摘要
目的 探讨微小RNA(miRNA,miR)-133b靶向基质金属蛋白酶-9(MMP-9)抑制结直肠癌细胞增殖的分子机制。方法 应用实时荧光定量聚合酶链反应(FQ-PCR)技术检测结直肠癌组织中MMP-9的表达水平;利用RNA干扰及重组质粒过表达技术在结直肠癌细胞株HCT116中调节MMP-9的表达水平,并用噻唑蓝(MTT)比色法及克隆形成实验检测细胞的增殖能力;借助生物信息学预测MMP-9的上游miRNA,并用FQ-PCR及双荧光素酶实验进行验证;通过转染技术在HCT116细胞中过表达miR-133b和MMP-9,并用MTT及克隆形成实验检测细胞的增殖能力。结果 MMP-9在癌组织中表达高于癌旁组织(3.17±0.79比1.00±0.25,P<0.01);MMP-9干扰组细胞增殖活力低于对照组(MTT:1.54±0.06比1.89±0.03,P<0.01;克隆形成:6.33±1.53比13.33±0.58,P<0.01);MMP-9过表达组细胞增殖活力高于对照组(MTT:2.10±0.12比1.82±0.04,P<0.05;克隆形成:23.33±2.08比13.67±1.53,P<0.01)。miR-133b过表达组MMP-9表达低于对照组(0.42±0.06比1.00±0.12,P<0.01);野生型MMP-9 3’端非编码区(3’UTR)质粒miR-133b过表达组荧光素酶活力低于对照组(0.49±0.08比0.97±0.08,P<0.01),突变型MMP-9 3’UTR质粒miR-133b过表达组与对照组比较荧光素酶活力差异无统计学意义(0.95±0.11比1.02±0.15,P>0.05)。miR-133b过表达组细胞增殖活力低于对照组(MTT:1.34±0.04比1.54±0.06,P<0.01;克隆形成:8.67±3.06比17.67±2.52,P<0.05);miR-133b/MMP-9同时过表达组细胞增殖活力高于miR-133b过表达组(MTT:1.66±0.05比1.34±0.04,P<0.01;克隆形成:22.00±4.00比8.67±3.05,P<0.05)。结论 miR-133b可以靶向下调MMP-9的表达并抑制结直肠癌细胞增殖。
Objective To investigate the molecular mechanism by which microRNA (miRNA, miR)-133b inhibits colorectal cancer cell proliferation through targeting matrix metalloproteinase-9 (MMP-9).Methods To examine the expression level of MMP-9 in clinic tissue specimens using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR);to achieve MMP-9 knockdown or overexpression using RNA interference or recombinant overexpressing plasmid, and then monitor the cell proliferation of colorectal cancer cell, HCT116, in response to MMP-9 knockdown or overexpression using methyl thiazol tetrazolium (MTT) and colony formation assays;to predict the upstream candidate miRNA which might regulate MMP-9 expression and to validate the prediction using FQ-PCR and luciferase reporter gene assays;to achieve miR-133b and MMP-9 overexpression in HCT116 cells, and then monitor the cell proliferation of colorectal cancer cells.Results The expression of MMP-9 significantly increased in colorectal tissues, compared with the paired adjacent normal tissues (3.17±0.79 vs. 1.00±0.25, P<0.01). After inhibition of MMP-9, the cell proliferation ability was significantly decrease than control group by MTT (1.54±0.06 vs. 1.89±0.03, P<0.01) and cloning efficiency (6.33±1.53 vs. 13.33±0.58, P<0.01), respectively. And the cell proliferation ability of MMP-9 overexpression group was significantly increased than control group by MTT (2.10±0.12 vs. 1.82±0.04, P<0.05) and cloning efficiency (23.33±2.08 vs. 13.67±1.53, P<0.01). Meanwhile overexpression of miR-133b, the expression level of MMP-9 was down-regulated (0.42±0.06 vs. 1.00±0.12, P<0.01). The luciferase activity of the MMP-9 3’untranslated regions (3’UTR) luciferase reporter vector was notably suppressed in response to miR-133b mimics transfection, compared to control groups (0.49±0.08 vs. 0.97±0.08, P<0.01). Additionally, repression of miR-133b mimics induced luciferase activity was offset by mutation in the MMP-9 3’UTR (0.95±0.11 vs. 1.02±0.15, P>0.05). After overexpre
作者
刘亚彬
孔德贤
李秉慧
Liu Yabin;Kong Dexian;Li Binghui(Department of Surgery, the Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang 050011, China;Department of Endocrinology, the Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang 050011, China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2019年第2期212-214,共3页
Chinese Journal of Experimental Surgery
基金
河北省卫生与计划生育委员会青年科技课题 (20170732).
