沉默miR-20a-5p的肝癌细胞恶性生物学行为及Zbed3、Axin蛋白表达情况观察

Effects of silencing miR-20a-5p on malignant biological behavior and expression of Zbed3 and Axin in hepatocellular carcinoma cells

目的观察沉默miR-20a-5p通过调控三结构域蛋白家族样1(TRIML1)对肝癌细胞恶性生物学行为及锌指蛋白3(Zbed3)、Axin表达的影响。方法在线数据库TargetScan及双荧光素酶报告基因实验证实TRIML1是miR-20a-5p的靶基因。选择肝癌细胞中miR-20a-5p、TRIML1表达明显高于人正常肝细胞7701的huh-7细胞进行实验,随机分为7组,其中模型组细胞正常培养,miR-20a-5p-NC组转染miR-20a-5p阴性对照,miR-20a-5p siRNA组转染miR-20a-5p siRNA,TRIML1-NC组转染TRIML1阴性对照,TRIML1 siRNA组转染TRIML1 siRNA,TRIML1-NC+miR-20a-5p-NC组转染TRIML1阴性对照+miR-20a-5p阴性对照,TRIML1 siRNA+miR-20a-5p siRNA组转染TRIML1 siRNA+miR-20a-5p siRNA,转染48 h。采用实时荧光定量PCR法检测各组细胞TRIML1、miR-20a-5p表达,流式细胞术、Transwell小室法检测细胞凋亡率及迁移、侵袭细胞数,Western blotting法检测Zbed3、Axin蛋白表达。结果模型组、miR-20a-5p-NC组、TRIML1-NC组、TRIML1-NC+miR-20a-5p-NC组细胞TRIML1、miR-20a-5p相对表达量、细胞凋亡率、迁移细胞数、侵袭细胞数及Zbed3、Axin蛋白相对表达量比较P均>0.05。与模型组、miR-20a-5p-NC组、TRIML1-NC组、TRIML1-NC+miR-20a-5p-NC组比较,miR-20a-5p siRNA组、TRIML1 siRNA组、TRIML1 siRNA+miR-20a-5p siRNA组细胞TRIML1、miR-20a-5p相对表达量及迁移细胞数、侵袭细胞数、Zbed3蛋白相对表达量均降低,细胞凋亡率及Axin蛋白相对表达量均升高,且TRIML1 siRNA+miR-20a-5p siRNA组变化更明显(P均<0.05)。结论沉默miR-20a-5p表达可抑制肝癌huh-7细胞侵袭、转移及Zbed3表达,并促进细胞凋亡及Axin蛋白表达,其机制可能与正向调控TRIML1表达有关。

Objective To observe the effects of silencing miR-20a-5p on malignant biological behavior and expression of zinc finger protein 3(Zbed3)and Axin in hepatocellular carcinoma cells by regulating Tripartite motif family-like 1(TRIML1).Methods Online database TargetScan and double luciferase reporter assay confirmed that TRIML1 was the target gene of miR-20a-5p.Huh-7 cells which had significantly higher expression of miR-20a-5p and TRIML1 than human normal liver cell 7701 were selected for the experiment,and were randomly divided into seven groups.The cells in the model group were cultured normally,and the cells in the miR-20a-5p-NC group were transfected with miR-20a-5p negative control.The cells in the miR-20a-5p siRNA group were transfected with miR-20a-5p siRNA,TRIML1-NC group with TRIML1 negative control,and TRIML1 siRNA group with TRIML1 siRNA,Triml1-NC+miR-20a-5p-NC group with TRIML1 negative control+miR-20a-5p negative control,and TRIML1 siRNA+miR-20a-5p siRNA group with TRIML1 siRNA+miR-20a-5p siRNA,and they were all transfected for 48 h.The expression levels of TRIML1 and miR-20a-5p were detected by real-time fluorescence quantitative PCR,the apoptosis rate and the number of migrating and invading cells were detected by flow cytometry and Transwell cell assay,and the expression levels of Zbed3 and Axin proteins were detected by Western blotting.Results No significant differeces were found in the relative expression levels of TRIML1,miR-20a-5p,apoptosis rate,number of migrating cells,number of invading cells or relative expression levels of Zbed3 and Axin proteins between the model group,miR-20a-5p-NC group,TRIML1-NC group and TRIML1-NC+miR-20a-5p-NC group(all P>0.05).Compared with the model group,miR-20a-5p-NC group,TRIML1-NC group,and TRIML1-NC+miR-20a-5p-NC group,the relative expression levels of TRIML1 and miR-20a-5p and the number of migrating cells,invading cells and Zbed3 protein decreased,the apoptosis rate and Axin protein relative expression increased,and the changes of TRIML1 siRNA+miR-20a-5p siRNA group were more obvious in the miR-20a-5p siRNA group,TRIML1 siRNA group,and TRIML1 siRNA+miR-20a-5p siRNA group(all P<0.05).Conclusion Silencing the expression of miR-20a-5p can inhibit the invasion and metastasis of Huh-7 cells and the expression of Zbed3,and promote apoptosis and Axin protein expression;the mechanism may be related to the positive regulation of TRIML1 expression.