Liu Shikai, Song Lili, Yao Hairong, Zhang Liang, Xu Dongkui, Gao Fangyuan, Li Qian
Department of Obstetrics & Gynaecology, Cangzhou Central Hospital, Hebei, 061001, China.
PLoS One. 2016 Sep 22;11(9):e0163460. doi: 10.1371/journal.pone.0163460. eCollection 2016.
Epigenetic modulation is an important mechanism of miRNA dysregulation in cervical cancer. In this study, we firstly studied how this mechanism contributes to miR-375 downregulation in cervical cancer cells. Then, we further studied the association between miR-375 and MALAT1 (metastasis associated lung adenocarcinoma transcript 1) in epithelial mesenchymal transition (EMT) of the cancer cells. HPV-16 positive SiHa and CaSki cells were used as in vitro model. Our data showed that HPV-16 E6 positively modulated DNMT1 expression in both SiHa and CaSki cells. Knockdown of DNMT1 partly restored miR-375 levels in the cells. The following methylation-specific PCR (MSP) assay and qRT-PCR analysis showed that methylation was common in the promoter region of miR-375 in both SiHa and CaSki cells and demethylation partly restored miR-375 levels in the cells. Therefore, we infer that miR-375 is downregulated partly due to promoter hypermethylation mediated by DNMT1 in HPV-16 positive cervical cancer cells. Our bioinformatics analysis showed that MALAT1 has three putative binding sites with miR-375 and the following dual luciferase assay confirmed two of them. QRT-PCR analysis showed that miR-375 overexpression significantly reduced MALAT1 expression, while MALAT1 overexpression reversely suppressed miR-375 levels. Therefore, we infer that there is a reciprocal regulation between miR-375 and MALAT1 in the cells. In SiHa cells, miR-375 overexpression or MALAT1 siRNA partly restored E-cadherin expression, significantly reduced N-cadherin and also reduced invasion capacity of SiHa cells. Therefore, these results suggest that miR-375 and MALAT1 form a functional axis modulating EMT in cervical cancer.
表观遗传调控是宫颈癌中miRNA失调的重要机制。在本研究中，我们首先研究了该机制如何导致宫颈癌细胞中miR-375的下调。然后，我们进一步研究了miR-375与转移相关肺腺癌转录本1（MALAT1）在癌细胞上皮-间质转化（EMT）中的关联。HPV-16阳性的SiHa和CaSki细胞用作体外模型。我们的数据表明，HPV-16 E6在SiHa和CaSki细胞中均正向调节DNMT1的表达。敲低DNMT1可部分恢复细胞中miR-375的水平。随后的甲基化特异性PCR（MSP）检测和qRT-PCR分析表明，SiHa和CaSki细胞中miR-375启动子区域的甲基化均很常见，去甲基化可部分恢复细胞中miR-375的水平。因此，我们推断在HPV-16阳性宫颈癌细胞中，miR-375下调部分是由于DNMT1介导的启动子高甲基化所致。我们的生物信息学分析表明，MALAT1与miR-375有三个假定的结合位点，随后的双荧光素酶检测证实了其中两个。qRT-PCR分析表明，miR-375过表达显著降低MALAT1的表达，而MALAT1过表达则反向抑制miR-375的水平。因此，我们推断细胞中miR-375与MALAT1之间存在相互调节关系。在SiHa细胞中，miR-375过表达或MALAT1 siRNA可部分恢复E-钙黏蛋白的表达，显著降低N-钙黏蛋白的表达，并降低SiHa细胞的侵袭能力。因此，这些结果表明，miR-375和MALAT1形成了一个调节宫颈癌EMT的功能轴。
