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Title: [Mechanisms of the suppression of proliferation and invasion ability mediated by microRNA-147b in esophageal squamous cell carcinoma].
Author: Tang Y, Li Z, Shi ZX.
Journal: Zhonghua Yi Xue Za Zhi; 2018 Jul 10; 98(26):2092-2098. PubMed ID: 30032507.
Abstract:
Objective: To explore the expression of microRNA(miR)-147b in esophageal squamous cell carcinoma (ESCC) and its regulatory roles in cell proliferation, cell cycle and invasion as well as its molecular mechanisms. Methods: Real-time quantitative PCR (qPCR) was used to investigate the expression of miR-147b in ESCC tissues and cells. Negative control (NC) and miR-147b inhibitor were transfected into ESCC EC1 and EC9706 cells, which were divided into two groups: NC group and miR-147b inhibitor group, and qPCR was employed to detect the miR-147 level and CCK-8. Flow cytometry and Transwell chamber were utilized to investigate the effects of miR-147b downregulation on cell proliferation, cell cycle and invasion in ESCC cells. Besides, target genes of miR-147b was confirmed by double luciferase reporter assay. Subsequently, qPCR and Western blot were used to examine the effects of miR-147b downregulation on NDUFA4 expression, and NDUFA4 expression and its correlation with miR-147b were investigated in ESCC tissues. Results: Relative level of miR-147b in ESCC tissues (3.03±0.27) and cells were markedly higher than that in para-carcinoma tissues (1.00±0.01) and normal esophageal epithelial cell, and the differences had statistical significance (P<0.01), and its high expression was closely associated with clinical staging, invasion depth, histological grading and lymph node metastasis(P<0.05). Importantly, clinical staging, lymph node metastasis and miR-147b may be an independent prognostic factor in ESCC. Moreover, miR-147b downregulation dramatically suppressed cell proliferation, arrested cell cycle in G0/G1 phase and reduced invasion ability in ESCC cells. Most importantly, NDUFA4 was a direct target gene of miR-147b, and miR-147b inhibitor evidently upregulated the expression of NDUFA4. Furthermore, NDUFA4 displayed low expression in ESCC tissues and its expression exhibited negative correlation with miR-147b expression. Conclusions: The downregulation of miR-147b expression significantly suppresses the proliferation and invasion abilities as well as alters cell cycle distribution in ESCC. 目的： 探讨微RNA(microRNA，miR)－147b在食管鳞状细胞癌(以下简称"鳞癌")中的表达及其在细胞增殖、细胞周期和侵袭中的分子机制。 方法： 采用实时荧光定量PCR(qPCR)检测食管鳞癌组织和细胞中miR－147b的表达。将阴性对照(NC)和miR－147b抑制剂转染食管鳞癌EC1和EC9706细胞。试验分为两组：NC组和miR－147b抑制剂组，采用qPCR检测上述细胞中miR－147b的表达，采用CCK－8、流式细胞术和Transwell小室检测分别检测miR－147b表达下调对食管鳞癌细胞增殖、细胞周期和侵袭的影响。利用双荧光素酶报告载体分析其直接作用的靶基因。采用qPCR和Western印迹检测miR－147b表达下调对靶基因NDUFA4表达的影响，进一步利用qPCR检测NDUFA4在食管鳞癌组织中的表达，并分析其表达与miR－147b表达的相关性。 结果： 食管鳞癌组织(3.03±0.27)和细胞中miR－147b的相对表达水平均显著高于癌旁组织(1.00±0.01)和正常食管上皮细胞，差异均有统计学意义(均P<0.01)，并且miR－147b高表达与食管鳞癌患者的临床分期、浸润深度、组织学分级及淋巴结转移均密切相关(均P<0.05)，临床分期、淋巴结转移和miR－147b是食管鳞癌患者的独立预后因素。miR－147b表达下调能显著抑制食管鳞癌细胞的增殖、静止细胞周期在G0/G1期和降低其侵袭能力。NDUFA4是miR－147b直接作用的靶基因，miR－147b抑制剂显著增加食管鳞癌EC1和EC9706细胞中NDUFA4的表达。NDUFA4在食管鳞癌组织中表达显著下调，其与miR－147b的表达负相关(r＝－0.765，P<0.001)。 结论： miR－147b表达的下调显著抑制食管鳞癌的增殖和侵袭能力，且可以改变其细胞周期分布。.

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