miR-491-5p靶向调控SHOC2对食管鳞癌细胞增殖、迁移及侵袭的影响

目的探讨miR-491-5p对食管鳞癌细胞增殖、迁移及侵袭的影响及作用机制。 方法培养永生化食管上皮细胞株HET-1A和人食管癌细胞株EC109，EC9706，KYSE510，qRT-PCR检测细胞中miR-491-5p和富含亮氨酸重复蛋白SHOC2 （SHOC2） mRNA水平。EC109细胞分为空白对照组、miR- 491-5p组、miR-NC组、miR-491-5p+pcDNA-SHOC2组和miR-491-5p+pcDNA组，MTT检测细胞增殖，Transwell检测细胞迁移和侵袭，Western Blot法检测CyclinD1、Vimentin、E-cadherin以及MAPK/ERK信号通路相关蛋白水平。双荧光素酶报告基因实验验证miR- 491- 5p与SHOC2之间调控关系。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，两两比较采用SNK-q检验。 结果食管鳞癌细胞EC109、EC9706和KYSE510中miR-491-5p表达水平低于HET-1A细胞（0.32±0.06、0.62±0.10、0.61±0.08比1.00±0.08），差异具有统计学意义（F = 106.340，P < 0.001）；SHOC2 mRNA表达水平高于HET- 1A细胞（2.85±0.16、1.73±0.10、1.45±0.06比1.02±0.09），差异具有统计学意义（F = 464.949，P < 0.001）。miR-491-5p组EC109细胞培养72 h后的OD值、细胞迁移数、侵袭数及CyclinD1、Vimentin、p-MEK和p-ERK蛋白水平均低于miR-NC组（0.70±0.06比1.42±0.08，65.01±10.36比150.01±12.48，70.03±10.26比140.02±11.85，0.30±0.03比0.93±0.16，0.41±0.05比0.86±0.08，0.32±0.06比0.95±0.11，0.40±0.06比0.92±0.13），差异具有统计学意义（F = 236.565、159.440、120.706、101.071、98.619、130.766、77.046，P均< 0.001），E-cadherin蛋白水平高于miR-NC组（0.89±0.13比0.48±0.08），差异具有统计学意义（F = 816.432，P < 0.001）。miR-491-5p在EC109细胞中负调控SHOC2表达，SHOC2过表达逆转了miR-491-5p过表达对EC109细胞增殖、迁移和侵袭及MAPK/ERK信号通路的影响。 结论miR-491-5p可抑制食管鳞癌细胞的增殖、迁移和侵袭，其作用机制可能与下调SHOC2表达抑制MAPK/ERK信号通路活性有关。

Effect of miR-491-5p's regulation of SHOC2 on the proliferation,migration and invasion of esophageal squamous cell carcinoma cells

ObjectiveTo investigate the effect of miR-491-5p on proliferation, migration and invasion of esophageal squamous cell carcinoma (ESCC) cells and its mechanism. MethodsThe immortalized esophageal epithelial cell line HET-1A and human esophageal cancer cell lines EC109, EC9706, KYSE510 were cultured, and the levels of miR-491-5p and SHOC2 mRNA were detected by qRT- PCR. EC109 cells were divided into control group, miR-491-5p group, miR-NC group, miR-491-5p+pcDNA-SHOC2 group and miR-491-5p+pcDNA group. MTT was used to detect cell proliferation, Transwell was used to detect cell migration and invasion, and Western Blot was used to detect the expression levels of CyclinD1, Vimentin, E-cadherin and MAPK/ERK signaling pathway-related protein. The dual luciferase reporter gene assay was used to verify the regulatory relationship between miR-491-5p and SHOC2. The comparison between the two groups was performed using independent sample t tests. One-way analysis of variance was used for comparison between multiple groups, and SNK-q test was used for further comparisons. ResultsThe expression levels of miR- 491-5p in esophageal squamous cells EC109, EC9706 and KYSE510 were lower than those in HET-1A cells (0.32±0.06, 0.62±0.10, 0.61±0.08 vs 1.00±0.08, F = 106.340, P < 0.001) ; The mRNA expression level of SHOC2 was higher than that of HET-1A cells (2.85±0.16, 1.73±0.10, 1.45±0.06 vs 1.02±0.09, F = 464.949, P < 0.001) . The OD value, cell migration number, invasion number, and CyclinD1, Vimentin, p-MEK, and p-ERK protein levels in miR-491- 5p group of EC109 cells after 72 h of culture were lower than those in miR-NC group (0.70±0.06 vs 1.42±0.08, 65.01±10.36 vs 150.01±12.48, 70.03±10.26 vs 140.02±11.85, 0.30±0.03 vs 0.93±0.16, 0.41±0.05 vs 0.86±0.08, 0.32±0.06 vs 0.95±0.11, 0.40±0.06 vs 0.92±0.13, F = 236.565, 159.440, 120.706, 101.071, 98.619, 130.766, 77.046, P < 0.001) , E-cadherin protein levels were higher than miR-NC group (0.89±0.13 vs 0.48±0.08, F = 816.432, P < 0.001) . miR-491-5p negatively regulated SHOC2 expression in EC109 cells. SHOC2 overexpression reversed the effect of miR-491-5p overexpression on proliferation, migration and invasion of EC109 cells and MAPK/ERK signaling pathway. ConclusionmiR-491-5p can inhibit the proliferation, migration and invasion of ESCC cells, and its mechanism may be related to the down-regulation of SHOC2 expression and inhibition of MAPK/ERK signaling pathway activity.