CircRNA hsa_circ_0001627 aggravates cervical cancer progression through upregulation of FNDC3B and activating PI3K/mTOR signaling pathway

Abstract

Circular RNAs (CircRNAs) are key regulators in the development and progression of human cancers. However, the biological roles and mechanisms of circRNAs in gastric cancer (GC) remain largely unknown. Analyzing circRNA microarray dataset (GSE102686) and clinical specimens, a novel circRNA termed hsa_circ_0001627, was identified and it was highly expressed in CC cancerous tissues and cells, and was associated with poor clinical outcomes. Functionally, hsa_circ_0001627 silencing impaired the malignant progression of CC cells and the growth of CC xenografts in nude mice. Mechanistically, hsa_circ_0001627 acted as a miR-1225-5p sponge, thus indirectly regulating FNDC3B and leading to the activation of PI3K/mTOR signaling pathway. Collectively, the present study indicates that hsa_circ_0001627 regulates miR-1225-5p/FNDC3B/PI3K/mTOR axis and functions as an oncogene in CC progression, suggesting the potential therapeutic use of hsa_circ_0001627 in CC treatment.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12079-022-00696-w

Introduction

Cervical cancer (CC), a common gynecological cancer, has a high morbidity and mortality (Buskwofie et al. 2020). Every year, approximately 500,000 women are diagnosed with CC, leading to far more than 300,000 deaths worldwide (Cohen et al. 2019). Scientific evidence clearly manifests that a great amount of cases of CC are caused by persistent infection of oncogenic genotypes of the human papillomavirus (HPV), such as HPV16 and HPV18 (Jee et al. 2020). In recent years, HPV screening programs have become effective measures to prevent CC (Eun and Perkins 2020). Radical surgery following radiotherapy and chemotherapy is the main strategy for the treatment of CC (Sala et al. 2020). However, the prognosis of CC patients is often less than expected due to tumor recurrence and metastasis (Kilic et al. 2021). Therefore, in-depth research of the molecular mechanism of CC etiology is of great importance for the development of cancer-related biomarkers.

Circular RNA (CircRNA) is a novel non-coding RNA with covalently closed loops derived from pre-mRNA back-splicing (Chen and Yang 2015). CircRNAs are resistant to RNA exonuclease degradation, and thus are more stable than related linear mRNAs (Qu et al. 2015). Other characteristics, including evolutionary conservatism and high abundance also give circRNAs distinct advantages in the development and application of new clinical diagnostic markers (Kristensen et al. 2019). Increasing circRNAs have been discovered, and their functions have been gradually reported, especially in promoting the physiological and pathological progression, such as cell proliferation, apoptosis, migration and carcinogenesis (Zhang et al. 2018, Han et al. 2018). Dysregulation of circRNAs have been associated with many types of human diseases particularly various types of cancer, such as prostate cancer (Taheri et al. 2021), pancreatic cancer (Sayad et al. 2022) and CC (Najafi 2022). Previous studies have shown that circRNAs are involved in many malignancies by acting as miRNA sponges (Meng et al. 2017; Li et al. 2020b). Nevertheless, their roles in CC are unascertained.

In the present study, we attempt to identify CC-related circRNAs. As identified by GSE102686 dataset, the upregulation of hsa_circ_0001627 is observed in CC tissues and cell lines. In the functional assays, we found that hsa_circ_0001627 might play an oncogenic role in CC cells in vitro and in vivo. In mechanism, hsa_circ_0001627 acts as a sponge of miR-1225-5p, which further activates fibronectin type III domain containing 3B (FNDC3B)-mediated PI3K/mTOR signal pathway. In summary, hsa_circ_0001627 promoted the progression of CC by upregulating FNDC3B and inducing the activation of PI3K/mTOR pathway via sponging miR-1225-5p.

Materials and methods

Bioinformatic analysis of circRNA expression profile in Gene expression Omnibus (GEO) datasets

We searched GEO (http://www.pubmed.com/geo) to identify dataset suitable for the analysis (Jiao et al. 2020). The GSE102686 matrix data file was downloaded from the GEO database, containing data from 5 cervical squamous cell carcinoma tissues and 5 paired-paracancerous cervical tissues and the GEO2R online tool was used to screen the differentially expressed circRNAs. P value < 0.05 was set as the threshold for significantly differential expression.

Tissue samples

The ethic approval was acquired from Shengjing Hospital of China Medical University. Tissue specimens were collected from 40 CC patients undergoing radical resection at Shengjing Hospital of China Medical University during January 2015 and January 2016. All patients had not received preoperative radiotherapy or chemotherapy before tissue resection. All tissues were collected immediately after specimen resection and stored in a refrigerator at -80℃. All histopathological types were confirmed by two pathologists with more than 10 years of experience and with the informed consent of the patients and their families.

Cell culture

Human cervical epithelial cell line (HCerEpiC), and CC cell lines (HeLa, SiHa, CaSki, C33A) were purchased from Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM (Gibco, Carlsbad, CA, USA) in an incubator with 5% CO₂ at 37℃. Meanwhile, 10% fetal bovine serum, 100U/mL penicillin and 100 µg/mL streptomycin were supplemented during incubation.

Transfection

HeLa and C33A cells (2 × 10⁵ cells/well) were seeded onto 6-well plates for 24 h and prepared for transfection using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Hsa_circ_0001627 small interfering RNA (siRNA), miR-1225-5p mimic, inhibitor, plasmid vector pcDNA3.1 used for FNDC3B overexpression and the corresponding negative controls were designed by Shanghai GenePharma (Shanghai, China). All oligonucleotide sequences were listed in Table S1.

Quantitative real-time PCR (QRT-PCR) assay

Total RNA of tissues and cells was extracted by Trizol (Invitrogen), and cDNA was synthesized according to the instructions of the First Strand cDNA Synthesis Kit purchased from Thermo Scientific (Shanghai, China). The procedure of real-time PCR was described as follows: 95 °C for 5 min; 45 cycles of 95 °C for 15 s and 60 °C for 30 s; 95 °C for 15 s, 60 °C for 1 min and 95 °C for 3 min. Primers sequences were designed using Primer Premier v5.0 and listed in Table S2. The results were calculated with the 2^−∆∆Ct method and normalized to either GAPDH or U6 snRNA.

CCK-8 assay

One hundred microliter cell suspension was prepared in a 96-well plate at 2 × 10³ cells/well, and incubated for 0, 24, 48 or 72 h. Then, 10 µl CCK-8 solution was added to each well and incubated for 1 h. The absorbance at 450 nm was measured with a microplate analyzer.

Colony formation assay

Transfected cells (1 × 10³ cells/well) were re-seeded into 6-well plates. Two weeks later, cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet solution for 30 min at room temperature. The number of colonies was counted under the microscope.

Transwell assay

Transwell chambers pre-coated with or without matrigel were used to examine cell migration and invasion. A total of 600 µl medium containing 10% FBS was transferred to the lower chamber, while 100 µl serum-free medium containing 2 × 10⁵ cells was added to the upper chamber. Twelve hours later, the cells that passed through the filter were fixed with paraformaldehyde for 30 min, followed by 0.1% crystal violet staining for 15 min at room temperature. The stained cells were finally counted under a microscope.

Bioinformatic prediction

The target miRNAs of hsa_circ_0001627 were predicted using CircInteractome (https://circinteractome.nia.nih.gov). The TargetScan website (http://www.targetscan.org/) was used to predict the target genes of miR-1225-5p.

Dual-luciferase reporter assay

The wild-type and mutant pmir-GLO vectors of hsa_circ_0001627 and FNDC3B were co-transfected into HeLa and C33A cells using Lipofectamine 3000 together with miR-1225-5p mimic or miR-NC. The luciferase activities were detected using Renilla-Firefly Luciferase Dual Assay Kit (Pierce Biotechnology, Rockford, IL, USA) after 48 h transfection.

RNA immunoprecipitation (RIP)

RIP assay was carried out in both HeLa and C33A cells using Dynabeads™ Protein G Immunoprecipitation Kit (Invitrogen). Briefly, cell lysate supernatant was treated with anti-Ago2/IgG-conjugated magnetic beads. The immunoprecipitated complex was digested with proteinase K, and then subjected to PCR analysis.

Western blot

After exacting from HeLa and C33A cells using RIPA (Thermo Scientific), 30 µg protein were subjected to electrophoresis using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was transferred to polyvinylidene fluoride (PVDF) membrane at 250 mA at 4℃ for 2 h and sealed with 5% skimmed milk powder at room temperature for 1 h. After incubation with these indicated antibodies (1:1000 dilution) overnight at 4 °C: FNDC3B (22605-1-AP; Proteintech Group, Wuhan, China), p-PI3K (#17,366; Cell Signaling Technology, Boston, MA, USA), PI3K (#4249; Cell Signaling Technology), p-mTOR (#2983; Cell Signaling Technology), mTOR (#5536; Cell Signaling Technology), and GAPDH (#5174; Cell Signaling Technology), the membranes were washed with tris buffered saline tween (TBST) and stained with secondary antibody (1:2000 dilution; #7074; Cell Signaling Technology) at room temperature for 2 h. After adding the Chemiluminescent Substrates (Invitrogen), the bands were scanned.

Xenograft mouse assay

Male BALB/c nude mice (4-week old) were randomly divided into 2 groups (si-NC group and hsa_circ_0001627 group, 6 mice per group). 1 × 10⁶ HeLa cells stably transfected with si-NC or hsa_circ_0001627 were subcutaneously injected into nude mice. Tumor volume was measured every 7 days. Four weeks later, mice were euthanized and tumors were excised. A part of tumors was kept at -80˚C in order to detect the expression levels of hsa_circ_0001627, miR-1225-5p, FNDC3B, matrix metalloproteinase-2 (MMP-2), and MMP-9 as well as PI3K/mTOR pathway. The other obtained tissues were subjected to hematoxylin and eosin (H&E) staining. The Animal Care and Use Committee of Shengjing Hospital of China Medical University approved this study.

Statistical analysis

All of the experiments were performed in triplicate. Values were expressed by the mean ± standard deviation (SD). GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA) was used for data analysis using Student’s t test or ANOVA followed by Tukey’s post hoc test. Survival analysis was plotted according to the Kaplan-Meier curves and log-rank test. The association between hsa_circ_0001627, miR-1225-5p and FNDC3B expression in CC tissues was analyzed by Pearson’s correlation analysis. P < 0.05 was regarded as statistically significance.

Results

Hsa_circ_0001627 is highly expressed in CC tissues and cell lines

CircRNA microarray profiling in CC were identified through GEO dataset (GSE102686). Analyzed by GEO2R analysis, hsa_circ_0001627 expression was markedly higher in CC samples than that in paracancerous samples (Fig. 1 A). We verified the high expression of hsa_circ_0001627 in CC clinical tissues (Fig. 1B). Furthermore, hsa_circ_0001627 was identified to be markedly increased in several CC cell lines (HeLa, SiHa, CaSki, C33A) compared to HCerEpiC cell line (Fig. 1 C). According to the median expression of hsa_circ_0001627 in CC tissues, the CC patients were classified into high expression group (n = 21) and low expression group (n = 19). Then, Kaplan-Meier survival analysis suggested that CC patients with high hsa_circ_0001627 expression had poor overall survival than those with low hsa_circ_0001627 expression (Fig. 1D). The relationship between hsa_circ_0001627 and the clinicopathological parameters was shown in Table S3, suggesting the correlations of hsa_circ_0001627 expression and FIGO stage, lymphatic metastasis and HPV infection. The above findings indicated that the upregulation of hsa_circ_0001627 may exert an oncogenic effect on the progression of CC.

Fig. 1.

Hsa_circ_0001627 is highly expressed in CC tissues and cell lines. (A) GEO2R analysis showing the expression of hsa_circ_0001627 in CC and paracancerous samples from GSE102686 dataset. (B) Relative expression of hsa_circ_0001627 was determined in CC (n = 40) and adjacent normal tissues (n = 40) by qRT-PCR. (C) Relative expression of hsa_circ_0001627 was examined in human cervical epithelial cell line (HCerEpiC) and CC cell lines (HeLa, SiHa, CaSki, C33A). (D) Kaplan-Meier analysis of overall survival rate of CC patients with low or high hsa_circ_0001627 expression. Data were represented as mean ± SD. ^*P < 0.05; ^***P < 0.001

Knockdown of hsa_circ_0001627 inhibits the proliferation, migration and invasion of CC cells in vitro

Three specific siRNAs were then used to silence the hsa_circ_0001627 expression in HeLa and C33A cells to further investigate the biological functions of hsa_circ_0001627 in CC. By means of qRT-PCR, high knockdown efficiencies of hsa_circ_0001627 siRNAs were confirmed in HeLa and C33A cells, especially siRNA#1 (Fig. 2 A). For the proliferation analysis using CCK-8 and colony formation assays, hsa_circ_0001627 siRNA resulted in a remarkable reduction of cell viability (Fig. 2B) and the number of colonies (Fig. 2 C) in HeLa and C33A cells. In addition, Transwell assays were performed to analyze the impact of hsa_circ_0001627 knockdown on the migration and invasion. As demonstrated in Fig. 2D and E, hsa_circ_0001627 interference alleviated the migratory and invasive abilities. These results revealed that hsa_circ_0001627 inhibition ameliorated the malignant behaviors of CC cells.

Fig. 2.

Knockdown of hsa_circ_0001627 inhibits the proliferation, migration and invasion of CC cells in vitro. A, Knockdown efficiencies of hsa_circ_0001627 siRNAs in HeLa and C33A cells were determined by qRT-PCR. B and C, Cell proliferation of HeLa and C33A cells transfected with si-NC or si-hsa_circ_0001627 were assessed via CCK-8 and colony formation assays. D and E, After hsa_circ_0001627 knockdown, the cell migration and invasion of HeLa and C33A cells were analyzed by Transwell assays. Data were represented as mean ± SD of at least three independent experiments. ^*P < 0.05, ^**P < 0.01; ^***P < 0.001

Hsa_circ_0001627 serves as a mir-1225-5p sponge in CC cells

To further investigate how hsa_circ_0001627 achieves its tumor-promoting effect in CC, we analyzed its potential target. The commonly used bioinformatics tool (Circinteractome) confirmed that hsa_circ_0001627 contains several binding sites with miR-1225-5p (Fig. 3 A). The results from luciferase reporter assay indicated that miR-1225-5p mimic which was proved to upregulate miR-1225-5p expression (Fig. 3B) could reduce the luciferase activity of HeLa and C33A cells after transfection with hsa_circ_0001627 wild-type plasmids; however, the same effect was never observed in cells transfected with mutant constructs (Fig. 3 C). We carried out RIP assay to validate the interplay between hsa_circ_0001627 and miR-1225-5p, and the results showed that hsa_circ_0001627 and miR-1225-5p were both enriched in anti-Ago2-treated cells (Fig. 3D). Besides, we found that miR-1225-5p was increased upon hsa_circ_0001627 silencing in HeLa and C33A cells (Fig. 3E). Meanwhile, the expression of miR-1225-5p was obviously reduced (Fig. 3 F) in CC cancerous tissues that that in the normal tissues. There was a negative correlation between miR-1225-5p expression and the hsa_circ_0001627 level in these CC tissues (Fig. 3G). The above results indicated that hsa_circ_0001627 reversely regulated the expression of miR-1225-5p in CC cells.

Fig. 3.

Hsa_circ_0001627 serves as a miR-1225-5p sponge in CC cells.

A, The predicted binding sites between hsa_circ_0001627 and miR-1225-5p. B, Overexpression efficiency of miR-1225-5p mimic in HeLa and C33A cells were determined by qRT-PCR. C, The luciferase reporter assays were performed in HeLa and C33A cells that were co-transfected with hsa_circ_0001627 WT/MUT luciferase vectors and miR-1225-5p mimic/miR-NC. D, RIP assay was performed in HeLa and C33A cells to further confirm the interaction between hsa_circ_0001627 and miR-1225-5p. E, The expression of miR-1225-5p was measured by qRT-PCR in HeLa and C33A cells after hsa_circ_0001627 silencing. F, Relative expression of miR-1225-5p was determined in CC (n = 40) and non-tumor tissues (n = 40) by qRT-PCR. G, Correlation between hsa_circ_0001627 and miR-1225-5p in CC tissues. Data were represented as mean ± SD of at least three independent experiments. ^**P < 0.01; ^***P < 0.001

FNDC3B is a downstream target of miR-1225-5p

Then, miR-1225-5p expression was silenced in HeLa and C33A cells by the transfection of the miR-1225-5p inhibitor (Fig. 4 A). The data in Fig. 4B C depicted that the mRNA and protein expression of FNDC3B was elevated after miR-1225-5p suppression in HeLa and C33A cells. Using an online bioinformatics database TargetScan, we found that FNDC3B might a target gene of miR-1225-5p (Fig. 4D). The results shown in Fig. 4E demonstrated that miR-1225-5p upregulation reduced luciferase activities of FNDC3B WT group instead of the mutant group in both HeLa and C33A cells. The FNDC3B mRNA level in CC tissues was found to be observably higher than that in normal non-tumor samples (Fig. 4 F). In CC tissues, the FNDC3B mRNA level was positively associated with the level of hsa_circ_0001627, while negatively correlating with miR-1225-5p (Fig. 4G). The si-hsa_circ_0001627 memorably decreased FNDC3B protein level, which was then reversed by miR-1225-5p knockdown (Fig. 4 H). Collectively, these results indicated that hsa_circ_0001627 sponged miR-1225-5p to regulate FNDC3B in CC cells.

Fig. 4.

FNDC3B is a downstream target of miR-1225-5p. A, Knockdown efficiency of miR-1225-5p inhibitor in HeLa and C33A cells were determined by qRT-PCR. B and C, The mRNA and protein levels of FNDC3B were measured in HeLa and C33A cells after miR-1225-5p knockdown. D, The binding sites of miR-1225-5p in the 3ʹ-UTR of FNDC3B were predicted by TargetScan. E, The interaction between FNDC3B and miR-1225-5p was determined by luciferase reporter assays in HeLa and C33A cells. F, Relative expression of FNDC3B was determined in CC (n = 40) and non-tumor tissues (n = 40) by qRT-PCR. G, Correlation between FNDC3B mRNA level and hsa_circ_0001627/miR-1225-5p level in CC tissues. H, The protein levels of FNDC3B were determined in HeLa and C33A cells after hsa_circ_0001627 and/or miR-1225-5p knockdown. Data were represented as mean ± SD of at least three independent experiments. ^**;##P < 0.01; ^***P < 0.001

Hsa_circ_0001627/miR-1225-5p/FNDC3B regulates CC cell progression by modulating the PI3K/mTOR signaling pathway

To determine whether hsa_circ_0001627 exerted a tumor-promoting effect via FNDC3B-mediated PI3K/AKT pathway, the following rescue experiment was performed. We overexpressed FNDC3B by overexpression vector, and the transfection efficiency was examined via western blotting (Fig. 5 A). In HeLa and C33A cells with silenced hsa_circ_0001627, upregulating FNDC3B promoted cell viability (Fig. 5B), colony forming rate (Fig. 5 C), migration (Fig. 5D) and invasion (Fig. 5E) capabilities. Furthermore, the protein expressions of the PI3K/mTOR signaling were analyzed. The silencing of hsa_circ_0001627 inhibited the protein levels of phosphorylated PI3K and mTOR in HeLa and C33A cells, which were restored by FNDC3B overexpression (Fig. 5 F). Taken together, these data demonstrate that hsa_circ_0001627 plays an oncogenic role in CC via the modulation of FNDC3B and PI3K/mTOR signaling pathway.

Fig. 5.

Hsa_circ_0001627/miR-1225-5p/FNDC3B regulates CC cell progression by modulating the PI3K/mTOR signaling pathway. A, Overexpression efficiency of FNDC3B in HeLa and C33A cells were determined by western blotting. B and C, Cell proliferation of HeLa and C33A cells transfected with si-hsa_circ_0001627 and FNDC3B were assessed via CCK-8 and colony formation assays. D and E, After hsa_circ_0001627 knockdown and FNDC3B overexpression, the cell migration and invasion of HeLa and C33A cells were analyzed by Transwell assays. F, The protein levels of p-PI3K, PI3K, p-mTOR and mTOR were assessed in HeLa and C33A cells transfected with si-hsa_circ_0001627 and FNDC3B using western blotting. Data were represented as mean ± SD of at least three independent experiments. ^#P < 0.05; ^**;##P < 0.01; ^***;###P < 0.001

Silencing of hsa_circ_0001627 inhibits tumor growth and metastatic activity in vivo

To verify the effect of hsa_circ_0001627 on the tumorigenesis of CC in vivo, hsa_circ_0001627-knockdown or si-NC-transfected HeLa cells were subcutaneously injected into the nude mice, respectively. As shown in Fig. 6 A-6 C, the silence of hsa_circ_0001627 remarkably lessened the tumor volume and weight. In addition, the expression levels of hsa_circ_0001627 (Fig. 6D) were lessened, and miR-1225-5p expression was upregulated (Fig. 6E), whereas, the protein levels of FNDC3B and PI3K/mTOR signal were raised (Fig. 6 F) in the tumor tissues of mice that were transplanted with hsa_circ_0001627-silenced cells. As demonstrated by H&E staining (Fig. 6G), xenograft tumors in the si-NC group displayed with several cancer nests and necrosis, which was alleviated in the si-hsa_circ_0001627 group. Moreover, the expressions of MMP2 and MMP9 in xenograft tumors were assessed by western blotting analysis, and the results showed the decreased levels of MMP2 and MMP9 upon the inhibition of hsa_circ_0001627 in xenograft tumors (Fig. 6 H). These data suggest that hsa_circ_0001627 silencing suppressed tumor growth and metastasis by regulating miR-1225-5p and FNDC3B-mediated PI3K/mTOR signaling pathway in vivo.

Fig. 6.

Silencing of hsa_circ_0001627 inhibits tumor growth and metastatic activity in vivo. HeLa cells transfected with si-NC or si-hsa_circ_0001627 were subcutaneously injected into nude mice (N = 6 per group), and then the representative photographs of xenograft tumors were shown (A); the tumor volume (B) and weight (C) were assessed. QRT-PCR and western blot were performed to illustrate the impacts of hsa_circ_0001627 silencing on the expression levels of hsa_circ_0001627 (D), miR-1225-5p (E), FNDC3B, p-PI3K, PI3K, p-mTOR and mTOR (F). (G) Representative H&E staining images for xenograft tumors in each group. (H) Western blotting analysis detected the expressions of MMP2 and MMP9 in xenograft tumors. Data were represented as mean ± SD of at least three independent experiments. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001

Discussion

So far, a large number of long non-coding RNAs and miRNAs have been found to play important roles in the occurrence and progression of CC (Aalijahan and Ghorbian 2019, Shen et al. 2020). However, the biological function of circRNAs in CC has rarely been reported. In this study, we demonstrated that a novel circRNA (hsa_circ_0001627) exhibited carcinogenic potencies in CC cells. Mechanically, hsa_circ_0001627 acted as a competitive endogenous RNA (ceRNA) via competitively binding to miR-1225-5p, thereby increasing the expression of FNDC3B and subsequently PI3K/mTOR signaling pathway. Altogether, these results suggest that hsa_circ_0001627 exerted a tumor-promoting role in the occurrence and progression of CC and may be a potential therapeutic target.

As a new kind of endogenous non-coding RNAs, circRNAs are gradually entering the field of tumor research. For instance, He et al. reported that high expression of tumorigenic circ_0018289 was positively associated with metastasis and clinical staging of CC and predicted a lower overall survival rate (He et al. 2020). Song et al. found that circRNA_101996 played a carcinogenic role in CC and promoted the proliferation, cell cycle progression, and metastasis of CC cells, thus it may be used as a potential biomarker and therapeutic target for CC (Song et al. 2021). Our study indicated an upregulation of hsa_circ_0001627 in CC tissue samples and cell lines. The tumor-promoting role of hsa_circ_0001627 has been identified in papillary thyroid carcinoma, which was confirmed to act as miR-139-5p sponge to stimulate LMO4 expression to promote cancer cell proliferation, migration, and invasion (Cai et al. 2019). Further analysis showed that the high expression of hsa_circ_0001627 was positively correlated with overall survival, suggesting that hsa_circ_0001627 has the potential to be a predictor of prognosis in patients with CC. In addition, hsa_circ_0001627 inhibition restrained the proliferation, migration and invasion of CC cells in vitro and tumor growth in vivo. These results indicated that hsa_circ_0001627 was a carcinogen that promoted the progression of CC and was a promising biomarker for the diagnosis of CC, as well as a prognostic predictor and therapeutic target.

A large number of studies have shown that circRNAs competitively interact with miRNAs, thus protecting target genes from inhibition or degradation (Huang et al. 2020). Our results support that hsa_circ_0001627 act as a “sponge” to sequester miR-1225-5p. First of all, bioinformatics analysis revealed that hsa_circ_0001627 had a response component binding to miR-1225-5p. Secondly, the luciferase reporter assay showed that miR-1225-5p could regulate the luciferase activity through hsa_circ_0001627. Thirdly, RIP experiment showed that hsa_circ_0001627 could directly bind to miR-1225-5p through AGO2. Fourthly, downregulation of hsa_circ_0001627 could lead to increased expression of miR-1225-5p. Finally, in CC tissues, hsa_circ_0001627 was reversely correlated with the expression level of miR-1225-5p. In conclusion, hsa_circ_0001627 expedited the malignant biological properties of CC cells through adsorbing miR-1225-5p.

MiR-1225-5p has shown to exert its tumor-inhibitory effects in various types of cancers, such as hepatocellular carcinoma (Liu et al. 2020), non-small cell lung cancer (Li et al. 2020a) and osteosarcoma (Zhang et al. 2020; Gong et al. 2021). A published study reported that miR-1225-5p negatively regulated FNDC3B and contributed to tumor inhibition in glioblastoma (Wang et al. 2020). Consistently, we confirmed that FNDC3B was a downstream target of miR-1225-5p in CC cells. FNDC3B has been demonstrated to be highly expressed in CC tissues, and its overexpression is associated with poor outcome in CC patients, and has a regulatory effects on the proliferation, apoptosis, migration, invasion and cell cycle of CC (Han et al. 2020, Li et al. 2021). Previous studies have indicated that the activation of PI3K/mTOR pathway was positively correlated with cancer progression, including CC (Qin et al. 2016; Hu et al, 2021). Moreover, it has been indicated that FNDC3B may be a tumor suppressor for colorectal cancer via targeting PI3K/mTOR pathway (Li et al. 2020c). Consistent with previous findings, the high expression of FNDC3B was shown in CC tissues. Moreover, FNDC3B overexpression reversed the inhibitory effects of hsa_circ_0001627 deficiency on CC malignant properties via activating the PI3K/mTOR pathway. Although this study has confirmed the regulatory function of the hsa_circ_0001627/miR-1225-5p/FNDC3B/PI3K/mTOR signal axis in CC, there are still some questions to be resolved, for example, the PI3K/mTOR signaling pathway activity is not intervened to see whether the function of hsa_circ_0001627 is dependent on this pathway.

Conclusion

Taken together, this study demonstrated that hsa_circ_0001627 facilitated CC cell progression by acting as a miR-1225-5p sponge, thereby upregulating FNDC3B and activating PI3K/mTOR pathway (Fig. 7). Our results suggest that hsa_circ_0001627-miR-1225-5p-FNDC3B-PI3K/mTOR axis plays a pivotal role in the progression of CC and may play an important role in the diagnosis and treatment of CC as a new marker and therapeutic target.

Fig. 7.

Schematic representation of the proposed mechanism of hsa_circ_0001627 in CC cells. Hsa_circ_0001627 promoted CC tumorigenesis as a sponge of miR-1225-5p to upregulate FNDC3B and activate PI3K/mTOR pathway

Electronic Supplementary Material

Below is the link to the electronic supplementary material

Abbreviations

CC

Cervical cancer

ceRNA

Competitive endogenous RNA

CircRNA

Circular RNA

FNDC3B

Fibronectin type III domain containing 3B

GEO

Gene Expression Omnibus

H&E

Hematoxylin and eosin

HPV

Human papillomavirus

MMP

Matrix metalloproteinase

PVDF

Polyvinylidene fluoride

QRT-PCR

Quantitative real-time PCR

SD

Standard deviation

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

siRNA

Small interference RNA

TBST

Tris buffered saline tween

Authors’ contributions

YL and DC conceived and designed the experiments, FM and CS analyzed and interpreted the results of the experiments, YW performed the experiments. All authors read and approved the final manuscript.

Funding

This project was supported by the Liaoning Province science and technology and People’s Livelihood project (2021JH2/10300047).

Data Availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Shengjing Hospital of China Medical University and the Animal Care and Use Committee of Shengjing Hospital of China Medical University.

Consent for publication

All the patients signed written informed consent.

Competing interests

The authors state that there are no conflicts of interest to disclose.