miR-106a靶向调控TIMP2对人胃癌细胞增殖、转移及EMT的影响

Effects of miR-106a targeting TIMP2 on proliferation,metastasis and EMT of human gastric cancer

目的探讨微小RNA-106a(miR-106a)在人胃癌组织中的表达及其与癌细胞增殖、转移的关系。方法收集人胃癌和配对癌旁福尔马林固定-石蜡包埋样本共50对,Real-time PCR法检测miR-106a在人胃癌组织中的表达;培养人低分化胃癌细胞系SGC-7901、BGC-823、MKN-45和永生化人胃黏膜上皮细胞GES-1,Real-time PCR法检测miR-106a在人胃癌细胞中的表达;MTT法检测细胞增殖,Transwell法检测细胞迁移和侵袭,生物信息学和双荧光素酶法鉴定miR-106a靶基因,Western blot检测靶蛋白TIMP2、MMP2、MMP9、E-cadherin、N-cadherin表达。结果Real-time PCR检测显示,与癌旁组织比较,miR-106a在人胃癌组织中高表达,差异有统计学意义(P<0.001);与GES-1细胞比较,miR-106a在人胃癌细胞SGC-7901、BGC-823、MKN-45中普遍高表达,差异有统计学意义(P<0.001)。MTT检测显示抑制miR-106a后胃癌细胞SGC-7901、BGC-823增殖能力下降(P<0.01)。Transwell显示胃癌细胞BGC-823迁移、侵袭能力下降(P<0.001)。双荧光素酶法显示TIMP2野生型报告基因与miR-106a mimic共转后,其荧光素酶活性较miR-106a NC组明显下降(P<0.001),但TIMP2突变型报告基因无明显变化。Western blot显示抑制miR-106a后TIMP2表达升高,MMP2、MMP9表达下降,E-cadherin表达升高,N-cadherin表达下降。结论miR-106a在人胃癌中的高表达可能通过靶向TIMP2而影响癌细胞的增殖、转移和上皮-间质转化。

Objective The aim of this study was to investigate the expression of small RNA-106a(miR-106a)in human gastric cancer tissues and its relationship with cancer cell proliferation and metastasis.Methods A total of 50 pairs of human gastric cancer and paired paracancerous formalin-fixed-embedded samples were collected,and real-time PCR was used to detect the expression of miR-106a in gastric cancer tissues,human low differentiated gastric cancer or poorly differentiated gastric cancer SGC-7901,BGC-823,MKN-45 cell lines and immortalized human gastric mucosal epithelial GES-1 cell line.MTT method was used to detect the cell proliferation,and transwell method was used to detect cell migration and invasion.The bioinformatics analysis and dual luciferase assay were used to identify target genes of miR-106a,and Western blot was used to detect the expression of target proteins TIMP2,MMP2,MMP9,E-cadherin and N-cadherin.Results Real-time PCR result showed that miR-106a was highly expressed in human gastric cancer tissues,and the difference was statistically significant when compared with adjacent tissues(P<0.001).MiR-106a was also highly expressed in human gastric cancer SGC-7901,BGC-823 and MKN-45 cells,and the difference was statistically significant when compared with GES-1 cells(P<0.001).MTT assay showed that the proliferation ability of SGC-7901 cells and BGC-823 cells were decreased after inhibiting miR-106a(P<0.01).Transwell assay showed that the migration and invasion abilities of BGC-823 cells were also decreased(P<0.001).The dual-luciferase assay showed that after co-transfection of TIMP2 wild type reporter gene and miR-106a mimic,its luciferase activity was significantly lower than that of the miR-106a NC group(P<0.001),but there was no significant change in the TIMP2 mutant reporter gene.Western blot showed that after inhibiting miR-106a,the expression of TIMP2 was increased,the expressions of MMP2 and MMP9 were decreased,the expression of E-cadherin was increased,and the expression of N-cadherin was decreased.Conclusion The high expression of miR-106a in human gastric cancer may affect the proliferation,metastasis and EMT of gastric cancer cells by targeting TIMP2.