Cervical cancer is the second most frequent malignancy-affecting women worldwide, with approximately over 570000 new cases diagnosed and 310000 deaths each year. Nearly 85% of the cases occur in underdeveloped and developing countries.1 Interactions between cervical cancer and the persistent infection of high-risk human papilloma virus (especially HPV16 and HPV18) have been indicated in epidemiological and molecular studies.2, 3, 4 Despite the improvement of diagnostic approaches and treatment strategies have been made, the morbidity and mortality rates remain high as well as the poor prognosis. Therefore, there is an urgent need to clarify the underlying molecular mechanisms of cervical cancer, which could improve the development of therapeutic strategies against cervical cancer.
Long noncoding RNAs (lncRNAs) are a group of noncoding RNAs longer than 200 nucleotides that participate in numerous biological and physiological processes including cell development, survival, differentiation and apoptosis.5, 6, 7 Accumulating evidence also proved that lncRNAs have pivotal roles in the progression of cervical cancer. Yang
MicroRNAs (miRNAs) are another group of noncoding RNAs with length between 17-25 nucleotides that influence the pathogenesis of many human diseases including cancer.14, 15, 16 MiRNAs are normally dysregulated in human cancers and could serve as either oncogenes or tumor suppressors.17 Previous studies showed that expressions of miR-140-5p were downregulated in cancer tissues, which were negatively related to the prognosis of cancer18, 19, 20, indicating the tumor-suppressor role of miR-140-5p in the progression of cancer formation. Li
In the present study, the role and the underlying molecular mechanism of lncRNA PVT1 in cervical cancer were illuminated. Our results showed that lncRNA PVT1 could promote the proliferation, migration and invasion through binding miR-140-5p, which may provide new insights into the therapeutic strategy against cervical cancer.
Human normal cervical epithelial cell line (End1/ E6E7) and human cervical cell lines (HeLa and SiHa) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco’s minimal Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 6% fetal bovine serum (FBS; Gibico, Grand Island, NY, USA) and 1% penicillin/ streptomycin at 37°C in a humidified atmosphere containing 5% CO2.
A bioinformatics website (
Sh-PVT1, negative control (sh-NC), miR-140-5p mimics and miR-140-5p inhibitor were synthesized by GenePharma (Shanghai, China). After reaching 60%-70% confluence, the cervical cancer cells were transfected by using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions.
After transfection, cells were seeded in 6-well plates at a density of 1×103 cells/well. The cells were then fixed and stained with 0.1% crystal violet after incubation for 14 days. The numbers of colonies were counted under microscope. This assay was performed at least three times.
Cells were incubated in 6-well plates at a density of 1 × 106 cells/well. After transfection, the cells were cultured with serum-free medium for 24 h. Wounds were created by using a sterile plastic pipette tip to scratch the cell layer. Then the cells were incubated with medium containing 10% FBS for 48 h. Images were acquired by microscope. This assay was performed at least three times.
The transwell assays were determined using transwell chambers (Corning, NY, USA) as previously described.22 Briefly, transfected cells were suspended in serum-free medium and seeded into the upper chamber, while medium containing 10% FBS was added in the bottom chamber. After incubation at 37°C for 24 h, the cells that invaded through matrix membrane were fixed with 4% paraform-aldehyde and stained with crystal violet. Then, stained cells were photographed and counted from five random fields per filter for analysis. This assay was performed at least three times.
Total RNA was extracted from cell samples using Trizol reagents (Thermo Fisher Scientific, Waltham, MA, USA) from cell lines, and cDNA was synthesized using PrimeScriptTM cDNA Kit (Takara, Dalian, China) according to the manufacture’s protocols. qRT-PCR was determined using an ABI 7000 Prism Step One plus detection system (Life Technologies, USA). The relative expression was normalized using GAPDH as an internal reference gene, and U6 was used as the endogenous control of miR-140-5p. Fold changes were calculated using the formula 2-ΔΔCt. All qRT-PCR reactions were performed three times independently. The primer sequences used for qRT-PCR as follow.
Cell samples were harvested and lysed using cell lysis buffer on ice. Total proteins were extracted and concentrations were detected using a BCA protein assay kit (Beyotime, China). Same amounts of protein samples were isolated by 12% SDS-PAGE gels and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with TBS-T containing 5% nonfat milk powder for 2 h at RT and incubated with primary antibodies at 4°C overnight. The membranes were then incubated with secondary antibody for 1 h at RT. The images were visualized using an enhanced chemiluminescence system. Signals were quantified and analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Sarasota, USA). The following primary antibodies were applied: anti-Smad3 (1:1000; #9513), anti-E-cadherin (1:1000; #14472), anti-vimentin (1:1000; #3932), anti-N-cadherin (1:1000; #4061) anti-Snail (1:1000; #3879) (All purchased from Cell signaling Technology). The corresponding HRP-conjugated antibody (1: 5000; Abcam, UK) was used as the secondary antibody.
Statistical analysis was performed using SPSS 22.0 software (IBM Corporation, NY, USA). All data were expressed as the mean ± standard deviation (SD). All experiments were performed triplicate independently. Comparisons were determined using one-way analysis of variance (ANOVA) or Student’s
PVT1 forward 5’-AAAACGGCAGCAGGAAATGT-3’ and reverse 5’-GGAGTCATGGGTGTCAGACA-3’.
miR-140-5p forward 5’-GGGCCAGTGGTTTTACCCTA-3’
and reverse 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC CTACCA-3’.
Smad3 forward 5’-CTCCAAACCTATCCCCGAAT-3’
and reverse 5’-CCTGTTGACATTGGAGAGCA-3’. U6 forward 5′-AAAGCAAATCATCGGACGACC-3′
and reverse 5′- GTACAACACATTGTTTCCTCGGA-3′.
GAPDH forward 5′-AGAAGGCTGGGGCTCATTTG -3′
and reverse 5′-AGGGGCCATCCACAGTCTTC-3’.
To determine the roles of lncRNA PVT1, miR-140-5p and Smad3 in cervical cancer metastasis, expressions of lncRNA PVT1, miR-140-5p and Smad3 in cervical cancer cell lines (HeLa and SiHa) and human normal cervical cell line (End1/E6E7) were determined by qRT-PCR and western blotting. The results in Figure 1A and 1B show that lncRNA PVT1 expression was significantly upregulated in cervical cancer cell lines compared with that in the normal cervical cell line by over 1.6-fold (
Bioinformatics analysis showed that miR-140-5p might be a target of PVT1 (Figure 2A). Dual luciferase assay was further conducted to determine whether PVT1 could regulate miR-140-5p expression by acting as a molecular sponge, showing that miR-140-5p mimics could significantly inhibit the luciferase activity of PVT1-WT in both cancer cell lines, but had no significant effect on that of PVT1-MUT (Figure 2C and 2D). Furthermore, bio-informatics analysis revealed miR-140-5p can also bind directly to Smad3 (Figure 2B). The results of Figure 2E and 2F showed that miR-140-5p mimics could remarkably suppress the luciferase activity of Smad3-WT in both cervical cancer cell lines, and had no effect on that of Smad3-MUT.
Since PVT1 was highly expressed in cervical cancer cells, revealing that lncRNA PVT1 could be a potential oncogene during the progression of cervical cancer. To figure out the function of lncRNA PVT1 during the progression of cervical cancer cells, PVT1 specific sh-RNA was applied to knockdown the expression of PVT1. As shown in Figure 3A and 3B, the expressions of PVT1 were significantly downregulated in sh-PVT1 transfected cervical cancer cells compared with that in cells transfected with sh-NC and the control group as their respective reductions were 39.8% and 43.7% (
reductions in protein levels, respectively (
The results showed that PVT1 knockdown could significantly decrease the migration activities of cervical cancer cells compared with the sh-NC groups, with an inhibition rate of 51.2% and 61.3% in both cells lines respectively. (Figure 4A-4D,
To determine whether miR-140-5p was implicated in the effect of lncRNA PVT1 on the progression of cervical cancer cells, sh-PVT1 transfected cervical cancer cells were transfected with or without miR-140-5p inhibitor. The results showed that miR-140-5p inhibitor could decline the PVT1 inhibition-mediated decreasing effect on the Smad3 expression in cervical cancer cells (Figure 5A-5D). Colony formation assay indicated that inhibition of miR-140-5p could reserve the inhibition effects of PVT1
downregulation on proliferation of cervical cancer cells (Figure 5E and 5F). Similar results could also be observed in wound healing and transwell assays, that decreased migration and invasion abilities induced by PVT1 downregulation could be rescued by miR-140-5p inhibitor (Figure 6A-6H). Besides, treatment with miR-140-5p inhibitor could suppress sh-PVT1-induced upregulation of E-cadherin expression and reverse sh-PVT1-induced down-regulation of N-cadherin, vimentin and Snail expressions (Figure 6I-6J). These data indicated that PVT1 knockdown could suppress the progression of cervical cancer cells via miR-140-5p.
Accumulating evidence has revealed that aberrantly expressed lncRNAs plays crucial role in tumor occurrence, invasion and metastasis including cervical cancer.23 By far, PVT1 is recognized as an oncogene in many cancers, however, the role of PVT1 in the progression of cervical cancer and the potential underlying mechanism by which PVT1 participate in remains uncertain.
Recent studies have shown that PVT1 is upregulated in many kinds of cancers and serves as an oncogenic lncRNA. Zhang
Despite much progress have been made in the research of lncRNAs, the underlying mechanism remains unclear. Recently, interactions between lncRNAs and miRNAs have proposed that lncR-NAs can serve as a sponge to bind miRNA and regulate the functions of miRNAs, which play a crucial role in the proliferation and tumorigenesis of many cancers.28, 29 So, we further focused on miR-140-5p, due to its critical role in the progression of cervical cancer.12, 13 Our data revealed that expressions of miR-140-5p were significantly downregulated in cervical cancer cells. Inhibition of PVT1 significantly increased the expressions of miR-140-5p in cervical cancer cells. In addition, luciferase assay was further applied to determine the correlation between PVT1 and miR-140-5p. Our results revealed that miR-140-5p upregulation could significantly inhibit the luciferase activities of PVT1-WT but did not affect that of PVT1-MUT, indicating that PVT1 binds to miR-140-5p and regulate its function. To further, clarify the interaction of PVT1 and miR-140-5p during the progression of cervical cancer cells. MiR-140-5p inhibitor were transfected into PVT1 knockdown cervical cancer cells then proliferation, migration and invasion assays of cervical cancer cells were performed. Zhang
Normally, miRNAs could regulate tumorigenesis by targeting a variety of protein-coding genes.31 Smad3 has been demonstrated increased and could act as an oncogenic role in the progression of many type of cancers. For instance, Yamazaki
Collectively, we found that PVT1 is upregulated in cervical cancer cells, and PVT1 might serve as a prognostic indicator for cervical cancer patients.34, 35, 36 Furthermore, PVT1 sponges directly to miR-140-5p and regulates its expression and function. In addition, Smad3 is also upregulated in cervical cancer cells and it is the downstream target of miR-140-5p. Moreover, PVT1 promotes the proliferation, migration and invasion of cervical cancer cells via regulating miR-140-5p and Smad3. Our findings provide new insights into the therapeutic strategy against cervical cancer.