Long noncoding RNA MEG3 is downregulated in cervical cancer and affects cell proliferation and apoptosis by regulating miR-21

Abstract

Recent research has found that long noncoding RNAs (lncRNAs) were involved in various human cancers. However, the role of these lncRNAs in cervical cancer remains unexplored. Therefore, we aimed to investigate the biological function of maternally expressed gene 3 (MEG3), a cancer-related lncRNA, and its underlying mechanism in cervical cancer. In this study, MEG3 expression of 108 patients' cervical cancer tissues and adjacent normal tissues was detected by quantitative real-time PCR analysis (qRT-PCR), and the functional effect of MEG3 was determined in vitro assays. We observed that MEG3 was downregulated in cervical cancer tissues, compared to the adjacent normal tissues, and was negatively related with FIGO stages, tumor size, lymphatic metastasis, HR-HPV infection and the expression of homo sapiens microRNA-21 (miR-21). Furthermore, we focused on the function and molecular mechanism of MEG3, finding that overexpression of MEG3 reduced the level of miR-21-5p expression, causing inhibition of proliferation and increased apoptosis in cervical cancer cells. In summary, our findings indicate that MEG3 function as a tumor suppressor by regulating miR-21-5p, resulting in the inhibition of tumor growth in cervical cancer. As a result, this study improves our understanding of the function of MEG3 in cervical cancer and will help to provide new potential target sites for cervical cancer treatment.

Keywords: Apoptosis; MEG3; cervical cancer; long noncoding RNA; miR-21; p53; proliferation.

Figure 1.

Relative MEG3 expression in cervical cancer tissues and its clinical significance. (A) Relative expression of MEG3 in cervical cancer tissues (n = 108) compared with adjacent normal tissues (n = 108). (B, C, D and E) MEG3 expression was significantly lower in patients with larger tumor size, lymph nodes metastasis, HR HPV positive and advanced FIGO stage. (F) Pearson's correlation coefficient analysis showed that miR-21-5p expression was negatively correlated with MEG3 in cervical cancer tissues. **P < 0.01.

Figure 2.

Effects of MEG3 on cervical cancer cells proliferation in vitro. (A) Upregulated MEG3 expression dramatically decreased the ability of Hela and CaSki cells proliferation by CCK-8 assay. (B) Downregulated MEG3 expression dramatically increased the ability of Hela and CaSki cells proliferation by CCK-8 assay. ^＊P < 0.05.

Figure 3.

Colony formation assay was performed to determine the colony formation ability of transfected Hela cells. (A, B) The colony number of pcDNA-MEG3 or si-MEG3 transfected Hela cells. (C, D) The colony number of Hela cells transfected with pcDNA- MEG3 and miR-21-5p mimic or transfected with si-MEG3 and miR-21-5p inhibitor. ^＊P < 0.05.

Figure 4.

Effects of MEG3 on cervical cancer cells apoptosis in vitro. (A) Overexpression of MEG3 promotes the apoptosis of Hela and CaSki cells. (B) Knockdown of MEG3 reduced the apoptosis of Hela and CaSki cells. ^＊P < 0.05.

Figure 5.

P53 and cleaved caspase 3 protein levels after the transfection. (A) P53 and cleaved caspase 3 protein levels was increased in HeLa and CaSki cell after the transfection of the pcDNA- MEG3. (B) P53 and cleaved caspase 3 protein levels was decreased after the transfection of the si- MEG3.

Figure 6.

The interaction between MEG3 and miR-21-5p. (A, B) Hela and CaSki cells were transfected with pcDNA-MEG3 or si-MEG3, and qRT-PCR was used to detect the miR-21-5p levels compared with controls. (C, D) Hela and CaSki cells were transfected with miR-21-5p mimic or inhibitor, and qRT-PCR was used to detect the MEG3 levels compared with controls. ^＊P < 0.05.

Figure 7.

MiR-21-5p mediated the effects of MEG3 on Hela and CaSki cells proliferation. (A) CCK8 assay to evaluate the effect of MEG3 overexpression and miR-21-5p stimulation on Hela and CaSki cells proliferation. (B) CCK8 assay to evaluate the effect of MEG3 knockdown and miR-21-5p inhibition on Hela and CaSki cells proliferation. ^＊P < 0.05.

Figure 8.

MiR-21-5p mediated the effects of MEG3 on Hela and CaSki cells apoptosis. (A) Effect of co-transfection of pcDNA- MEG3 and miR-21-5p mimic on apoptosis of Hela and CaSki cells. (B) Effect of co-transfection of si-MEG3 and miR-21-5p inhibitor on apoptosis of Hela and CaSki cells. ^＊P < 0.05.

Figure 9.

MiR-21-5p mediated the effects of MEG3 on regulating P53 and cleaved caspase 3 protein levels. (A) The level of p53 and cleaved caspase 3 protein was decreased in pcDNA- MEG3 + miR-21-5p mimic group. (B) The level of p53 and cleaved caspase 3 protein was increased in si-MEG3 + miR-21-5p inhibitor groups.