Guo Ying, Liao Ying, Jia Chunyan, Ren Jianlin, Wang Jianchao, Li Ting
Department of Gynaecology, Shanghai Traditional Chinese Medicine Hospital, Shanghai, China.
Cell Physiol Biochem. 2013;32(3):581-90. doi: 10.1159/000354462. Epub 2013 Sep 6.
BACKGROUND/AIMS: Endometrial carcinoma (EC) is the most common gynecological malignancy among women worldwide. Despite its prevalence, the molecular mechanisms underlying endometrial carcinogenesis are poorly understood. The purpose of this study was to examine the role of microRNA-182 and its target gene transcription elongation factor A-like 7 (TCEAL7) in EC.
The expression of miR-182 in human normal endometrial epithelial cells (NEEC) and in three human endometrial carcinoma cell lines (HEC-1B, RL95-2 and AN3CA) was measured by qRT-PCR, and the mRNA and protein expression of TCEAL7 were assessed in the same three endometrial carcinoma cell lines and NEEC by qRT-PCR and western blotting, respectively. Subsequently, the target of miR-182 was predicted by bioinformatics and confirmed using a luciferase assay. Cell proliferation and colony formation of RL95-2 cells were examined by MTT assay and crystal violet staining, respectively. The expression of NFκB-p65, c-Myc and cyclin D1 proteins was determined by Western blot analysis.
MiR-182 was significantly upregulated and TCEAL7 was downregulated in EC cell lines compared to NEEC. We showed that MiR-182 binds directly to a conserved 8 bp sequence in the 3'-UTR of TCEAL7, and inhibition of miR-182 upregulated TCEAL7 mRNA and protein expression to levels comparable to those induced by lentiviral-mediated overexpression of TCEAL7. MiR-182 inhibition decreased cell proliferation and colony formation ability, downregulated the expression of the pro-proliferative genes c-Myc and cyclin D1, and inhibited NFκB activation, and these effects were mimicked by TCEAL7 overexpression.
miR-182 acts as an oncogenic miRNA in EC, promoting cell proliferation by targeting the tumor suppressor gene TCEAL7 and modulating the activity of its downstream effectors c-Myc, cyclin D1 and NFκB.
背景/目的：子宫内膜癌（EC）是全球女性中最常见的妇科恶性肿瘤。尽管其发病率较高，但子宫内膜癌发生的分子机制仍知之甚少。本研究旨在探讨微小RNA-182及其靶基因转录延伸因子A样7（TCEAL7）在子宫内膜癌中的作用。
采用qRT-PCR检测人正常子宫内膜上皮细胞（NEEC）及三种人子宫内膜癌细胞系（HEC-1B、RL95-2和AN3CA）中miR-182的表达，分别采用qRT-PCR和蛋白质印迹法检测同一三种子宫内膜癌细胞系和NEEC中TCEAL7的mRNA和蛋白质表达。随后，通过生物信息学预测miR-182的靶标，并使用荧光素酶测定法进行验证。分别采用MTT法和结晶紫染色法检测RL95-2细胞的增殖和集落形成情况。通过蛋白质印迹分析确定NFκB-p65、c-Myc和细胞周期蛋白D1蛋白的表达。
与NEEC相比，miR-182在EC细胞系中显著上调，而TCEAL7下调。我们发现miR-182直接与TCEAL7 3'-UTR中的一个保守8bp序列结合，抑制miR-182可将TCEAL7 mRNA和蛋白质表达上调至与慢病毒介导的TCEAL7过表达诱导的水平相当。抑制miR-182可降低细胞增殖和集落形成能力，下调促增殖基因c-Myc和细胞周期蛋白D1的表达，并抑制NFκB激活，而TCEAL7过表达可模拟这些作用。
miR-182在子宫内膜癌中作为一种致癌性微小RNA发挥作用，通过靶向肿瘤抑制基因TCEAL7并调节其下游效应物c-Myc、细胞周期蛋白D1和NFκB的活性来促进细胞增殖。
