circEPSTI1 promotes tumor progression and cisplatin resistance via upregulating MSH2 in cervical cancer

Abstract

CircRNAs (circRNAs) are a kind of non-coding RNAs which are extensively distributed in tissues. Previous investigations reported that circRNAs harbor indispensable roles in modulating the progress of multiple cancers. Nevertheless, the function along with the molecular mechanism of most circRNAs in cervical cancer progression was still not clear. Herein, we illustrated that circEPSTI1 is a remarkably upregulated circRNA, which we validated in tissues with cervical cancer along with cell lines. The biological role of circEPSTI1 in the advancement of cervical cancer was probed via loss-of function assessments. Silencing circEPSTI1 could diminish the proliferative capacity of the cervical cancer cells to spread. In cervical cancer cells, silencing circEPSTI1 dramatically elevated drug responsivity to cisplatin. Mechanically, RNA immuno-precipitation experiments and dual luciferase enzyme reporter experiments were conducted to reveal the molecular mechanism of circEPSTI1 in cervical cancer. In conclusion, this research premise identified the biological function of circEPSTI1-miR-370-3p-MSH2 axis in cervical cancer progression. Our result is significant for slowing the progress of and overcoming drug resistance of cervical cancer.

Keywords: MSH2; cervical cancer; circEPSTI1; circular RNAs; cisplatin resistance.

Figure 1

circEPSTI1 is upregulated in cervical cancer with circRNA features. (A) The contents of circEPSTI1 in ten paired cervical cancer samples and neighboring non-malignant tissues. (B) The relative contents of circEPSTI1 in normal HcerEpic cell line and cervical cancer cell lines. (C, D) Circular transcripts of circEPSTI1 were more stable, in contrast with that of linear EPSTI1 mRNA assessed via actinomycin D treated assay in SiHa and HT-3 cervical cancer cell lines. (E, F) The circular structure of circEPSTI1 was assessed via RNase R assay in SiHa, as well as HT-3 cell lines of cervical cancer. The analyzed gene was selected from the host genes of circEPSTI1 in cervical cancer tissues.

Figure 2

Inhibition of circEPSTI1 attenuates the progress and cisplatin resistance of cervical cancer cells. (A) The effect of shRNA knockdown of circEPSTI1 was assessed in SiHa, as well as HT-3 cell lines of cervical cancer, assessing via qRT-PCR. (B) CCK-8 assay was adopted to test the cell growth rate in SiHa along with HT-3 cell lines of cervical cancer. (C, D) Colony-formation experiments were conducted in SiHa, as well as HT-3 cell lines of cervical cancer. (E, F) Transwell assay for assessment of the metastasis potential of SiHa cervical cancer cell line. (G, H) Inhibition of circEPSTI1 remarkably increased the responsivity of cervical cancer to cisplatin treatment in SiHa along with HT-3 cervical cancer cell lines.

Figure 3

circEPSTI1 sponges miR-370-3p in cervical cancer. (A, B) U6, ACTB, circEPSTI1 and EPSTI1 mRNA contents in nuclear as well as cytoplasmic fractions were assessed via qRT-PCR assays in SiHa and HT-3 cell lines of cervical cancer. (C) Predicted docking sites of miR-370-3p within the circEPSTI1 sequence. (D) The contents of miR-370-3p in ten paired cervical cancer samples and vicinal non-malignant tissues. (E) The relative contents of miR-370-3p in non-malignant HcerEpic cell line and cervical cancer cell lines. (F, G) Dual luciferase reporting experiments revealed that the relative amount of fluorescence value was reduced after transfected with miR-370-3p in SiHa, as well as HT-3 cervical cancer cell lines. (H) MS2-based RNA immunoprecipitation assays were conducted to prove the docking of miR-370-3p on circEPSTI1 in SiHa along with HT-3 cell lines of cervical cancer.

Figure 4

MSH2 was the downstream target of miR-370-3p in cervical cancer. (A) On the basis of the TargetScan data resource, MSH2 mRNA was the putative downstream target of miR-370-3p. (B) The contents of MSH2 mRNA in ten paired cervical cancer samples and vicinal non-malignant tissues. (C) The relative contents of MSH2 mRNA in normal HcerEpic cell line and cervical cancer cell lines. (D, E) Dual luciferase reporting experiments revealed that the relative amount of fluorescence value was reduced after transfected with miR-370-3p in SiHa, as well as HT-3 cervical cancer cell lines. (F, G) Abundance of circEPSTI1, MSH2 mRNA and miR-370-3p on the AGO2 target protein, assessed via RIP assays. (H, I) Enrichment of AGO2 protein to circEPSTI1 was diminished whilst MSH2 mRNA was increased after silencing of circEPSTI1 in SiHa along with HT-3 cervical cancer cell lines.

Figure 5

circEPSTI1 facilitates cervical cancer progression and cisplatin resistance through circEPSTI1-miR-370-3p-MSH2 axis. (A, B) CCK-8 assay for assessment of rate of cell proliferation in SiHa, as well as HT-3 cervical cancer cell lines. (C, D) Inhibition of circEPSTI1 remarkably increased the responsivity of cervical cancer to cisplatin treatment, whilst this effect could be rescues by miR-370-3p inhibitors in SiHa along with HT-3 cell lines of cervical cancer. (E) Expression of MSH2 mRNA transcript was reduced after circEPSTI1 inhibition and increased following the transfection with inhibitors of miR-370-3p, detected by qPCR analysis. (F) Knockdown of circEPSTI1 downregulated the MSH2 protein expression, which could be reversed through the miR-370-3p inhibitors in SiHa cervical cancer cells, illustrated by the western blot analysis.