAGGCTGAAGAAAAGGAAGACAAAGAAGAAGAAAAAGAAAAAGAAGAGAAAGAGTCGGAAGACAAACCTGAAATTGAAGATGTTGGTTCTGATGAGGAAGAAGAAAAGAAGGATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTCAACAAAACAAAGCCCATCTGGACCAGAAATCCCGACGATATTACTAATGAGGAGTACGGAGAATTCTATAAGAGCTTGACCAATGACTGGGAAGATCACTTGGCAGTGAAGCATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033396	GACTGCGCAGGCGTGCTCACCTGGCGTGCTCCACCCGACTGGGCGTCCGCAGGCTCCTCCCCCGGGTGTGGCCTCCGGGCGGCATGGCTGCTTCCCAGGTGATGCCGGCTTCAGCTAGTGGGGTCTAGTTGACCGTTCCGCAGCCGCCAGGGCCAGCGGAAAGCCGGTCAGGGGGAACCGCGGCGGGGCTGGTGTCATGAGCCTGAGGTGAACTTGAGGGTGCCTCCTCAGCGGTCTCCCGCCCTGCCCTGAGGGGCGCCGGGACCCCAAAGAGCGGAGGAAGAGCGCCACCCCGACGGCCACCGCTTCGGAGCCAGCACGCGGGGTACCCTACGGGGAGCGCGGATGCCCCCGTGTTCGGGCGGGGACGGCTCCACCCCTCCTGGGCCCTCCCTTCGGGACAGGGACTGTCCCGCCCAGAGTGCTGAATACCCGCGCGACCGTCTGGATCCCCGCCCAGGAAGCCCCTCTGAAGCCTCCTCGCCGCCGTTTCTGAGAAGCAGGGCACCTGTTAACTGGTACCAAGAAAAGGCCCAAGTGTTTCTCTGGCATCTGATGGTGTCTGGATCCACCACTCTACTCTGTCTCTGGAAACAGCCCTTCCACGTCTCTGCATTCCCTGTCACCGCGTCACTGGCCTTCAGACAGAGCCAAGGTGCAGGGCAACACCTCTACAAGGATCTGCAGCCATTTATATTGCTTAGGCTACTGATGCCTGAGGAAACCCAGACCCAAGACCAACCGATGGAGGAGGAGGAGGTTGAGACGTTCGCCTTTCAGGCAGAAATTGCCCAGTTGATGTCATTGATCATCAATACTTTCTACTCGAACAAAGAGATCTTTCTGAGAGAGCTCATTTCAAATTCATCAGATGCATTGGACAAAATCCGGTATGAAAGCTTGACAGATCCCAGTAAATTAGACTCTGGGAAAGAGCTGCATATTAACCTTATACCGAACAAACAAGATCGAACTCTCACTATTGTGGATACTGGAATTGGAATGACCAAGGCTGACTTGATCAATAACCTTGGTACTATCGCCAAGTCTGGGACCAAAGCGTTCATGGAAGCTTTGCAGGCTGGTGCAGATATCTCTATGATTGGCCAGTTCGGTGTTGGTTTTTATTCTGCTTATTTGGTTGCTGAGAAAGTAACTGTGATCACCAAACATAACGATGATGAGCAGTACGCTTGGGAGTCCTCAGCAGGGGGATCATTCACAGTGAGGACAGACACAGGTGAACCTATGGGTCGTGGAACAAAAGTTATCCTACACCTGAAAGAAGACCAAACTGAGTACTTGGAGGAACGAAGAATAAAGGAGATTGTGAAGAAACATTCTCAGTTTATTGGATATCCCATTACTCTTTTTGTGGAGAAGGAACGTGATAAAGAAGTAAGCGATGATGAGGCTGAAGAAAAGGAAGACAAAGAAGAAGAAAAAGAAAAAGAAGAGAAAGAGTCGGAAGACAAACCTGAAATTGAAGATGTTGGTTCTGATGAGGAAGAAGAAAAGAAGGATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTCAACAAAACAAAGCCCATCTGGACCAGAAATCCCGACGATATTACTAATGAGGAGTACGGAGAATTCTATAAGAGCTTGACCAATGACTGGGAAGATCACTTGGCAGTGAAGCATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
hsa_circ_0033397	GTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGG
hsa_circ_0033398	GTGGAGAAGGAACGTGATAAAGAAGTAAGCGATGATGAGGCTGAAGAAAAGGAAGACAAAGAAGAAGAAAAAGAAAAAGAAGAGAAAGAGTCGGAAGACAAACCTGAAATTGAAGATGTTGGTTCTGATGAGGAAGAAGAAAAGAAGGATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTCAACAAAACAAAGCCCATCTGGACCAGAAATCCCGACGATATTACTAATGAGGAGTACGGAGAATTCTATAAGAGCTTGACCAATGACTGGGAAGATCACTTGGCAGTGAAGCATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAGGTGAGACCAAGGACCAGGTAGCTAACTCAGCCTTTGTGGAACGTCTTCGGAAACATGGCTTAGAAGTGATCTATATGATTGAGCCCATTGATGAGTACTGTGTCCAACAGCTGAAGGAATTTGAGGGGAAGACTTTAGTGTCAGTCACCAAAGAAGGCCTGGAACTTCCAGAGGATGAAGAAGAGAAAAAGAAGCAGGAAGAGAAAAAAACAAAGTTTGAGAACCTCTGCAAAATCATGAAAGACATATTGGAGAAAAAAGTTGAAAAGGTGGTTGTGTCAAACCGATTGGTGACATCTCCATGCTGTATTGTCACAAGCACATATGGCTGGACAGCAAACATGGAGAGAATCATGAAAGCTCAAGCCCTAAGAGACAACTCAACAATGGGTTACATGGCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTGGAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGG
hsa_circ_0033399	GTGAACCTATGGGTCGTGGAACAAAAGTTATCCTACACCTGAAAGAAGACCAAACTGAGTACTTGGAGGAACGAAGAATAAAGGAGATTGTGAAGAAACATTCTCAGTTTATTGGATATCCCATTACTCTTTTTGTGGAGAAGGAACGTGATAAAGAAGTAAGCGATGATGAGGCTGAAGAAAAGGAAGACAAAGAAGAAGAAAAAGAAAAAGAAGAGAAAGAGTCGGAAGACAAACCTGAAATTGAAGATGTTGGTTCTGATGAGGAAGAAGAAAAGAAGGATGGTGACAAGAAGAAGAAGAAGAAGATTAAGGAAAAGTACATCGATCAAGAAGAGCTCAACAAAACAAAGCCCATCTGGACCAGAAATCCCGACGATATTACTAATGAGGAGTACGGAGAATTCTATAAGAGCTTGACCAATGACTGGGAAGATCACTTGGCAGTGAAGCATTTTTCAGTTGAAGGACAGTTGGAATTCAGAGCCCTTCTATTTGTCCCACGACGTGCTCCTTTTGATCTGTTTGAAAACAGAAAGAAAAAGAACAACATCAAATTGTATGTACGCAGAGTTTTCATCATGGATAACTGTGAGGAGCTAATCCCTGAATATCTGAACTTCATTAGAGGGGTGGTAGACTCGGAGGATCTCCCTCTAAACATATCCCGTGAGATGTTGCAACAAAGCAAAATTTTGAAAGTTATCAGGAAGAATTTGGTCAAAAAATGCTTAGAACTCTTTACTGAACTGGCGGAAGATAAAGAGAACTACAAGAAATTCTATGAGCAGTTCTCTAAAAACATAAAGCTTGGAATACACGAAGACTCTCAAAATCGGAAGAAGCTTTCAGAGCTGTTAAGGTACTACACATCTGCCTCTGGTGATGAGATGGTTTCTCTCAAGGACTACTGCACCAGAATGAAGGAGAACCAGAAACATATCTATTATATCACAG
hsa_circ_0033340	GTCCATAGGAAGTACACAGGGGAAGACTTTGATGAAGATCTACGGACAGTGTTGAGACGTTCTGGCTGTAAAAATGAAAAGATAGCATTTATAATGGATGAATCTAATGTGTTAGATTCTGGATTCCTGGAGCGAATGAATACCCTTCTGGCCAATGGAGAGGTGCCTGGTCTCTTTGAAGGAGACGAGTATGCCACCTTGATGACGCAGTGCAAAGAGGGGGCACAGAAGGAAGGCCTGATGCTGGACTCGCACGAGGAGCTCTACAAGTGGTTCACTAGCCAGGTTATCCGCAACCTCCACGTCGTGTTCACCATGAACCCGTCCTCGGAGGGACTCAAGGACCGGGCAGCTACATCACCAGCACTTTTCAACAG
hsa_circ_0033385	GTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGATGGAAATGTAGTGCTCGAGTCACATTCTGCTTAAAGTTGTAACAAATACAGATGAGTTAAAAGATATTGTGTGACAGTGTCTTATTTAGGGGGAAAGGGGAGTATCTGGATGACAGTTAGTGCCAAAATGTAAAACATGAGGCGCTAGCAGGAGATGGTTAAACACTAGCTGCTCCAAGGGTTGACATGGTCTTCCCAGCATGTACTCAGCAGGTGTGGGGTGGAGCACACGTAGGCACAGAAAACAGGAATGCAGACAACATGCATCCCCTGCGTCCATGAGTTACATGTGTTCTCTTAGTGTCCACGTTGTTTTGATGTTATTCATGGAATACCTTCTGTGTTAAATACAGTCACTTAATTCCTTGGCCTTA
sh-NC	CCGGGTGGTTGTGT TAAGGCCAAGCTCGAGCTTGGCCTTAACACAACCACTTTTTG
sh-circ-HSP90A#1	CCGGTTCCTTGGCCTTAGTGGTTGTCTCGAGACAACCACTAAGGCCAAGGAATTTTTG
sh-circ-HSP90A#2	CCGGTGGCCTTAGTGGTTGTGTCAACTCGAGTTGACACAACCACTAAGGCCATTTTTG
sh-HSP90A#1	CACCATCAAACGAGCATGTCTACACCTCGAGGTGTAGACATGCTCGTTTGA
sh-HSP90A#2	CACCGTGTTTGTGCGAAGATGTTGCTCTCGAGAGCAACATCTTCGCACAAACA
pcDNA3.1/HSP90A	atggcttccactgtgacacccgaggttcgctgggcccagcgctcttctgctaccgatcccgagaacaacttcatctacctcaccattagcgttcccgacgtccctacttccaaccttaagctcgaccttaagcccactggtctcgtcttcaccggccactccgacaccctcaagaagacctaccatgtcgagcttgagttctacgccgaaattgaccctgctgcgagccaggtccaccacacggcccgtgatgtcgagatgaagctccgcaagaaggagcttgatgctcattactggccccgtctcctcaaggagcccaagaaggttcacttcctcaagaccgactttgacaagtgggttgatgaggacgagcagcacgaggccgctgaggatgacttcagcaactttggcggtatggaagggatgggtggaatgcctggtatgggtggcatgaatggcatgggtggtatgggtggtgacttcggcggtatcgacttctccaagttgggcggtgctggtttgcccggcggcgacgatgaggaagactccgacgatgacatgcctcctctcgagggtgagggcgaggctgataaggccgaggaggcaaaggatgccaaggagactgtcgacacgtcaaaggaggctgcgtaa
pcDNA3.1/PD-L1	atggagcagacttttctcttggttttacatgtagtcttatggccaactcttgcagccttgttcacagtggaggtggacagtctctcacatgtggcagagttctacggtgatgtcaccatgggctgcaggttccaacctgggagctgggacccgaacctgtcagtgatatggcaacgggtccagcctcttccagatgtagaggtgtacaggctggataacggacaggagaacctcacttctcagaatttccagtaccgcggcagggcacggttggtgtcggaggagctgaccaatggctgggccaagctacatgtgtccaggctgaggatcaacgactctggggtgtacagatgtcttgtggaaatggggggagctgattataaacagaccactttgactgtcaaagctacctataagactatcattaaaagcatgcagagacgtgggggaggtgaggtggagctggcctgtgagtctgagggctatcctcttgccacaatcaactggagagacaagagtctcaggaacatcaaatccaacgacaccgttgtgaaaactccaaaccagcttttccatgtcaccagcaaaataacggtcaaatactctgagaaaaacaactatacatgtgccttcgtggagaaaggggaagctccaaagggtccatcagccaggtttgacatcccagatgaaatacctgtgattgagagtaaacctaacaccctttctattgtattgggcacgacattgacggtggctatgatcattgtagctaccatcttcgggtatcgcagacagaaaggtaggctcaggaccctcaaactctaa
bio-circ-HSP90A	ACACAACCACTAAGGCCAAG-biotin
NC mimics	acacagcaguugucaugauauu
miR-424-5p mimics	cagcagcaauucauguuuugaa
NC inhibitor	cacauguuaauaugcugcugaa
miR-424-5p inhibitor	uucaaaacaugaauugcugcug

Cell transfection

Specific shRNAs against circ-HSP90A or HSP90A or USP30, sh-NC, pcDNA3.1 vector targeting HSP90A or PD-L1, and the empty vector were all procured from Genechen (Shanghai, China). MiR-424-5p mimics/inhibitor and NC mimics/inhibitor were offered by GenePharma (Shanghai, China). Plasmid transfection was performed by means of Lipotransfectamine 3000 (Thermo Fisher Scientific). All experiments were performed at least three times.

Cell counting kit-8 (CCK-8)

Transfected cells were planted in 96-well plates for incubation. Next, cells were treated with CCK-8 solution (Dojindo, Tokyo, Japan). The absorbance was observed at 450 nm. Three experiments were required.

Colony formation

Transfected cells were grown in 6-well plates and cultured for 8 days. Afterward, the number of cells was counted after fixation and staining. This experiment was in triplicate.

Wound healing

Transfected cells were plated into 24-well plates for incubation. Then, a scratch was made with a plastic tip. The time of wound infliction was considered as 0 h, and wound closure was photographed after 24 h using a microscope. Three experiments were required.

Transwell

A 24-well transwell chamber (BD Biosciences, USA) was adopted for cell invasion assay and performed three times. Transfected cells in serum-free medium (Gibco) were placed into upper chambers pre-coated with Matrigel (BD Biosciences). The lower chambers were placed with medium containing 20% FBS (Gibco). After 48 h, the non-invaded cells were wiped off, and the invaded cells through the Matrigel were fixed and stained and then observed via a microscope (Olympus, Japan). Three experiments were required.

Sphere formation

Transfected cells were plated on ultra-low attachment plates (Corning, NY, USA) in serum-free medium containing EGF (20 ng/mL, Invitrogen, USA), FGF (20 ng/mL, Invitrogen, USA), heparin (4 mg/mL, Sigma-Aldrich, Germany) and B27 (2%, Invitrogen, USA). After 14 days of incubation, the sphere number and size were analyzed. All experiments were performed for 3 times.

Luciferase reporter assay

Cignal Finder Reporter Array (336,841, QIAGEN, Dusseldorf, Germany) was used to detect the possible signaling pathway downstream of circRNA. Transfected cells were treated with the various luciferase reporter constructs to examine the luciferase activities of the Notch pathway, the Wnt pathway, the STAT3 pathway, the NF-κB pathway, the PI3K/AKT pathway, the NRF2 pathway, the Hedgehog pathway, the MAPK/JNK pathway, and the MAPK/ERK pathway, respectively. Besides, transfected cells were incubated with the pGL3-basic reporter vectors (Promega, Madison, WI, USA) including PD-L1 promoter. Moreover, the pmirGLO dual-luciferase vectors (Promega, Madison, WI, USA) of circ-HSP90A or PD-L1 3′UTR containing miR-424-5p sequences were co-transfection with miR-424-5p mimics or NC mimics into cells. Luciferase activities were measured via a luciferase reporter assay kit (Promega). The experiment was in triplicate.

Western blot

RIPA lysis buffer (Beyotime) was applied to extract total protein. Then, the protein was separated by 10% SDS-PAGE gel and moved onto PVDF membranes (Millipore, MA, USA). The membranes were incubated with primary antibodies including anti-p-STAT3 (Abcam, 1/2000), anti-STAT3 (Abcam, 1/1000), anti-c-Myc (Abcam, 1/1000), anti-MMP2 (Abcam, 1/1000), anti-MMP9 (Abcam, 1/1000), anti-HSP90 (Abcam, 1/10000), anti-USP30 (Abcam, 1/500), anti-PD-L1 (Abcam, 1/1000) and anti-GAPDH (Abcam, 1/1000) after blocking. Next, the membranes were incubated with secondary antibodies. The immunoblots were visualized through ECL chemiluminescence detection system (Thermo Scientific). The experiment was performed thrice.

Ubiquitination assay

Ubiquitin and the indicated plasmids were transfected into cells for 48 h. Then cells were immunoprecipitated with the HSP90A antibodies together with protein A/G beads (Life Technologies) with rotation and then examined by western blot. Anti-Ubiquitin (ab134953), anti-HSP90A (ab203126), and anti-GAPDH (ab181602) were all purchased from Abcam (Cambridge, MA, USA). Three experiments were required.

RNA pull down assay

Using a Pierce Magnetic RNA Protein Pull-Down Kit (Thermo Fisher Scientific), we explored circ-HSP90A-interacting proteins. Protein extracts from cells were processed with biotinylated circ-HSP90A probes and beads for 1 h of incubation. The RNA-complex was monitored by western blot.

For detecting the interaction between circ-HSP90A and miRNAs, cell lysates were transcribed with biotinylated circ-HSP90A and then treated with streptavidin beads. RT-qPCR was employed to examine the enrichment of selected miRNAs after washing. All experiments were performed in triplicate.

RNA immunoprecipitation (RIP)

This assay was implemented through a RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). Transfected cells were lysed and incubation with RIP buffer which contained magnetic beads. The beads were conjugated with anti-Ago2 antibody or anti-USP30 or anti-IgG. Then, the cells samples were treated with Proteinase K and the immunoprecipitated RNA was purified and then detected by RT-qPCR. The experiment was taken for three times.

Flow cytometry analysis

Transfected cells were co-cultured with CD8⁺ T cells, and flow cytometry was then conducted for assessing the percentage of CD8⁺ T cells or CD8⁺ T-cell apoptosis. All experiments were performed thrice.

Subcellular fractionation

Cytoplasmic or nuclear RNA was isolated from cells and purified employing a PARIS Kit (Ambion). The expression level of circ-HSP90A was examined by RT-qPCR. All experiments were performed thrice.

Fluorescent in situ hybridization (FISH)

The RNA FISH probe designed for circ-HSP90A was purchased from RiboBio. Cell nuclei were counterstained with DAPI for observation under a microscope. All experiments were performed thrice.

Statistical analysis

Data were exhibited as mean ± SD and analyzed using SPSS 22.0. Variance analyses were implemented via Student’s t test or one-way ANOVA. P < 0.05 was considered statistically significant. All assays were implemented thrice.

Results

Circ-HSP90A expression and function in NSCLC cells

A current study has indicated that HSP90A is abundant in NSCLC patients [20]. To search for circRNAs linked to NSCLC, we selected 16 circRNAs derived from HSP90A and applied RT-qPCR to measure their expression levels in five NSCLC cells (H1299, A549, PC9, CAL-12T, and H1915) and normal human bronchial epithelial cells (BEAS-2B). As revealed in Figure S1A-O and Fig. 1A, only up-regulation of hsa_circ_0033393 (named circ-HSP90A) was observed in all selected NSCLC cells compared with BEAS-2B cells, mirroring a potential role in NSCLC. As the expression was most obviously shown in H1299 and A549 cells, these two cell lines were chosen as the subject. Before probing circ-HSP90A function in NSCLC, we firstly validated the character of circ-HSP90A through several assays. Firstly, the head-to-tail splicing of circ-HSP90A is displayed in Figure S2A. Then, we adopted divergent primers to amplify circ-HSP90A and adopted cDNA and genomic DNA (gDNA) as templates. The circ-HSP90A magnification product was only discovered in cDNA by divergent primers rather than in gDNA (Figure S2B). Moreover, the fragment of linear-HSP90A was reduced after RNase R treatment, whereas circ-HSP90A was unchanged after RNase R treatment (Figure S2C). In addition, we discovered that circ-HSP90A was more stable than the linear form of HSP90A after treating actinomycin D (Act D) (Figure S2D). All above data suggested that circ-HSP90A was stably overexpressed in NSCLC cells. Subsequently, we established stable circ-HSP90A knockdown vectors and applied RT-qPCR to verify circ-HSP90A silence efficiency (Fig. 1B). Followed by CCK-8 and colony formation experiments, we found that circ-HSP90A depletion obviously inhibited two NSCLC cells proliferation (Fig. 1C, D). Besides, the migratory and invasive capacities were repressed when circ-HSP90A was down-regulated in two NSCLC cells, as evidenced by wound healing and transwell assays (Fig. 1E, F). At the same time, we tried to examine the stemness of H1299 and A549 cells under circ-HSP90A silence. Sphere formation assays depicted that circ-HSP90A deficiency reduced the sphere formation efficiency (Fig. 1G).

Fig. 1.

Circ-HSP90A promotes NSCLC cell proliferation, migration, invasion and stemness. A Circ-HSP90A expression in NSCLC cells and BEAS-2B cells was detected via RT-qPCR. B Depletion of circ-HSP90A was confirmed by RT-qPCR in two NSCLC cells. C, D Proliferation was evaluated via CCK-8 and colony formation experiments in circ-HSP90A-shRNAs transfected cells. E, F migration and invasion were evaluated via wound healing and transwell assays in circ-HSP90A-shRNAs transfected cells. G Stemness was evaluated via sphere formation assays in circ-HSP90A-shRNAs transfected cells. *P < 0.05, **P < 0.01

Circ-HSP90A stabilizes HSP90A protein in NSCLC cells

Increasing reports have indicated that circRNAs can activate the signaling pathways to promote the progression of cancers, NSCLC included [21]. To assess whether circ-HSP90A facilitates NSCLC progression via regulating some certain pathways, we adopted a luciferase reporter assay kit to examine the activities of several common pathways in HEK293T and A549 cells transfected with circ-HSP90A-shRNAs. Compared to the control group, circ-HSP90A silence decreased the activity of the STAT3 pathway, whereas had no impacts on that of other pathways (Fig. 2A). Western blot analysis further validated that circ-HSP90A deletion reduced phosphorylation level of STAT3 as well as the levels of downstream genes such as c-Myc, MMP2 and MMP9 (Fig. 2B), suggesting that circ-HSP90A could activate the STAT3 pathway in NSCLC cells. Besides, recent studies have revealed that circRNAs exhibit diverse biological functions via regulating their host genes [22]. Thus, we assumed that circ-HSP90A might influence the STAT3 pathway and NSCLC progression via regulating HSP90A. We firstly measured HSP90A expression in five NSCLC cells. In contrast to BEAS-2B cells, HSP90A presented a high expression level in all NSCLC cells (Fig. 2C). We then detected HSP90A mRNA and protein levels in two NSCLC cells after transfection with circ-HSP90A-shRNAs. Intriguingly, we found that circ-HSP90A depletion could not influence HSP90A mRNA level but significantly decreased the protein level of HSP90A (Fig. 2D, E). We next treated both NSCLC cells with the protein synthesis inhibitor CHX and found that the stability of HSP90A protein was restrained after circ-HSP90A knockdown (Fig. 2F). Besides, we confirmed that proteasome inhibitor MG132 could weaken this phenomenon (Fig. 2G). Moreover, the ubiquitination of HSP90A protein was increased when circ-HSP90A was silenced in two NSCLC cells (Fig. 2H). To verify whether HSP90A promotes NSCLC cells proliferation, migration, invasion and stemness, we constructed HSP90A-knockdown H1299 and A549 cells (Figure S3A). It was manifested that HSP90A silence hindered two NSCLC cells proliferation (Figure S3B-SC). Consistently, the migratory and invasive abilities were inhibited in wound healing and transwell assays when HSP90A was down-regulated (Figure S3D, E). Additionally, sphere formation assays further validated that HSP90A depletion repressed the stemness of H1299 and A549 cells (Figure S3F). Moreover, the correlation between circ-HSP90A and HSP90A is detected and shown in Figure S3G.

Fig. 2.

Circ-HSP90A stabilizes HSP90A protein. A Luciferase activity of several pathways including Notch, Wnt, STAT3, NF-κB, PI3K/AKT, NRF2, Hedgehog, MAPK/JNK and MAPK/ERK was assessed in HEK293T and A549 cells transfected with circ-HSP90A-shRNAs. B Western blot detected the levels of p-STAT3, total STAT3, c-Myc, MMP2 and MMP9 in circ-HSP90A-shRNAs transfected H1299 and A549 cells. C RT-qPCR and western blot detected HSP90A expression in two NSCLC cells. D, E RT-qPCR and western blot measured HSP90A expression in circ-HSP90A-shRNAs transfected cells. F Two NSCLC cells with stable circ-HSP90A knockdown were treated with CHX, and HSP90A protein levels were analyzed by western blot. G Stable circ-HSP90A silence two NSCLC cells were treated with MG132 and then HSP90A protein levels were analyzed via western blot. H Cell lysates form stable circ-HSP90A deletion cells were immunoprecipitated with either control IgG or HSP90A antibody and then immunoblotted for ubiquitin and HSP90A. *P < 0.05, **P < 0.01, n.s. meant no significance

Circ-HSP90A recruits USP30 to de-ubiquitinate and stabilizes HSP90A protein

To unveil the molecular mechanism that why circ-HSP90A could stabilize HSP90A protein in NSCLC cells, RNA pull down assay was implemented to search out circ-HSP90A-interacting proteins. A specific band exhibited on the electrophoretic gel at 59 kDa in bio-circ-HSP90A WT compared to bio-circ-HSP90A-MUT (Fig. 3A). We then identified that this protein was USP30 after mass spectrometry. We further confirmed the combination between circ-HSP90A and USP30 by an independent immunoblot and a RIP assay (Fig. 3B, C). Besides, we also proved the combination of HSP90A and USP30 via RIP assays (Fig. 3D). More importantly, this bond was attenuated when circ-HSP90A expression was declined in H1299 and A549 cells (Fig. 3E). USP30 is an enzyme that de-ubiquitinates proteins [23]. Thus, we conjectured that circ-HSP90A might recruit USP30 to de-ubiquitinate and stabilize HSP90A protein. To validate our assumption, we reduced USP30 expression and measured the stability of HSP90A protein in H1299 and A549 cells treated with CHX (Fig. 3F, G). The results showed that USP30 silence obviously decreased the stability of HSP90A protein (Fig. 3H). At the same time, this phenomenon was reversed after MG132 treatment (Fig. 3I). As expected, USP30 reduction increased the ubiquitination of HSP90A protein (Fig. 3J).

Fig. 3.

Circ-HSP90A recruits USP30 to de-ubiquitinate and stabilizes HSP90A protein. A biotinylated wild-type and mutant-type of circ-HSP90A were transcribed in vitro for RNA pull down assays, followed by sliver staining. B immunoblotting for specific relation of USP30 with circ-HSP90A. C, D Interaction of USP30 with circ-HSP90A and HSP90A were detected by RIP assays. E RIP assays assessed the interaction of USP30 and HSP90A in two NSCLC cells with stable circ-HSP90A knockdown. F, G RT-qPCR and western blot verified the depletion efficiency of USP30 in two NSCLC cells. H Two NSCLC cells with stable USP30 knockdown were treated with CHX, and HSP90A protein levels were measured via western blot. I Stable USP30 silence cells were treated with MG132 and then HSP90A protein levels were analyzed via western blot. J Cell lysates form USP30 silence two NSCLC cells were immunoprecipitated with either control IgG or HSP90A antibody and then immunoblotted for ubiquitin and HSP90A. **P < 0.01

Circ-HSP90A influences the STAT3 pathway and NSCLC progression via HSP90A

After that, we investigated whether circ-HSP90A influences the STAT3 pathway and NSCLC progression via regulating HSP90A. We stably overexpressed HSP90A (Fig. 4A) and found that HSP90A overexpression reversed the reduced phosphorylation level of STAT3 as well as the levels of downstream genes such as c-Myc, MMP2, and MMP9 caused by circ-HSP90A silence (Fig. 4B). Besides, the reduced proliferation in circ-HSP90A-depleted H1299 and A549 cells was almost restored after HSP90A elevation together (Fig. 4C, D). Moreover, when HSP90A was up-regulated simultaneously, the repressive migration and invasion in two NSCLC cells after circ-HSP90A down-regulation were offset (Fig. 4E, F). Additionally, the inhibited stemness in circ-HSP90A-shRNA transfected cells was abolished after co-transfection of pcDNA3.1/HSP90A (Fig. 4G).

Fig. 4.

Circ-HSP90A influences the STAT3 pathway and NSCLC progression via HSP90A. A up-regulation efficiency of USP30 in two NSCLC cells was detected using RT-qPCR and western blot. Rescue assays were performed in two NSCLC cells transfected with sh-NC, sh-circ-HSP90A#1 and sh-circ-HSP90A#1 + pcDNA3.1/HSP90A, respectively. B Western blot detected the levels of p-STAT3, total STAT3, c-Myc, MMP2, and MMP9. C, D CCK-8 and colony formation assays detected proliferation. E, F Migration and invasion were assessed by wound healing and transwell assays. G Stemness was measured by sphere formation assays. **P < 0.01

Circ-HSP90A triggers CD8⁺T cells apoptosis through PD-L1

As report goes, tumor cells interact with and hinder the cytotoxic functions of CD8⁺ T cells in tumor microenvironment [24]. Hence, we speculated that circ-HSP90A might affect CD8⁺ T cells in NSCLC. To simulate the tumor microenvironment, the NSCLC cells were co-cultured with CD8⁺ T cells. The results from flow cytometry analysis showed that the percentage of CD8⁺ T cells was enhanced while CD8⁺ T cells apoptosis was declined after circ-HSP90A was down-regulated in H1299 and A549 cells (Fig. 5A, B). Besides, due to the fact that PD-1/PD-L1 interaction can repress CD8⁺ T cells activity and reinforce the immune evasion in tumors [25], we further supposed that circ-HSP90A might influence the alteration of CD8⁺ T cells via PD-1/PD-L1 checkpoint. In order to certify our assumption, we firstly detected PD-L1 mRNA and protein levels in circ-HSP90A-shRNAs transfected cells using RT-qPCR and western blot. As a result, we found that the mRNA and protein levels of PD-L1 were reduced upon circ-HSP90A silence (Fig. 5C, D). Subsequently, we overexpressed PD-L1 and severally co-transfected them together with sh-circ-HSP90A#1 into the co-culture system (Fig. 5E, F). We found that the reduced ratio of CD8⁺ T cells as well as the induced CD8⁺ T cells apoptosis caused by circ-HSP90A silence was reversed after co-transfection of pcDNA3.1/PC-L1 (Fig. 5G, H).

Fig. 5.

Circ-HSP90A riggers CD8⁺ T cells apoptosis through PD-1/PD-L1 immune checkpoint. A, B CD8⁺ T cells were co-cultured with two NSCLC cells with circ-HSP90A down-regulation. Flow cytometry analysis assessed CD8⁺ T cell percentage and apoptosis. C, D RT-qPCR and western blot detected PD-L1 mRNA and protein levels in two NSCLC cells with circ-HSP90A silence. E, F RT-qPCR and western blot verified the elevation efficiency of PD-L1 in two NSCLC cells. G, H CD8⁺ T cells were co-cultured with two NSCLC cells and then transfected with sh-NC, sh-circ-HSP90A#1, and sh-circ-HSP90A#1 + pcDNA3.1/PD-L1. Flow cytometry analysis assessed CD8⁺ T cell percentage and apoptosis. **P < 0.01

Circ-HSP90A promotes the immune evasion of NSCLC via sponging miR-424-5p to up-regulate PD-L1

Furthermore, we investigated how circ-HSP90A regulates PD-1/PD-L1 immune checkpoint in NSCLC cells. Many studies have evidenced that the localization of circRNAs within cells is the main determinant of their molecular functions [26]. Based on subcellular fractionation and FISH experiments, we identified that the cellular location of circ-HSP90A was in the cytoplasm (Figure S4A, B). As is known to all, cytoplasmic circRNAs can interact with miRNAs to regulate mRNA expression. To test whether circ-HSP90A regulates PD-L1 expression via binding to miRNAs, RIP assays were carried out. It demonstrated that circ-HSP90A and PD-L1 were both enriched in Ago2 antibody (Figure S4C). At the same time, we ruled out the transcriptional regulation of PD-L1 influenced by circ-HSP90A, for that circ-HSP90A silence could not affect the luciferase activity of PD-L1 promoter (Figure S4D). Therefore, we continued to explore the possible miRNAs combined with both circ-HSP90A and PD-L1. Through starBase (http://starbase.sysu.edu.cn) database, 12 miRNAs were observed (Fig. 6A). To confirm the interaction between circ-HSP90A and miRNAs, a biotinylated circ-HSP90A-specific probe was designed to perform RNA pull down assay in two NSCLC cells. The result displayed that only miR-424-5p was markedly enriched, while other irrelevant miRNAs were not (Fig. 6B). Thus, miR-424-5p was chosen for subsequent analyses. The separate binding sites of miR-424-5p and circ-HSP90A along with the mutant sequences are shown in Fig. 6C. To validate the effectiveness of these binding sites, we increased miR-424-5p expression (Figure S5A) and constructed wild-type and mutant pmirGLO-circ-HSP90A vectors incorporating miR-424-5p binding sites for dual luciferase reporter assays. The results manifested that enforced miR-424-5p expression lessened the activity of circ-HSP90A-WT but not circ-HSP90A-MUT (Fig. 6D). Similarly, the base pairing of miR-424-5p and PD-L1 along with the mutant fragments is presented in Fig. 6E, and luciferase reporter assays further proved that miR-424-5p overexpression significantly reduced the luciferase activity of PD-L1 3′UTR-WT, but barely changed that of PD-L1 3′UTR-MUT (Fig. 6F). We further performed rescue assays to certify whether miR-424-5p is necessary for circ-HSP90A modulation on the immune evasion of NSCLC. Before this, we down-regulated miR-424-5p in two NSCLC cells (Figure S5B), and we found that in the co-culture system, the effects of enhanced ratio of CD8⁺ T cells and reduced CD8⁺ T cells apoptosis caused by circ-HSP90A silence were abrogated after co-transfecting of miR-424-5p inhibitor (Figure S5C, D).

Fig. 6.

Circ-HSP90A sponges miR-424-5p to up-regulate PD-L1. A Common miRNAs of circ-HSP90A and PD-L1 were predicted by starBase. B Combination between circ-HSP90A and miRNAs was measured by RNA pull down assay. C Binding sites of circ-HSP90A and miR-424-5p. D Luciferase activities of circ-HSP90A-WT and circ-HSP90A-MUT were assessed in miR-424-5p overexpressed cells. E Binding sites of PD-L1 and miR-424-5p. F Luciferase activities of PD-L1 3′UTR-WT and PD-L1 3′UTR-MUT were assessed in miR-424-5p overexpressed cells. **P < 0.01

Discussion

Emerging studies have demonstrated that circRNAs are momentous participants in the progression of human cancers, NSCLC included [27]. CircRNA_100876 predicts poor prognosis of NSCLC patients [28]. CircFOXM1 promotes proliferation of NSCLC cells via acting as a ceRNA to increase FAM83D [29]. Circ_0067934 contributes to NSCLC development by activating Wnt/β-catenin pathway [30]. Herein, we discovered that circ-HSP90A expression was elevated in NSCLC cells. This research for the first time discussed the relation between circ-HSP90A and NSCLC. Functional assays indicated that circ-HSP90A facilitated cell proliferation, migration, invasion, and stemness in NSCLC.

Mechanistically, this study unveiled that circ-HSP90A activated the STAT3 pathway in NSCLC. Excessive activation of the STAT3 signaling is strongly involved in many cancers, making activated STAT3 a promising possible therapeutic target supported by clinical studies [31]. Besides, many reports have displayed that circRNAs regulate the signaling pathways via affecting their host gene expression (32). To uncover the possible mechanism of STAT3 activation regulated by circ-HSP90A, our study investigated the interaction between circ-HSP90A and HSP90A and found that circ-HSP90A could stabilize HSP90A protein in NSCLC cells. HSP90A has been registered to work as an oncogene in hepatocellular carcinoma [33]. Likewise, our study also proved that HSP90A expression was increased in NSCLC cells and facilitated NSCLC progression.

A further finding in our study was that both circ-HSP90A and HSP90A bound to USP30 in NSCLC cells. USP30 is a type of deubiquitinase that can reverse ubiquitin modifications and is therefore considered as an essential modulator of the ubiquitin system [34]. Our study indicated for the first time that USP30 was physically associated with and stabilized USP30 by its deubiquitinase activity. Additionally, rescue assays were carried out to prove that circ-HSP90A promoted the STAT3 signaling and NSCLC progression via recruiting USP30 to stabilize HSP90A expression.

For all we know, tumor cells can modify the T cell activity to get out from the anti-tumor immune response and thereby promoting tumor cells survival [35]. Moreover, recent studies have proposed that tumor cells can cooperate and stimulate CD8⁺ T cells apoptosis to expedite tumor progression [36]. In the current study, we disclosed that circ-HSP90A down-regulation could induce CD8⁺ T cells activity while attenuate CD8⁺ T cells apoptosis. Moreover, accumulating evidences have demonstrated that PD-L1 and PD-1 are expressed in NSCLC and are related to the density of CD8⁺ T-cells, implying an adaptive immune resistance mechanism in NSCLC [37]. PD-L1 expression is majorly presented in tumor and immune cells and is correlated with PD-1 expression (38). In our study, we found that circ-HSP90A promoted CD8⁺ T cells apoptosis via up-regulating PD-L1 expression. As reported previously, circRNAs can modulate mRNA expression through sequestering miRNAs [39]. Also, circRNA-controlled ceRNA mechanism has been widely reported in NSCLC [40]. Consistently, our study found that circ-HSP90A promoted PD-L1 expression via sponging miR-424-5p. MiR-424-5p has been registered to be lowly expressed in NSCLC and inhibits the radiosensitivity of NSCLC [41]. Our study firstly showed that circ-HSP90A altered CD8⁺ T cell activity through binding to miR-424-5p. Taken together, targeting circ-HSP90A potentially promoted the effect of immunotherapy in NSCLC via PD-1/PD-L1.

Our research firstly disclosed that circ-HSP90A facilitated NSCLC cell proliferation, migration, invasion, and stemness via recruiting USP30 to stabilize HSP90A and activate the STAT3 pathway. At the same time, circ-HSP90A regulated miR-424-5p to activate PD-L1, resulting in the suppression of CD8⁺ T cells and the immune evasion of NSCLC cells (Fig. 7). All above findings implied that HSP90A might be a hopeful target for the immunotherapy via PD-L1/PD-1 blockade in NSCLC. However, our study also presented some limitations. First, the detail regulatory mechanism of why circ-HSP90A was up-regulated in the NSCLC is still unclear. More, the specific STAT3 phosphorylation site activated by circ-HSP90A is still not explained. In the future, both deficiencies will be studied in depth.

Fig. 7.

Graphical Abstract illustrates the functional mechanism of circ-HSP90A in NSCLC

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

JL, JZ, and BH contributed to the conceptualization. JL, JZ, BH, CJ, XY, TJ, and XW were involved in the data curation. JL, JZ, BH, CJ, XY, TJ, and XW helped in the formal analysis. TJ and XW were involved in the investigation. TJ and XW contributed to the methodology. JL, JZ, and BH were involved in the project administration. TJ and XW contributed to software. JL, JZ, BH were involved in the supervision. JL, JZ, and Bengang Hui were involved in the validation. JL, JZ, and BH contributed to the visualization. JL, JZ, and BH were involved in writing—original draft preparation. JL, JZ, and BH contributed to writing—review and editing.

Funding

None.

Data availability

Data sharing is not applicable.

Declarations

Competing interests

No potential conflict of interest was reported by the authors.

Consent for publication

We hope it to be considered for publication in your journal. All authors agreed upon the publication of this manuscript.

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors.