A novel positive feedback regulation between long noncoding RNA UICC and IL-6/STAT3 signaling promotes cervical cancer progression

Abstract

Long noncoding RNAs (lncRNAs), a novel class of transcripts that have critical roles in carcinogenesis and progression, have emerged as important gene expression modulators. However, the pathophysiological contributions and the underlying mechanisms of specific lncRNAs in cervical cancer remain largely unknown. Here, using transcriptome microarray analysis, we identified a novel lncRNA termed lncRNA upregulated in cervical cancer (lnc-UICC) that was highly expressed in cervical cancer tissue. lnc-UICC expression in cervical cancer was associated with FIGO stage, lymph node metastasis and prognosis. Through gain- and loss-of-lnc-UICC expression, we found lnc-UICC could significantly promote tumor growth and metastasis in vitro and in vivo. Mechanistically, lnc-UICC promoted STAT3 activation through two complementary ways. lnc-UICC could regulate the IL-6 transcription through binging to IL-6 promoter. lnc-UICC also directly interacted with the phospho-STAT3, and increased its protein stability by protecting it from proteasome-dependent degradation. Moreover, we revealed that lnc-UICC was a STAT3-responsive lncRNA, as STAT3 could bind to the lnc-UICC promoter to enhance its transcription, suggesting that there exists a positive feedback loop between lnc-UICC and IL-6/STAT3 signaling. In sum, therefore, we have identified an lncRNA-based IL-6/STAT3 signaling regulatory circuit that promotes tumorigenesis and metastasis in cervical cancer cells, highlighting the role that lncRNAs can play in tumor progression.

Introduction

As one of the most malignant cancers, cervical cancer accounts for a large proportion of cancer-associated mortalities among women worldwide. It has been reported that there are more than 530,000 new cases annually, and approximately 275,000 deaths due to cervical cancer per year [1]. Even though the patients suffering from cervical cancer can receive relevant treatments such as surgery, chemotherapy, radiotherapy and targeted therapy, the overall survival rates of them are still unsatisfactory [2]. Therefore, a deepgoing study on the molecular and functional mechanism of cervical cancer is urgently necessary.

Long noncoding RNAs (lncRNAs), more than 200 nucleotides in length, do not have any apparent protein-coding ability and are important members of the noncoding RNA family. LncRNAs are evolutionarily conserved and act as crucial regulators in modulating cellular proliferation, migration, invasion and cancer stem cells’ self-renewal [3,4]. lncRNAs regulate gene expression through diverse mechanisms, such as chromatin modification, epigenetic regulation, DNA methylation, gene imprinting, and RNA splicing [5,6]. Accumulating evidence has reported the relationship between lncRNAs and the initiation and progression of many cancers [4]. The aberrant expression of lncRNAs has been observed in multiple malignancies, including cervical cancer [7]. However, the pathophysiological contributions and the underlying molecular mechanisms of specific lncRNAs in cervical cancer remain largely unknown. In the current study, we report the identification of lncRNA upregulated in cervical cancer (lnc-UICC, GeneSymbol: BC038578), and elucidated its oncogenic role in cervical cancer progression via IL-6/STAT3 signaling.

Materials and methods

Cell culture

Caski, SiHa, HeLa, Ms751 and C33a cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in DMEM/F-12 (Gibco, USA) with 10% FBS (Gibco, USA) at 37°C in a humidified 5% CO₂.

Clinical samples

A total of 68 cervical cancer tissues and adjacent non-tumor tissues were obtained from The First Affiliated Hospital of Zhengzhou University between 2012 and 2017. Samples were selected based on the diagnosis of cervical cancer, determined by at least two pathologists and no patients had received chemotherapy or radiotherapy prior to surgery. All patients signed informed consent. All procedures involving human participants were in accordance with the ethical standards of the Human Research Ethics Committee at The First Affiliated Hospital of Zhengzhou University.

Microarray analysis

The general profiles of human lncRNAs and protein-coding transcripts from the three cases of cervical cancer and adjacent noncancerous tissues were detected using the Arraystar Human lncRNA Microarray V3.0 (Arraystar, Inc., Rockville, MD, USA). Array images were analyzed by Agilent Feature Extraction software (version 11.0.1.1; Agilent Technologies, Inc.). Differentially expressed genes were identified through the random variance model. A P value was calculated using the paired t-test. The threshold set for up- and down-regulated genes was a fold change ≥ 3.0 and a P value <0.05.

5’ and 3’ Rapid Amplification of cDNA Ends (RACE)

To determine the transcriptional initiation and termination sites of lnc-UICC, 5’-RACE and 3’-RACE analyses were performed by using a SMARTer™ RACE cDNA Amplification Kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions.

Lentiviral vector construction, production and transfection

The shRNAs targeting lnc-UICC sequences were designed. The target sequence for shUICC-1 (KD1) is 5’-CTCCGAACACATCTGAACA-3’; the sequence for shUICC-2 (KD2) is 5’-CACCAATTCCGGACACAAT-3’. Scramble shRNA was used as the negative control (NC). Human lnc-UICC full-length cDNA was amplified by PCR from the mRNA of HeLa cells. The objective products were cloned into pLV plasmid. The constructed vectors and the lentivirus packaging vectors were cotransfected into 293T cells. Lentiviruses were produced, harvested, and purified. Cells were transfected with lentivirus by using 8 μg/mL polybrene (Sigma, USA). Stable expression cells were selected in 1 μg/mL puromycin.

RNA isolation and quantitative real-time PCR (qRT-PCR)

The total RNA was isolated using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. After detection of RNA concentration, 1 μg of total RNA was reversely transcribed with random primers, using the ReverTra Ace^® qPCR RT kit (Toyobo, Osaka, Japan). qRT-PCR was performed using the SYBR-Green PCR Master Mix kit (Takara, Japan). Primers for lnc-UICC were as follows: forward: 5’-TCTTGCTACTGCTCACTCTTTG-3’; reverse: 5’-GTGTTCGGAGTTTCTTCCTTCT-3’. Primers for IL-6 were as follows: forward: 5’-GGAGACTTGCCTGGTGAAA-3’; reverse: 5’-CTGGCTTGTTCCTCACTACTC-3’. Primers for GAPDH were as follows: forward: 5’-CAAGAGCACAAGAGGAAGAGAG-3’; reverse: 5’-CTACATGGCAACTGTGAGGAG-3’. Relative expression was calculated using the 2^-ΔΔCt method.

Cell viability and colony formation

For cell viability detection, 3000 cells per well were seeded in a 96-well plate and were incubated at 37°C. At indicated time point, 10 μL of CCK8 solution (Dojindo, Japan) was added to each well. After 1.5 h, the absorbance was measured by using a micro-plate reader (BioTek Instruments, USA) at 450 nm. For colony formation detection, 2000 cells were planted in 6-well plates and cultured for 2 weeks. Cells were then fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The assay was performed three times for each treatment.

Flow cytometric analysis of the cell cycle and apoptosis

For cell cycle detection, cells were harvested, fixed in 70% ethanol, and stained with propidium iodide solution (50 mg/mL propidium iodide, 50 mg/mL RNase A, 0.1% Triton-X, 0.1 mmol/L EDTA). For cell apoptosis detection, cells were stained using an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). The images of cell cycle and apoptosis were obtained and analyzed, using the FACS Calibur (BD Biosciences, USA) and the Flowjo software (Tree Star Corp, USA).

Migration and invasion assays

Transwell chambers (Corsta) were used to perform the migration and invasion assays. For the migration assay, 1 × 10⁵ cells in a serum-free medium were added to the upper chamber, and 500 μL of full medium were added to the lower chamber. For the invasion assay, the diluted matrigel (BD Biosciences, USA) paved the upper well of the transwell chamber. After 24 h of incubation, the migratory and invasive cells were fixed with 4% paraformaldehyde, and then stained with crystal violet solution.

Western blot

Cells were lysed using a RIPA buffer, including a protease inhibitor cocktail (Beyotime, Beijing, China). The proteins were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). After blocking, the membranes were incubated with primary antibodies at 4°C overnight. The HRP-conjugated secondary antibodies were used to incubate the membranes for 1 h at room temperature. The primary antibodies used were the anti-STAT3 (1:1000, Cell Signaling), the anti-p-STAT3 (1:1000, Cell Signaling), and anti-GAPDH antibody (1:4000, Beverly, USA).

Immunofluorescence (IF) staining

Cells were cultured on a coverslip and fixed with 4% paraformaldehyde. 0.2% Triton X-100 solutions in PBS were used to permeabilize cells for 10 min. After blocking by 1% bovine serum albumin (BSA) for 1 hr, cells were incubated with anti-p-STAT3 antibody (1:200, Cell Signaling) overnight at 4°C. The fluorescence-conjugated secondary antibody (goat-anti-rabbit-Alexa 594-conjugated antibodies, Life Technologies) was used to incubate the treated coverslips for 1 h at room temperature. The treated coverslips were then incubated with 4’,6-Diamidine-2’-phenylindole dihydrochloride (DAPI; Life Technologies) for 3 min at room temperature. The images were obtained using a confocal fluorescence microscope (Olympus, USA).

RNA immunoprecipitation (RIP)

To determine the interaction between lnc-UICC and STAT3, RIP assay was conducted by using Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions.

RNA pull-down

RNA pull-down was performed as previously described [8]. In vitro biotin-labeled RNAs (lnc-UICC and its antisense RNA) were transcribed with the biotin RNA labeling mix (Roche) and T7 RNA polymerase (Roche) treated with RNase-free DNase I (Promega) and purified with RNeasy Mini Kit (QIAGEN). Biotinylated RNA was incubated with nuclear extracts of breast cancer cells, and pull-down proteins were run on SDS-PAGE gels. Western blot followed.

Statistical analysis

All data were expressed as the mean ± SD and analyzed by SPSS Statistics 21. Comparison between groups was analyzed by a Student’s t test or one-way analysis of variance (ANOVA). The survival rates were analyzed by the Kaplan-Meier estimator. The statistical significance was set at P<0.05.

Results

lncRNAs expression profile in cervical cancer

To identify functional lncRNAs involved in cervical cancer pathogenesis, lncRNA-mRNA co-expression microarray detection was conducted. Hierarchical clustering showed the differential expression levels of lncRNAs between cervical cancer tissues and adjacent non-tumor tissues (Figure 1A). To validate the results of microarray analysis, we randomly selected five lncRNAs among these differential lncRNAs and then examined their expression by using qRT-PCR) in 63 pairs of cervical cancer tissues and adjacent non-tumor tissues. These data confirmed that BC041856, CDKN2B-AS1, and RP11-443P15.2 were overexpressed in cervical cancer, whereas the expression of LOC100506229 and HHIP-AS1 was decreased (Figure 1B). Thus, our data indicate that a set of lncRNAss are frequently aberrantly expressed in cervical cancer.

Figure 1.

lncRNAs expression profile in cervical cancer. A. Hierarchical clustering reveals distinguishable lncRNA expression profiles between cervical cancer tissues and adjacent non-tumor tissues. B. Five lncRNAs (BC041856, CDKN2B-AS1, and RP11-443P15.2, LOC100506229 and HHIP-AS1) were selected on the basis of the microarray results. The relative expression of indicated lncRNAs in 63 pairs of cervical cancer tissues and adjacent non-tumor tissues was determined by qRT-PCR. *P<0.05.

lnc-UICC is overexpressed in cervical cancer and correlates with poor prognosis

We used gene coexpression networks to cluster thousands of transcripts into phenotypically relevant coexpression modules. We focused on coexpression modules that have a high rate of protein-coding RNAs in the cervical cancer coexpression network. Among the differentially expressed lncRNAs, lnc-UICC expression was one of the most significantly overexpressed in the cervical cancer tissues compared to adjacent non-tumor tissues. The role of lnc-UICC in cervical cancer can be characterized by this method. Transcriptional initiation and termination sites of lnc-UICC were then identified by rapid amplification of cDNA ends (RACE) analysis (Figure 2A).

Figure 2.

lnc-UICC is overexpressed in cervical cancer and correlates with poor prognosis. A. A representative image of PCR products from the 5’-RACE and 3’-RACE procedure. The major PCR product is marked by an arrow. B. The lnc-UICC expression in 63 pairs of cervical cancer tissues and adjacent non-tumor tissues was examined by qRT-PCR. C. The Kaplan-Meier method was used to determine the survival of 63 patients with cervical cancer and log-rank test to compare survival between the lnc-UICC-high, and lnc-UICC-low groups. The median expression level of lnc-UICC in cervical cancer was used as the cutoff.

Then, we evaluated lnc-UICC expression in 63 paired tissue samples and confirmed the higher levels of lnc-UICC expression in cervical cancer tissue (Figure 2B). To investigate the relationship between lnc-UICC expression and cervical cancer clinical features, 68 patients with cervical cancer were divided into high and low lnc-UICC expression level groups based on the median value. As shown in Table 1, statistical analysis of clinical characteristics and lnc-UICC expression indicated that overexpression of lnc-UICC was significantly associated with FIGO stage, lymph node metastasis. Moreover, we performed Kaplan-Meier analysis to determine the association between lnc-UICC expression and prognosis of patients with cervical cancer. We found that patients with high lnc-UICC levels had shorter overall survival time (Figure 1C). These findings suggest that upregulation of lnc-UICC is associated with cervical cancer progression and poor prognosis.

Table 1.

The correlation between lnc-UICC expression and clinical features of cervical cancer patients

Clinical fertures	lnc-UICC		P value
	High	Low
Age (years)
≤40	16	18	0.455
>40	18	14
Size (cm)
≥ 4	21	15	0.145
<4	13	19
FIGO stages
I-II	9	20	0.007
III-IV	25	14
Distant metastasis
Present	23	18	0.215
Absent	11	16
Lymphatic metastasis
Present	21	12	0.029
Absent	13	22

FIGO, International Federation of Gynecology and Obstetrics. The median expression level of lnc-UICC in cervical cancer tissue was used as the cutoff.

lnc-UICC promotes cell growth in vitro and in vivo

After detecting lnc-UICC expression in 5 cervical cancer cell lines (Figure 3A), we developed HeLa cells with stably overexpressed lnc-UICC (Figure 3B), and SiHa cells with stably silenced lnc-UICC expression (Figure 3C). To detect the effect of lnc-UICC on the proliferation of cervical cancer cells, CCK-8 and colony formation assays were performed. The results showed that lnc-UICC overexpression increased the proliferative capacity of HeLa cells, compared to that of control cells (Figure 3D, 3E). In contrast, deletion of endogenous lnc-UICC expression significantly decreased the proliferative capacity of SiHa cells (Figure 3F, 3G).

Figure 3.

lnc-UICC promotes cell growth in vitro and in vivo. (A) qRT-PCR detected lnc-UICC levels in cervical cancer cells. (B) The validation of stably overexpressing lnc-UICC in HeLa cells via qRT-PCR. Con indicates control group, and OE indicates lnc-UICC overexpression. (C) The validation of stably silencing lnc-UICC in SiHa cells via qRT-PCR. Con indicates control group, and KD indicates lnc-UICC knockdown. (D) Proliferation assay of HeLa cells with lnc-UICC overexpression via CCK-8 detection. (E) Proliferation assay of HeLa cells with lnc-UICC overexpression via colony formation assay. (F) Proliferation assay of SiHa cells with lnc-UICC knockdown via CCK-8 detection. (G) Proliferation assay of SiHa cells with lnc-UICC knockdown via colony formation assay. (H and J) Flow cytometry was performed to assess cell cell cycle progression (H) and apoptosis (J) of HeLa cells after lnc-UICC overexpression. (I and K) Flow cytometry was performed to assess cell cell cycle progression (I) and apoptosis (K) of SiHa cells after lnc-UICC knockdown. (L) lnc-UICC overexpression in HeLa cells significantly increases tumor growth in a mouse xenograft model (left). Tumor weights of corresponding mouse xenograft models (right). (M) lnc-UICC knockdown in SiHa cells significantly inhibits tumor growth in a mouse xenograft model (left). Tumor weights of corresponding mouse xenograft models (right). (N and O) Immunohistochemistry (IHC) staining with antibody against proliferation marker Ki-67 in each group. *P<0.05.

We then analyzed differences in cell cycle distributions and apoptosis following lnc-UICC overexpression or knockdown by flow cytometry. We found that lnc-UICC overexpression drove progression beyond the G1/S transition in HeLa cells, whereas lnc-UICC-knockdown induced G1/S arrest in SiHa cells (Figure 3H, 3I). lnc-UICC overexpression inhibited the apoptosis of HeLa cells (Figure 3J). In contrast, lnc-UICC-knockdown SiHa cells had a significantly higher percentage of Annexin V-positive cells than control cells (Figure 3K).

To validate the role of lnc-UICC in the tumorigenesis of cervical cancer cells, we next injected the above stable cells into nude mice. The result showed that xenograft tumors grown from HeLa cells overexpressing lnc-UICC had larger mean volumes and weights than tumors grown from control cells (Figure 3L). Knockdown of lnc-UICC expression dramatically inhibited the tumor growth in both weight and size in nude mice (Figure 3M). Moreover, immunohistochemistry analysis indicated that Ki-67 staining was significantly reduced in lnc-UICC knockdown xenograft tumors, while increased in lnc-UICC overexpressed tumors (Figure 3N, 3O). Together, our results indicate that lnc-UICC promotes the growth of cervical cancer.

lnc-UICC facilitates cell migration, invasion and metastasis in cervical cancer

To evaluate whether lnc-UICC contributes to the progression of cervical cancer, we determined the effect of lnc-UICC on the migration and invasion of HeLa and SiHa cells. Transwell assays showed that overexpression of lnc-UICC dramatically increased cell migration and invasion in HeLa cells (Figure 4A). Conversely, lnc-UICC knockdown suppressed the migratory and invasive ability of the cervical cells (Figure 4B).

Figure 4.

lnc-UICC facilitates cell migration, invasion and metastasis in cervical cancer. A. Transwell migration and invasion assays were performed in HeLa cells with lnc-UICC overexpression. B. Transwell migration and invasion assays were performed in SiHa cells with lnc-UICC knockdown. C. An experimental metastasis animal model was performed by injecting lnc-UICC stable overexpressing HeLa cells into the tail vein of nude mice. The number of tumor nodules on lung surfaces are shown. D. An experimental metastasis animal model was performed by injecting lnc-UICC stable knockdown SiHa cells into the tail vein of nude mice. The number of tumor nodules on lung surfaces are shown. *P<0.05.

To validate the in vitro results, control and lnc-UICC overexpressed HeLa cells were injected into nude mice. As shown in Figure 4C, upregulation of lnc-UICC resulted in an increase in metastatic nodules on the mice lungs when compared with those in the control group (Figure 4C). On the contrary, knockdown of lnc-UICC suppressed pulmonary metastasis of SiHa cells (Figure 4D). These results indicate that lnc-UICC also affects cancer cell migration, invasion and metastasis.

lnc-UICC activates STAT3 signaling

Given the evident effects of lnc-UICC on cervical cancer cell growth and metastasis, signaling pathways involved in tumor growth and metastasis that might be activated by lnc-UICC were analyzed by examining the expression of phosphorylated forms of STAT3, AKT and ERK using western blot assay (Figure 5A). The results indicated that only phospho-STAT3 (p-STAT3) level was observed to be significantly changed by lnc-UICC. lnc-UICC knockdown decreased the phosphorylation of STAT3, whereas lnc-UICC overexpression increased it (Figure 5A). STAT3 activation by lnc-UICC was further confirmed by STAT3 reporter assay. STAT3 response element did be activated with lnc-UICC overexpression, but was clearly inhibited with lnc-UICC depletion (Figure 5B, 5C). Considering that the p-STAT3 in nucleus is the main executor to activate the transcription of its downstream target genes, we then examined whether the nuclear p-STAT3 accumulation was altered by lnc-UICC. The cellular immunofluorescence assay demonstrated that lnc-UICC induced the nuclear translocation of p-STAT3 protein (Figure 5D). Moreover, the treatment of the STAT3 specific inhibitor S3I-201 diminished the the capacities of cellular proliferation, migration and invasion between lnc-UICC overexpressing HeLa cells and control cells (Figure 5E, 5F), suggesting that lnc-UICC promoted malignant progression of cervical cancer cells by activating STAT3 signaling. These results suggested that STAT3 signaling played a crucial role in mediating lnc-UICC’s function.

Figure 5.

lnc-UICC activates STAT3 signaling. (A) The effect of lnc-UICC overexpression and knockdown on activation of STAT3, AKT and ERK was detected by western blot. (B) Luciferase activity in the lysates of HeLa-Con and HeLa-OE cells transfected with STAT3 response element-luciferase reporter plasmid was measured (normalized by Renilla luciferase activity). (C) Luciferase activity in the lysates of SiHa-Con and SiHa-KD cells transfected with STAT3 response element-luciferase reporter plasmid was measured (normalized by Renilla luciferase activity). (D) p-STAT3 nuclear translocation in HeLa-Con and HeLa-OE cells was analyzed by immunofluorescence assay and representative images were shown. (E and F) The STAT3 inhibitor (INH) S3I-201 (20 μM) was added to lnc-UICC-overexpressing HeLa cells and the proliferation (E), migration and invasion (F) was detected. *P<0.05.

lnc-UICC increases IL-6 transcription

We next sought to elucidate whether STAT3 activation induced by lnc-UICC was dependent on IL-6. The IL-6 mRNA level of subcutaneous xenograft tumors was dramatically increased in the lnc-UICC overexpressed group (Figure 6A) compared with that in the control group. Meanwhile, IL-6 mRNA was significantly decreased in the lnc-UICC knockdown subcutaneous xenograft tumors compared with that in the control group (Figure 6B). To validate that lnc-UICC could regulate the IL-6 level, the protein in the cell culture supernatants of the cultured cervical cancer cells with lnc-UICC alteration was examined by ELISA assay. IL-6 protein level was increased in HeLa-OE cells compared with that in the vector control cells (Figure 6C), whereas it was significantly decreased in SiHa-KD cells compared with that in control cells (Figure 6D). Consistently, IL-6 mRNA level was also affected by lnc-UICC in HeLa and SiHa cells (Figure 6E, 6F).

Figure 6.

lnc-UICC increases IL-6 transcription. (A and B) mRNA level of IL-6 in lnc-UICC overexpressed (A) or knockdown (B) xenograft. (C and D) Media from the indicated cultured lnc-UICC overexpressed (C) or lnc-UICC knockdown (D) cervical cancer cells was collected and subjected to IL-6 ELISA assays. (E and F) mRNA level of IL-6 in lnc-UICC overexpressed (C) or knockdown (D) cervical cancer cells. (G) The sequence of the predicted lnc-UICC binding locus in IL-6 promoter is shown. The sequences of oligoribonucleotides (RNA oligo) and oligodeoxyonucleotides (DNA decoy) are shown. (H) qRT-PCR analysis of lnc-UICC pulled down by biotin-labeled antisense oligodeoxynucleotide (Biotin-AS) or sense oligodeoxynucleotide (Biotin-S) control in HeLa cells. (I) Transcript of IL-6 was measured in HeLa-Con and HeLa-OE cells transfected with 200 nM oligodeoxynucleotides negative control (ODN NC), lnc-lnc-UICC oligodeoxynucleotides decoy (UICC decoy) or mutated oligodeoxynucleotides (ODN mut) was measured by qRT-PCR analysis. (J) STAT3 activity was measured in HeLa-Con and HeLa-OE cells transfected with 200 nM oligodeoxynucleotides negative control (ODN NC), lnc-lnc-UICC oligodeoxynucleotides decoy (UICC decoy) or mutated oligodeoxynucleotides (ODN mut) by luciferase activity analysis. (K) Transcript of IL-6 in SiHa-Con and SiHa-KD1 cells transfected with oligoribonucleotide negative control (oligo NC), oligo mimics or oligo mut (200 nM) by qRT-PCR analysis. (L) STAT3 activity in SiHa-Con and SiHa-KD1 cells transfected with oligoribonucleotide negative control (oligo NC), oligo mimics or oligo mut (200 nM) was measured by luciferase activity analysis. *P<0.05.

Next, we identified one putative lnc-UICC complementary binding locus within the IL-6 promoter region (671 bp from the transcription start site) using Basic Local Alignment Search Tool (BLAST) (Figure 6G). A biotin-labeled antisense oligodeoxynucleotide (Biotin-AS), complementary to the sequence of human lnc-UICC at the binding locus in IL-6 promoter, was synthesized for the biotin pull down assay. As shown in Figure 6H, lnc-UICC was significantly enriched in the AS-biotin pull down group compared with the sense oligodeoxynucleotide (Biotin-S) control group of SiHa cells. To further confirm the interaction between lnc-UICC and IL-6 promoter, an oligoribonucleotide (oligo mimics) and an oligodeoxynucleotide (ODN) decoy as well as their corresponding mutated controls (Oligo mut/ODN mut) were designed according to the sequence of the binding locus (Figure 6G). As shown in Figure 6I, 6J, the increase of IL-6 transcription and STAT3 activation of lnc-UICC-OE cells was abolished by the ODN decoy but not ODN mut. Consistently, the oligo mimics abrogated the suppression of IL-6 transcription and STAT3 inactivation of lnc-UICC-KD cells compared with control cells, but the mutated control exhibited no influence (Figure 6K, 6L).

lnc-UICC interacts with STAT3 protein

To further seek out the underlying mechanism of lnc-UICC inducing STAT3 activation, we performed RNA pull-down and mass spectrometry (MS) analysis. Intriguingly, we found p-STAT3 was a potential lnc-UICC-interacting candidate. To verify the interaction between lnc-UICC and p-STAT3, RNA immunoprecipitation (RIP) assay was used. The result showed that lnc-UICC transcript was robustly enriched by p-STAT3 antibody (Figure 7A). RNA pull-down assay was further used to validate the interaction between lnc-UICC and p-STAT3 (Figure 7B).

Figure 7.

lnc-UICC interacts with p-STAT3 protein. A. The RIP assay was performed to determine the interaction between lnc-UICC and p-STAT3. The IgG was taken as negative control. B. The RNA pull down assay was performed to determine the interaction between lnc-UICC and p-STAT3. Antisense (AS) lnc-UICC was was taken as negative control. C. HeLa cells with or without lnc-UICC overexpression were treated with CHX (100 mg/ml) for the indicated time points. The cell lysates were examined by immunoblotting (up panel). A plot of normalized amount of p-STAT3 protein is shown (down panel). D. HeLa cells with or without lnc-UICC knockdown were treated with CHX (100 mg/ml) for the indicated time points. The cell lysates were examined by immunoblotting (up panel). A plot of normalized amount of p-STAT3 protein is shown (down panel). E. Western blot of p-STAT3-associated ubiquitination in cervical cancer cells with lnc-UICC overexpression (left panel) or knockdown (right panel). F. Western blot of p-STAT3 expression in SiHa-Con and SiHa-KD1 cells treated with vehicle control or MG132. *P<0.05.

Furthermore, to determine the mechanism by which lnc-UICC regulated p-STAT3, we detected the effect of lnc-UICC on pSTAT3 protein stability. After treatment of protein synthesis inhibitor cycloheximide (CHX), p-STAT3 protein halflife was markedly prolonged by lnc-UICC overexpression (Figure 7C), while the stability of p-STAT3 was markedly suppressed by lnc-UICC knockdown (Figure 7D). Moreover, lnc-UICC overexpression dramatically suppressed ubiquitination level of p-STAT3 protein, whereas depletion of lnc-UICC exerted opposite function (Figure 7E). Subsequently, we found the upregulation of p-STAT3 protein levels induced by lnc-UICC overexpression were abolished after proteasomal degradation was blocked by MG132 (Figure 7F). These results demonstrate that lnc-UICC can associate with p-STAT3 to increase its protein stability.

IL-6/STAT3 signaling activates lnc-UICC transcription to form a positive feed-back regulatory loop

As IL-6/STAT3 signaling shares a longstanding association with cervical cancer carcinogenesis, we investigated whether IL-6 could regulate lnc-UICC. Recombinant human IL-6 (rhIL-6) treatment increased the lnc-UICC expression both in HeLa and SiHa cells (Figure 8A). Moreover, we transfected IL-6 siRNA into cervical cancer cells and found that IL-6-siRNA significantly decreased lnc-UICC expression (Figure 8B), suggesting IL-6 could indeed regulate lnc-UICC expression.

Figure 8.

IL-6/STAT3 activates lnc-UICC transcription to form a positive feed-back regulatory loop. A. The cervical cancer cells were treated with 10 ng/mL rhIL-6 for 12 hours. The lnc-UICC was then detected by qRT-PCR. B. The cervical cancer cells were transfected with IL-6 siRNA. After 48h, the lnc-UICC was then detected by qRT-PCR. C. Schematic diagram of canonical STAT3-binding motif (JASPAR Database) and three potential STAT3 responsive elements (E1, E2, and E3) in the lnc-UICC promoter region. D. lnc-UICC RNA levels when 20 μM STAT3 inhibitor and 10 ng/mL rhIL-6 was used to treat cells. E. lnc-UICC RNA levels when STAT3 siRNA and 10 ng/mL rhIL-6 was used to treat cells. F. lnc-UICC promoter luciferase activity when 20 μM STAT3 inhibitor and 10 ng/mL rhIL-6 was used to treat cells. G. lnc-UICC promoter luciferase activity and 10 ng/mL rhIL-6 was used to treat cells. H. Three predicted STAT3-binding sites of the lnc-UICC promoter was individually deleted and named E1-Del, E2-Del, and E3-Del. Luciferase assay was employed to detect transcriptional activities of the three lnc-UICC promoter deletion mutants when STAT3 expression was enforced in HeLa cells. FL indicates Full-length lnc-UICC promoter sequence. I. ChIP assays showed that STAT3 bound to the E2 element of lnc-UICC promoter. IgG served as a negative control. *P<0.05.

Of note, we discovered three putative STAT3-binding elements within lnc-UICC promoter region through JASPAR bioinformatics analysis (Figure 8C). We further investigated whether IL-6/STAT3 could regulate lnc-UICC expression in turn. STAT3 inhibitor or siRNA significantly decreased lnc-UICC transcription in HeLa cells treated with or without rhIL-6 (Figure 8D, 8E). Luciferase assay indicated the lnc-UICC promoter was repressed by STAT3 inhibitor or siRNA (Figure 8F, 8G). To clarify which element was necessary for STAT3-mediated lnc-UICC expression, the three predicted STAT3-binding sites were individually deleted. We found STAT3 nearly failed to promote lnc-UICC transcriptional activity without the E2 element, while E1 and E3 absence only downregulated lnc-UICC promoter activity in part (Figure 8H), indicating that the E2 element was essential for STAT3 to activate lnc-UICC transcription. To further confirm this result, ChIP assay was performed with p-STAT3 antibody, followed by qRT-PCR detection with specific primer for E2 element. As shown in Figure 8I, STAT3 could associate with lnc-UICC promoter and were enriched within the E2 region. These findings suggest that there is a regulatory feed-back loop between lnc-UICC and IL-6/STAT3 signaling, which may continuously activate their oncogenic functions.

Discussion

Emerging evidence has indicated the regulatory role of lncRNAs in various cancers including cervical cancer. However, the mechanism of specific lncRNA remains largely unclear. Here, we report that lncUICC acts as an oncogene in cervical cancer progression. As described in Figure 9, lnc-UICC bound to IL-6 promoter to activate its transcription. lnc-UICC also binds to p-STAT3 protein and protects it from degradation. As a result, STAT3 signaling pathway is activated by lnc-UICC, thereby inducing growth and metastasis. Meanwhile, the activated STAT3 reciprocally triggers lnc-UICC transcription to sustain the oncogenic effect of the lnc-UICC/IL-6/STAT3 axis continuously.

Figure 9.

Schematic diagram of lnc-UICC regulating progression via activating the IL-6/STAT3 signaling in cervical cancer.

Increasing evidence has demonstrated that lncRNAs regulate biological behavior of malignant tumors. For instance, increase of lncRNA HOTAIR in cervical cancer cells upregulated VEGF, MMP-9 and genes associated with epithelial mesenchymal transition for enhancement of tumor invasion [9]. Overexpression of lncRNA MALAT-1 in cancer tissues was linked to tumor cell differentiation, lymph node metastasis and prognosis [10]. Moreover, lncRNA LET downregulation was associated with prognosis of cervical cancer and inhibited the progression of bladder cancer and gastric cancer [11-13]. This study, for the first time, reported that lnc-UICC expression significantly increased in cervical cancer tissue. lnc-UICC expression was correlated with FIGO stage, lymph node metastasis. The Kaplan-Meier analysis illustrated that high levels of lnc-UICC correlated with worse overall survival of cervical cancer patients, revealing that lnc-UICC might be an important regulator in the pathogenesis of cervical cancer.

STAT3 plays central roles in cancer development, which can regulate a number of genes expression to promote cell proliferation, metastasis, immunosuppression, and angiogenesis [14]. Currently, several lncRNAs have been documented to affect the activation of STAT3. For example, lncRNA-DILC and lncRNA-SRLR can indirectly modulate STAT3 signaling through regulating IL-6 expression [15,16], and IL-6 is the most well-established upstream activator of STAT3. Our in vitro and in vivo assays demonstrated that IL-6/STAT3 signaling was responsible for lnc-UICC mediated growth and metastasis of cervical cancer. lnc-UICC could increase the phosphorylation of STAT3. We further showed that the lnc-UICC promoted STAT3 activation through two different ways. lnc-UICC could regulate the IL-6 transcription through binging to IL-6 promoter. A recent study demonstrated that nuclear localized lncRNAs can participate in the regulation transcription processes through binding to active chromatin sites [17]. Intriguingly, we identified a putative binding locus of lnc-UICC within the IL-6 promoter region through bioinformatics prediction, which was further confirmed by pull down assay. The alteration of IL-6 expression and STAT3 activation induced by lnc-UICC overexpression and lnc-UICC knockdown were abolished by the ODN decoy and oligo mimics of lnc-UICC, respectively, which further indicates the interaction of lnc-UICC with IL-6 promoter. On the other hand, lnc-UICC also has the capacity to regulate STAT3. We found the binding of lnc-UICC can protect p-STAT3 protein from proteasomal degradation, which increases its stability and induces the activation of STAT3. In future, it would be of interest to identify their binding fragments, and determine the underlying mechanism of p-STAT3 stability mediated by lnc-UICC.

In conclusion, our study showed that lnc-UICC was overexpressed in cervical cancer tissue, and upregulation of lnc-UICC could promote growth and metastasis of cervical cancer. Our study elucidated a novel pathway in lnc-UICC-mediated growth and metastasis, which su-ggested new therapeutic targets, including lnc-UICC/IL-6/STAT3 signaling pathway, in the prevention and treatment of cervical cancer. Further work should be performed to obtain more information about lnc-UICC, and to explore the clinical diagnosis value and develop new drug therapy for cervical cancer.

Disclosure of conflict of interest

None.