The long noncoding RNA PCGEM1 promotes cell proliferation, migration and invasion via targeting the miR-182/FBXW11 axis in cervical cancer
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Cervical cancer (CC) is the fourth leading cause of cancer-associated death in women worldwide. Recently, long noncoding RNA (lncRNA) prostate cancer gene expression marker 1 (PCGEM1) has been demonstrated to involve in the initiation and progression of human cancers. However, to date, the clinical and functional significance of PCGEM1 expression in CC progression remains unknown.
qRT-PCR was performed to investigate PCGEM1 expression levels in CC tissues and cell lines. The effect of PCGEM1 on CC cells was assessed by gain- and loss-of-function assays. MS2-binding sequences-MS2-binding protein-based RIP assay (MS2-RIP), RNA pull-down and Luciferase reporter assays were performed to investigate the interaction between PCGEM1 and miR-182. The association between miR-182 and F-box and WD repeat domain containing 11 (FBXW11) was verified by luciferase reporter assay. The effect of PCGEM1 on the NF-κB and β-catenin/TCF signaling pathways was determined by luciferase reporter assay.
Our present study showed that PCGEM1 was significantly upregulated in CC tissues and cell lines. Overexpression of PCGEM1 was correlated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node, distant metastasis and poor prognosis in CC patients. Functionally, PCGEM1 promoted cell proliferation, cell cycle progression, migration and invasion, while suppressed cell apoptosis in CC cells. Further mechanistic investigation revealed that PCGEM1 associated with miR-182 and suppressed its expression. PCGEM1 could act as a competing endogenous (ceRNA) of oncogene F-box and WD repeat domain containing 11 (FBXW11) for miR-182 in CC cells. Additionally, PCGEM1 was capable to activate the NF-κB and β-catenin/TCF signaling pathways, which was reversed by inhibition of FBXW11.
In conclusion, our findings demonstrated that PCGEM1-miR-182-FBXW11 axis play an important role in CC progression, and indicated a promising therapeutic target for CC patients.
KeywordsceRNA FBXW11 lncRNA miR-182
long noncoding RNAs
prostate cancer gene expression marker 1
competing endogenous RNA
F-box and WD repeat domain containing 11
Dulbecco’s Modified Eagle Medium
quantitative real-time PCR
cell counting kit-8 reagent
MS2-binding sequences-MS2-binding protein-based RIP assay
International Federation of Gynecology and Obstetrics
Cervical cancer (CC) is the fourth leading cause of cancer-associated death in women worldwide . Approximately 530,000 new cases are diagnosed with CC every year, and 85% of the deaths occur in underdeveloped or developing countries . Most early CC could be cured by surgical resection. However, for those CC patients in advanced stages, there are no effective therapeutic strategies . Hence, further investigation are needed to reveal the molecular mechanism responsible for CC initiation and progression, which may be greatly helpful for identifying effective therapeutic approaches for CC patients.
Long noncoding RNAs (lncRNAs) are a heterogeneous class of transcripts larger than 200 nucleotides without protein-coding ability . LncRNAs play crucial roles in controlling gene expression involving several cellular processes of human cancers, such as proliferation, migration, invasion, drug resistance, autophagy and angiogenesis [5, 6]. The mechanisms of lncRNAs include gene regulation in cis or in trans, and regulation of their interacting proteins [7, 8, 9]. Previous studies have provided evidence suggesting that the deregulation of lncRNAs participate in the initiation and progression of CC, including that of GAS5, CRNDE, SPRY-IT1 and CCAT1 [10, 11, 12, 13]. Recently, lncRNA prostate cancer gene expression marker 1 (PCGEM1) has been identified as an oncogenic gene in human cancers. PCGEM1 was first found to be highly expressed in prostate cancer and promotes cell proliferation [14, 15]. PCGRM1 exerts oncogenic effects in prostate cancer cells through acting as a competing endogenous RNA (ceRNA) for some microRNAs, such miR-145 and miR-148a [16, 17]. Besides, PCGEM1 expression level is overexpressed in epithelial ovarian cancer tissues. PCGEM1 enhances ovarian cancer cell proliferation, migration, and invasion, but decreased cell apoptosis through upregulating RhoA, YAP, MMP2, Bcl-xL, and P70S6K expression . In endometrial carcinoma, PCGEM1 upregulates STAT3 expression by acting as a ceRNA for miR-129 . Moreover, PCGEM1 is capable to induce epithelial–mesenchymal transition (EMT) and metastasis via increasing SNAI1 expression in gastric cancer cells . However, it is unclear whether PCGEM1 exerts similar function in CC tumorigenesis and development.
In present study, we first reported that lncRNA PCGEM1 was upregulated in CC tissues and cells, which may serve as a potential prognostic indicator for CC patients. We further explored the effects of PCGEM1 on the phenotypes of CC cells. Moreover, mechanistic investigation revealed that PCGEM1 could act as a ceRNA to regulate oncogene F-box and WD repeat domain containing 11 (FBXW11) expression by sponging miR-182 in CC cells. Taken together, our study provides the first evidence of the existence of a PCGEM1-miR-182-FBXW11 axis, which may be utilized as a promising therapeutic target for CC.
Material and method
Sixty-eight fresh CC tissues and their adjacent normal cervical tissues were obtained from patients diagnosed with cervical cancer in The First Affiliated Hospital of Jinzhou Medical University. All the tissue specimens were stored at − 80 °C until use. RNA later solution (Invitrogen™) was used to avoid the degradation of RNA, and all of the tissues were detect in a short time after resection from patients. This study was conducted with the approval of the Ethics committee of The First Affiliated Hospital of Jinzhou Medical University. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki. Informed consent was obtained from all patients.
A normal human cervix epithelial cell line (Ect1/E6E7) and four cervical cancer cell lines (C33A, HeLa, SiHa, and CaSki) were purchased from American Type Culture Collection (Manassas, USA). The STR profiling and mycoplasma testing in all cervical cancer cell line was checked. Cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 °C.
siRNAs targeting PCGEM1, FBXW11 and a negative control (siNC) were purchased from Ribobio Company (Guangzhou, China). miR-NC (control), miR-182 mimics, miR-182 inhibitors (inh-182) were also obtained from GenePharma Technology (Shanghai, China). PCGEM1 overexpression plasmids (pcDNA3.1-PCGEM1) were purchased from Genearray Biotechnology (Shanghai, China). Transfections were performed using lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. After 48 h, the cells were used for further experiments.
Western blot analysis
Cells were lysed using RIPA buffer (Beyotime, Beijing, China). A total of 20 μg protein samples were separated using SDS-PAGE, and then transfer onto PVDF membranes (Millipore). The membranes were blocked by 5% non-fat milk, and incubated with antibodies: anti-FBXW11 (Abcam), or anti-GAPDH (Proteintech), at 4 °C overnight. After wash, the membrane was probed with matched secondary antibody, and then the proteins were visualized using Immobilon Western Chemiluminescent HRP substrate (Millipore).
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA from CC tissues and cells was isolated by using Trizol reagent (Invitrogen, CA). The cDNA was synthesized from total RNA using the cDNA Reverse Transcription Kit (Takara). qRT-qPCR was performed using a StepOne Real-Time PCR System (Applied Biosystems, USA). Relative expression was normalized to GAPDH and was calculated by 2−ΔΔCt method. The primer sequences were shown as follow: GAPDH (forward: GGTGTGAACCATGAGAAGTATGA, reverse: GAGTCCTTCCACGATACCAAAG), PCGEM1 (forward: CTGTGTCTGCAACTTCCTCTAA, reverse: TCCCAGTGCATCTCGTAGTA), cyclinD1 (forward: CCTCTCCCATGACCACAATATC, reverse: GAGAATCCCAAAGGACCAGAC), IL-6 (forward: GGAGACTTGCCTGGTGAAA, reverse: CTGGCTTGTTCCTCACTACTC), MMP9 (forward: GAACTTTGACAGCGACAAGAAG, reverse: CGGCACTGAGGAATGATCTAA), CD44 (forward: AATGGTCGCTACAGCATCTC, reverse: GCAAACTGCAGGTCTCAAATC), Bcl-xL (forward: GGTGGTTGACTTTCTCTCCTAC, reverse: TCTCCGATTCAGTCCCTTCT), myc (forward: GCTGCTTAGACGCTGGATTT, reverse: GAGTCGTAGTCGAGGTCATAGTT), MMP7 (forward: CACTGTTCCTCCACTCCATTTA, reverse: GACATCTACCCACTGCAAGTATAG), Axin-2 (forward: CTTATCGTGTGGGCAGTAAGA, reverse: GTTCTCGGGAAATGAGGTAGAG), TCF-1 (forward: ACCTATGACTCCGTCGATCTAT, reverse: TCAGCCCATCTCTGACCTAT).
Cell proliferation assay
3 × 103 cells were seeded into 96-well plates. At indicated time point, cell proliferation was measured using the Cell Counting Kit-8 reagent (CCK-8, Dojindo, Kyushu, Japan). Briefly, 10 μL CCK-8 reagent was added into each well of cell culture, and the plates were further incubated for another 2 h. The absorbance at 450 nm was evaluated by a microplate reader. For colony formation assay, 2 × 103 cells were plated in six-well plates for 2 weeks. Cells were fixed in 4% paraformaldehyde for 10 min and then stained by 0.1% crystal violet for 30 min. Experiments were repeated at least three times.
Cell apoptosis detection
Apoptotic cells were evaluated by ANXA5 and propidium iodide (PI) staining (Invitrogen, A13201) according to the manufacturer’s instructions, and analyzed by flow cytometry (Beckman Coulter, USA).
Cell cycle detection
Cells were trypsinized and resuspended, fixed overnight at 4 °C in 70% ethanol, stained with propidium iodide, and then analyzed by flow cytometry (Beckman Coulter, USA).
Cell migration and invasion assays
To detect cell migration and invasion, the cells were pretreated with mitomycin C (10 μg/mL) for 1 h to eliminate the influence of cell proliferation. The transwell chambers with 8-μm pores were obtained from Corning (Corning, NY). For cell migration detection, the transfected cells were harvested and resuspended in 100 μL serum-free medium and then transferred to the upper chambers. 500 μL DMEM supplemented with 10% FBS was added to the lower chamber. After incubation for 24 h, the Transwell membrane was fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 30 min, and then counted under a light microscope. For the invasion assay, the Transwell membrane (filter) was precoated with Matrigel BD Biosciences) at 37 °C overnight; the remaining experimental procedures were similar to the migration assay.
RNA immunoprecipitation (RIP) and MS2-binding sequences-MS2-binding protein-based RIP assay (MS2-RIP)
For RIP assay, cells were transfected with miR-182 or miR-NC. After 48 h, cells were used to perform RIP experiments by using an AGO2 antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit according to the according to the manufacturer’s instructions.
MS2-RIP assay was performed as previously described . Cells were transfected with pcDNA3.1-MS2, pcDNA3.1-PCGEM1-MS2, or pcDNA3.1-PCGEM1-mut-MS2 (mutation in miR-182 binding sites). After 48 h, cells were used to perform RIP by using an anti-GFP antibody (Abcam) and the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. Then the co-precipitated RNAs level was analyzed by qRT-PCR.
RNA pull-down assay
RNA pull-down assay was performed as previously described . In brief, PCGEM1 or PCGEM1-mut were in vitro transcribed, respectively, and biotin-labeled with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA). 1 mg of whole-cell lysates were incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C, then the complexes were isolated with streptavidin agarose beads (Invitrogen). The RNA present in the pull-down material was detected by qRT-PCR analysis.
Luciferase reporter assays
The wild-type or mutant PCGEM1 was cloned into pmirGLO plasmid (named pmirGLO-PCGEM1 and pmirGLO-PCGEM1-mut). Cells were co-transfected with pmirGLO-PCGEM1 or pmirGLO-PCGEM1-mut and miR-NC or miR-182. After 48 h, Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, USA). To detect the effect of PCGEM1 on FBXW11 3′UTR, the FBXW11 3′UTR was cloned into pmirGLO plasmid (named pmirGLO-FBXW11). Cells were co-transfected with pmirGLO-FBXW11 and miR-NC or miR-182. After 48 h, Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, USA). NF-κB and β-catenin/TCF firefly luciferase reporter construct (TOPflash) and pRL-TK reporter plasmid encoding Renilla luciferase was purchased from Promega. Cells were plated in a 24-well plate and cotransfected with PCGEM1, NF-κB or β-catenin/TCF firefly luciferase reporter, and pRL-TK (Promega) by using Lipofectamine 2000. The pRL-TK vector was used as an internal control. 48 h later, cells were collected and analyzed using the Dual-Luciferase Reporter Assay System (Promega).
All experiments were performed at least three times. Data are presented as the mean ± standard error of mean (SEM). Statistical analysis was performed by using the SPSS 23.0 software. Student’s t test (for two groups comparison) or one-way analysis of variance (ANOVA; for more than two groups comparison) was utilized to analyze significant difference. The χ2 test was carried out to explore the correlation between PCGEM1 levels and clinical features. Kaplan–Meier method and log-rank test was used to assess overall survival. p < 0.05 was considered statistically significant.
PCGEM1 is highly expressed in CC tissues and predicts poor prognosis
The correlation between PCGEM1 expression and clinicopathological parameters in cervical cancer patients
PCGEM1 promotes CC cell proliferation, inhibits cell apoptosis and induces cell cycle progression
We then detected apoptosis by flow cytometry analysis in CC cells. The results showed that deletion of endogenous PCGEM1 expression significantly promoted apoptosis in HeLa cells (Fig. 2g), while overexpression of PCGEM1 inhibited apoptosis in SiHa cells (Fig. 2h). Consistent with the flow cytometry data, the protein levels of Bax, cleaved PARP1, and cleaved caspase 3, the apoptosis protein markers, increased in PCGEM1 knockdown HeLa cells, but decreased in PCGEM1 overexpressing SiHa cells (Fig. 2i).
To further gain insights into the mechanism by which PCGEM1 promotes CC cell proliferation, flow cytometry was used to analyze differences in cell cycle distributions following PCGEM1 alteration. It was found that PCGEM1 knockdown significantly induced G1/S arrest in HeLa cells (Fig. 2j), while PCGEM1 overexpression drove progression beyond the G1/S transition in SiHa cells (Fig. 2k). Consistent with the flow cytometry data, the expression of G1/S phase checkpoint protein, such as CDK4, CDK6, and cyclinD1, was significantly markedly downregulated in HeLa cells with PCGEM1 knockdown and increased when PCGEM1 was overexpressed (Fig. 2l). Together, these data strongly demonstrated that PCGEM1 promotes cell proliferation by facilitating cell cycle progression and inhibiting cell apoptosis in CC.
PCGEM1 enhances CC cell migration and invasion, and induces EMT
PCGEM1 directly interacts with miR-182
PCGEM1 exerts oncogenic effects through miR-182
FBXW11 is a target of PCGEM1 via miR-182
PCGEM1 activates NF-κB and β-catenin/TCF signaling pathways
Herein, we first demonstrated a tumor-promotive function of lncRNA PCGEM1 in CC cells. PCGEM1 was dramatically upregulated in both CC tissues and cell lines. High levels of PCGEM1 expression were associated with advanced FIGO stage, lymph node, distant metastasis, and worse overall survival of patients with CC. The similar prognostic value of PCGEM1 was also found in gastric cancer, ovarian cancer and prostate cancer [18, 20, 26]. Overall, our findings provided first clinical evidence that PCGEM1 may be used as a novel prognostic biomarker for patients with CC.
Recently, emerging evidence provided a novel regulatory mechanism between microRNA and lncRNA. lncRNAs associate with microRNAs, acting as ceRNAs by competitively binding common microRNAs . Specifically, many miRNAs involved in CC progression could be regulated by lncRNAs. For instance, lncRNA DLG1-AS1 promotes cell proliferation by competitively binding with miR-107 and upregulating ZHX1 expression in CC . LncRNA C5orf66-AS1, as a ceRNA, regulated the effect of RING1 on the proliferation, apoptosis and cell cycle of CC cells through sponging miR-637 . In the present study, our group used bioinformatics assays and found that PCGEM1 contained binding sites for miR-182. The results of MS2-RIP, RNA pull-down and luciferase assays revealed a direct interaction between PCGEM1 and miR-182. Our findings also showed that miR-182 expression was suppressed by PCGEM1 and negatively correlated with PCGEM1 expression in CC tissues. Moreover, miR-182 is responsible for PCGEM1-miediated CC cell proliferation, migration and invasion. These data suggested that PCGEM1 exerted oncogenic effect on CC progression by negatively regulating miR-182 expression.
FBXW11 belongs to the FBXW subfamily of the F-box protein family, which is crucial for embryonic development and plays pivotal roles in various signaling pathways by regulating the ubiquitination of phosphorylated substrates . Recent studies demonstrated that deregulation of FBXW11 contributes to carcinogenesis. For example, FBXW11 plays an important role in controlling the IκB-dependent apoptotic pathway in human melanoma and colorectal cancer . FBXW11 facilitates skin tumor progression by activating the NF-κB signaling pathway . Moreover, FBXW11 promotes the activation of the NF-κB and β-catenin/TCF signaling pathways, inducing cell cycle progression and tumor formation in lymphocytic leukemia . However, the role of FBXW11 in CC progression remains elusive. Here, we demonstrated a dramatically increase of FBXW11 expression in CC tissues. Consistent with previous research [33, 34], our results identified PCGEM1 as a direct target of miR-182. Interestingly, lncRNA PCGEM1 could upregulate FBXW11 through acting as a ceRNA for miR-182. We also found that PCGEM1-mediated upregulation of FBXW11 activated NF-κB and β-catenin/TCF signaling. Overall, our results indicated that PCGEM1 regulated FBXW11 in the carcinogenesis of CC by acting as a molecular sponge to modulate miR-182.
The research was supported by Liaoning Science and Technology Innovation Fund (No. 201102134).
LL: design the study, write and revise the manuscript. QZ, JZ: performed all the experiments. All authors read and approved the final manuscript.
The research was supported by Liaoning Science and Technology Innovation Fund (No. 201102134).
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of The First Affiliated Hospital of Jinzhou Medical University. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki. All patients provided written informed consent.
Consent for publication
The authors declare that they have no competing interests.
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