摘要
目的探讨miR-223对胃癌细胞生长和侵袭的影响,并研究miR-223的靶基因及其功能。方法利用实时定量PCR检测原发性胃癌和转移性胃癌间miR-223的表达水平。利用脂质体LipofectamineTM2000在胃癌细胞中瞬时转染miR-223的ASO以及对照寡核苷酸,运用平板克隆形成和transwell侵袭实验检测细胞生长和侵袭。生物信息学方法预测miR-223的靶基因,分别利用GFP报告载体实验和Western blot检测转染miR-223 ASO和对照细胞中靶基因3'UTR的荧光强度以及靶基因的蛋白水平。在细胞中转染靶基因的siRNA和对照,利用Western blot检测靶基因的蛋白表达情况,并检测细胞的克隆形成和侵袭能力。结果实时定量PCR结果显示miR-223在转移性胃癌中高表达(P<0.05)。与对照组细胞相比,转染miR-223 ASO的细胞克隆数目减少(P<0.05),发生侵袭的细胞减少(P<0.05)。生物信息学显示EPB41L3 3’UTR含有miR-223的结合位点。与对照组相比,miR-223 ASO降低EPB41L3 3'UTR的荧光强度(P<0.05),而对突变的3'UTR没有明显影响。miR-223 ASO组细胞中EPB41L3的蛋白水平较对照组升高(P<0.05)。转染EPB41L3 siRNA的细胞中EPB41L3的蛋白水平较对照组降低,而克隆形成和侵袭能力较对照组明显升高(P<0.05)。结论 miR-223通过抑制miR-223的表达而调控胃癌细胞的生长和侵袭,暗示miR-223可能与胃癌的生长和转移相关。miR-223具有作为胃癌分子治疗和预后靶标的潜能。
Objective To investigate the function and regulation mechanism of miR-223 in gastric carcinoma cell growth and invasion.Methods Real-time PCR was performed to detect miR-223 expression in primary and metastatic gastric carcinoma tissues .The gastric carcinoma cells were transfected with miR-223 ASO or controls using Lipofectamine TM 2000, and cell growth and invasion abilities were tested by colony formation and transwell invasion assay . Bioinformatics,Western blot and GFP reporter assays were used for the prediciton and valida -tion of the target of miR-223.The cells were transfected with the siRNA of the target gene, and Western blot was used for the detection of target expression.The cell colony formation and in-vasion were determined in siRNA-tranfected cells.Results Compared to primary gastric carci-noma tissues, miR-223 was upregulated in matastatic gastric carcinoma.The cells with miR-223 ASO had a low number of colonies and invading cells（P〈0.05）.EPB41L3 3′UTR had binding sites with miR-223, and miR-223 ASO increased the GFP intensity controlled by EPB41L3 3′UTR.However, miR-223 had no effect on the mutant EPB41L3 3′UTR.In addi-tion, the cells with miR-223 ASO had a high protein level of EPB41L3.EPB41L3 expression was knockdown by the siRNA of EPB41L3.Knocking down of EPB41L3 resulted in the inhib-tion of cell colony formation and invasion abilities （P〈0.05）.Conclusion miR-223 modu-lates gastric carcinoma cell growth and invasion by suppressing EPB41L3.Our data suggest that miR-223 has potential of a miRNA-based therapeutic target for gastric carcinoma.
出处
《哈尔滨医科大学学报》
CAS
北大核心
2014年第5期381-385,共5页
Journal of Harbin Medical University
基金
国家自然科学基金资助项目(31360431)
