Long non-coding RNA MEG3 inhibits cervical cancer cell growth by promoting degradation of P-STAT3 protein via ubiquitination

Abstract

Background: Maternally expressed 3 (MEG3) plays an important role in cervical cancer development, but its exact role remains unclear. Here, we explored the specific regulatory mechanism of MEG3 and its downstream proteins in cervical cancer cells.

Methods: The effect of MEG3 on tumor formation ability of cervical cancer cells was determined in nude mice. The direct binding of MEG3 to phosphorylated signal transducer and activator of transcription 3 (P-STAT3) was detected by RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays. Cycloheximide (CHX)-chase and ubiquitination assays were performed to determine the regulatory effect of MEG3 on P-STAT3 ubiquitination. Clone formation assay and flow cytometry were used to evaluate the effect of the MEG3-STAT3 regulatory axis on cell proliferation and apoptosis.

Results: In vivo tumor formation experiments showed that MEG3 inhibited the tumor formation ability of cervical cancer cells. RNA pull-down and RIP assays demonstrated that MEG3 bound directly to P-STAT3 protein. CHX-chase and ubiquitination assay results showed that MEG3 promoted P-STAT3 degradation via ubiquitination. Clone formation assay and flow cytometry analysis results revealed that the inhibitory effect of MEG3 on P-STAT3 promoted apoptosis and inhibited proliferation of cervical cancer cells.

Conclusion: MEG3 binds to P-STAT3 in cervical cancer cells, resulting in P-STAT3 ubiquitination and degradation and apoptosis and inhibition of proliferation of tumor cells. The in-depth elaboration of the MEG3-STAT3 regulatory axis in cervical cancer may clarify the mechanism of action of MEG3 and provide new ideas for cervical cancer treatment.

Keywords: Cervical cancer; LncRNA; MEG3; STAT3.

Fig. 1

MEG3 inhibits the tumor formation ability of cervical cancer cells. The rate of tumor formation and tumor size in MEG3 group were significantly lower than those in the control group. In contrast, the tumor formation rate and tumor size were significantly higher in MEG3 shRNA group than in the control group (a, b). Immunohistochemistry examination of tumors showed that the expression of Ki67 and P-STAT3 proteins was significantly lower and the number of TUNEL-positive cells was significantly higher in MEG3 group than in the control group; the expression of Ki67 and P-STAT3 proteins was significantly higher and the number of TUNEL-positive cells was significantly lower in MEG3 shRNA group than in the control group. The difference of STAT3 expression was not significant. Scale bar = 50 μm (c). *P < 0.05

Fig. 2

The mutual regulation between MEG3 and P-STAT3. The expression of P-STAT3 and MEG3 in cervical cancer tissues(n = 22)was detected by ELISA and RT-qPCR assays. Then Pearson correlation coefficient was calculated (a). RT-qPCR and western blot analysis results showed that the expression of P-STAT3 and c-MYC proteins decreased while that of caspase 3 and cleaved caspase 3 increased in Siha cells with high-level expression of MEG3. On the contrary, HeLa cells with low-level expression of MEG3 showed an increase in the expression of P-STAT3 and c-MYC proteins and a decrease in the expression of caspase 3 and cleaved caspase 3 (b, c). Immunofluorescence results confirmed that P-STAT3 protein was highly expressed in the cells from MEG3 group, while its expression was low in the cells from MEG3 shRNA group. Scale bar = 50 μm (d). RT-qPCR and luciferase assays showed that MEG3 had no significant effect on STAT3 gene expression (e). RT-qPCR and western blot analysis results revealed the absence of any significant change in the expression of MEG3 after upregulation or interference of STAT3 expression in cervical cancer cells (f, g). *P < 0.05

Fig. 3

MEG3 promotes the degradation of P-STAT3 via ubiquitination. The RNA pull-down assay results suggest that MEG3 may bind to P-STAT3 protein (a). The results of RIP assay suggest that P-STAT3 protein may enrich MEG3 (b). CHX-chase assay results suggest that MEG3 may promote P-STAT3 protein degradation (c, d). All groups of cells were treated with 20 μM of an ubiquitination inhibitor MG132 for 4 h or 5 mM of an autophagy inhibitor 3-methyladenine (3-MA, Selleck Chemicals) for 2 h, followed by western blot analysis to determine changes in P-STAT3 expression (e). Ubiquitination assay was used to detect P-STAT3 ubiquitination (f) in all groups of cells. *P < 0.05

Fig. 4

The effect of the MEG3-STAT3 regulatory axis on the proliferation and apoptosis of cervical cancer cells. Cells were treated with nifuroxazide (STAT3 phosphorylation inhibitor, Selleck Chemicals) at a final concentration of 20 mM in dimethyl sulfoxide (DMSO). Western blot analysis of P-STAT3 protein expression suggested that nifuroxazide may effectively decrease the level of P-STAT3 protein and reverse the regulation of P-STAT3 protein by MEG3 shRNA (a). The clone formation experiment results suggest that nifuroxazide may reverse the effect of MEG3 shRNA on the promotion of the proliferation of cervical cancer cells (b, d). Flow cytometry results indicate that nifuroxazide may reverse the effect of MEG3 shRNA on the inhibition of apoptosis of cervical cancer cells (c, e). *P < 0.05