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中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (06) : 333 -339. doi: 10.3877/cma.j.issn.2095-1221.2019.06.003

所属专题: 文献

论著

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
徐烨1,()   
  1. 1. 273500 济宁,山东省兖矿集团总医院消化内科二病区
  • 收稿日期:2019-07-29 出版日期:2019-12-01
  • 通信作者: 徐烨

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

Ye Xu1,()   

  1. 1. Department of Gastroenterology, Yankuang Group General Hospital, Jining 273500, China
  • Received:2019-07-29 Published:2019-12-01
  • Corresponding author: Ye Xu
  • About author:
    Corresponding author: Xu Ye, Email:
引用本文:

徐烨. 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[J/OL]. 中华细胞与干细胞杂志(电子版), 2019, 09(06): 333-339.

Ye Xu. Effect of miR-491-5p's regulation of SHOC2 on the proliferation, migration and invasion of esophageal squamous cell carcinoma cells[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(06): 333-339.

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。

Objective

To investigate the effect of miR-491-5p on proliferation, migration and invasion of esophageal squamous cell carcinoma (ESCC) cells and its mechanism.

Methods

The 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.

Results

The 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.

Conclusion

miR-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.

表1 miR-491-5p与SHOC2在食管鳞癌细胞中的表达(±sn = 3)
表2 miR-491-5p过表达对食管鳞癌EC109细胞增殖、迁移及侵袭的影响(±sn = 3)
图1 miR-491-5p过表达对EC109细胞迁移和侵袭的影响(结晶紫染色,×400)
图2 Western blot法检测各组细胞增殖、迁移及侵袭相关蛋白表达
表3 双荧光素酶报告实验(±sn = 3)
表4 miR-491-5p靶向调控SHOC2表达(±sn = 3)
图3 SHOC2的3'UTR中含有与miR-491-5p互补的核苷酸序列
图4 miR-491-5p负向调控SHOC2蛋白表达
表5 SHOC2过表达逆转miR-491-5p过表达对食管鳞癌EC109细胞增殖、迁移及侵袭的抑制作用(±sn = 3)
图5 Western Blot法检测各组细胞增殖、迁移及侵袭相关蛋白表达
图6 食管鳞癌EC109细胞迁移及侵袭观察(结晶紫染色,×400)
表6 SHOC2过表达逆转miR-491-5p过表达对食管鳞癌EC109细胞MAPK/ERK信号通路的抑制作用(±sn = 3)
图7 Western Blot法检测各组细胞MAPK/ERK信号通路相关蛋白表达
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