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Title: [MicroRNA-133b suppresses cell proliferation and invasion of esophageal squamous cell carcinoma via downregulating TAGLN2 expression].
Author: Tang Y, Liu JH, Shi ZX, Li Z, Liu HT, Lu P.
Journal: Zhonghua Zhong Liu Za Zhi; 2019 Feb 23; 41(2):91-96. PubMed ID: 30862136.
Abstract:
Objective: To investigate the expression of microRNA-133b (miR-133b) in esophageal squamous cell carcinoma (ESCC), and explore its effect and the underlying molecular mechanisms on cell proliferation and invasion. Methods: Real-time quantitative PCR (qPCR) was used to examine miR-133b expression in 63 ESCC tissues and paired adjacent non-cancerous tissues, several ESCC cells (Eca109, EC9706, EC1, TE1, KYSE70) and normal esophageal epithelial cell Het-1A. MiR-133b mimic, inhibitor and negative control (NC) were transfected into TE1 cells. The effect of miR-133b on cell proliferation and invasion were determined by CCK-8 and Transwell assays, respectively. Subsequently, the target gene of miR-133b was predicted by online tools TargetScan and miRDB, which was verified by dual luciferase reporter assays. Finally, Western blot was utilized to detect the effects of miR-133b overexpression on expression of target gene TAGLN2 as well as EMT-related proteins E-cadherin, N-cadherin, Snail, Slug and Vimentin. Results: Relative levels of miR-133b in ESCC tissues (0.295±0.040) were significantly lower than those in adjacent non-cancerous tissues (1.002±0.011, P<0.001). The expression of miR-133b was tightly associated with clinical staging, lymph node metastasis and prognosis. Moreover, relative levels of miR-133b in ESCC cells Eca109, EC9706, EC1, TE1 and KYSE70 (0.679±0.031, 0.391±0.008, 0.236±0.016, 0.031±0.005 and 0.099±0.020) were evidently lower than that in normal esophageal epithelial cell Het-1A (1.005±0.016, all P<0.001). In TE1 cells, miR-133b mimic significantly increased the level of miR-133b to 6.199±0.627, and suppressed cell proliferation and invasion, whereas miR-133b inhibitor obviously decreased its expression to 0.182±0.023, and promoted cell proliferation and invasion. Most notably, the relative luciferase activities of miR-133b-mimic group (0.320±0.018) in TE1 cells transfected with TAGLN-3'UTR-WT were markedly lower than that in NC group (1.010±0.036, P<0.001), whereas those in TAGLN-3'UTR-MUT transfection cells were 1.019±0.056 and 1.008±0.021, respectively, showing no significantly statistical difference (P>0.05). Furthermore, miR-133b overexpression markedly downregulated TAGLN2, N-cadherin, Snail, Slug and Vimentin levels, and increased E-cadherin expression. Conclusion: MiR-133b plays an important role in the proliferation and invasion of ESCC cells by regulating TAGLN2 expression, and it may be a potential therapeutic target for ESCC patients. 目的： 观察miR－133b在食管鳞癌中的表达，并探讨其对食管鳞癌细胞增殖和侵袭能力的影响及分子机制。 方法： 采用实时荧光定量PCR方法检测63例食管鳞癌组织及对应的癌旁组织、食管鳞癌细胞(Eca109、EC9706、EC1、TE1、KYSE70)和正常食管上皮细胞Het－1A中miR－133b的表达。将miR－133b类似物、miR－133b抑制剂及阴性对照转染TE1细胞，分别采用细胞计数试剂盒8(CCK－8)法和Transwell小室实验检测miR－133b对TE1细胞增殖和侵袭能力的影响。采用TargetScan和miRDB在线软件预测miR－133b的靶基因，并利用双荧光素酶报告载体证实其潜在的靶基因。采用Western blot方法检测miR－133b过表达对转胶蛋白2(TAGLN2)及上皮－间充质转化(EMT)相关蛋白上皮型钙黏附素(E－cadherin)、神经型钙黏附素(N－cadherin)、Snail、Slug和波形蛋白(Vimentin)表达的影响。 结果： 63例食管鳞癌组织miR－133b的相对表达水平为0.295±0.040，显著低于正常癌旁组织(1.002±0.011，P<0.001)。miR－133b mRNA的表达与食管鳞癌分期、淋巴结转移和预后均有关。食管鳞癌细胞Eca109、EC9706、EC1、TE1和KYSE70中miR－133b的相对表达水平分别为0.679±0.031、0.391±0.008、0.236±0.016、0.031±0.005和0.099±0.020，均显著低于正常食管上皮细胞Het－1A(1.005±0.016，均P<0.001)。miR－133b类似物转染能上调TE1细胞中的miR－133b水平达6.199±0.627，并抑制TE1细胞的增殖和侵袭能力，而miR－133b抑制剂转染能下调TE1细胞中的miR－133b水平至0.182±0.023，并增强TE1细胞的增殖和侵袭能力。最为显著的是，TAGLN－3′UTR－WT转染的细胞中，miR－133b类似物转染组的相对荧光素酶的活性(0.320±0.018)显著低于阴性对照组(1.010±0.036，P<0.001)；而TAGLN－3′UTR－MUT转染的细胞中，miR－133b类似物转染组和阴性对照组的相对荧光素酶活性分别为1.019±0.056和1.008±0.021，差异无统计学意义(P>0.05)。miR－133b上调能显著抑制食管鳞癌TE1细胞中TAGLN2蛋白的表达，并且上调E－cadherin的表达，下调N－cadherin、Snail、Slug和Vimentin的表达。 结论： miR－133b可能通过调控TAGLN2的表达在食管鳞癌细胞的增殖和侵袭中发挥作用，可能成为食管鳞癌潜在的分子治疗靶点。.

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