摘要
目的探讨易洛魁族同源盒基因5(IRX5)对肝癌细胞侵袭与迁移的影响,并验证其与miR-136-5p的靶向关系。方法取对数生长期肝癌SMMC7721细胞,过表达IRX5实验设空载质粒(pcDNA3.1)组和过表达IRX5(pcDNA3.1-IRX5)组,敲低IRX5实验设阴性对照(sh-NC)组和敲低IRX5(sh-IRX5)组。采用Western blot验证IRX5过表达和敲低效率;采用划痕实验和Transwell实验检测IRX5对肝癌细胞迁移与侵袭的影响;采用miRanda、Targetscan生物信息学软件预测IRX5结合的miRNA和位点;构建野生型和突变型IRX53′UTR双荧光素酶报告基因质粒,采用酶切、基因测序方法鉴定重组质粒是否构建成功。取对数生长期293T细胞,设野生型质粒+阴性对照(IRX5-3′UTR-Wt+NC)组和野生型质粒+miR-136-5p(IRX5-3′UTR-Wt+miR-136-5p)组,突变型质粒+阴性对照(IRX5-3′UTR-Mut+NC)组和突变型质粒+miR-136-5p(IRX5-3′UTR-Mut+miR-136-5p)组,双荧光素酶报告系统检测各组荧光素酶活性。结果过表达IRX5组IRX5蛋白表达水平、细胞迁移及侵袭数量均高于空载质粒组,敲低IRX5组IRX5蛋白表达水平、细胞迁移及侵袭数量均低于阴性对照组(均P<0.05)。成功构建IRX5基因3′UTR野生型和突变型双荧光素酶报告质粒;双荧光素酶实验结果显示,IRX5-3′UTR-Wt+miR-136-5p组双荧光素酶活性低于IRX5-3′UTR-Wt+NC组(P<0.01),IRX5-3′UTR-Mut+miR-136-5p组与IRX5-3′UTR-Mut+NC组差异无统计学意义。结论IRX5可促进肝癌细胞的侵袭与迁移,IRX5是miR-136-5p的一个靶基因,miR-136-5p可能通过IRX5的3′UTR抑制肝癌细胞的侵袭与迁移。
ObjectiveTo investigate the effect of IRX5 on the invasion and migration of hepatocellular carcinoma(HCC)cells and verify the targeted relationship between mi R-136-5 p and IRX5.MethodsThe SMMC7721 cells weredivided into empty plasmid(pc DNA3.1)group and overexpress IRX5(pc DNA3.1-IRX5)group in the overexpression IRX5 experiment.For knockdown IRX5 experiment a negative control(sh-NC)group and knockdown IRX5(sh-IRX5)group wereset up.The overexpression and knockdown efficiency rates of IRX5 were detected by Western blot assay.The effects of IRX5 on the invasion and migration of HCC cells were detected by wound healing assay and Transwell assay.miRNA and binding sites of IRX5 were predicted by mi Randa and Targetscan.The 3′untranslated regions(UTR)of IRX5 were amplified by PCRand connected to p GL3-Control vector.To verified recombinant plasmid by enzyme digestion and gene sequencing methods.The 293 T cells were divided into four groups:IRX5-3′UTR-Wt+NC group,IRX5-3′UTR-Wt+mi R-136-5 p group,IRX5-3′UTR-Mut+NC group and IRX5-3′UTR-Mut+mi R-136-5 p group in dual luciferase assay.The luciferase activity wasdetected by dual luciferase reporter system.ResultsThe expression level of IRX5 was significantly higher in theoverexpression IRX5 group than that of the empty plasmid group(P<0.05).Compared with the negative control group,theexpression level of IRX5 was significantly reduced in the knockdown IRX5 group(P<0.01).IRX5 promoted the invasionand migration of HCC cells(P<0.05).The IRX5-3′UTR-Wt and IRX5-3′UTR-Mut were successfully constructed.Theresults of dual luciferase assay showed that mi R-136-5 p can reduce IRX5-3′UTR-Wt luciferase activity(P<0.01),whichshowed no effect on IRX5-3′UTR-Mut luciferase activity(P>0.05).ConclusionIRX5 promotes the invasion andmigration of HCC cells.IRX5 acts as a target gene of miR-136-5 p.miR-136-5 p may inhibit the invasion and migration ofHCC cells through the 3′UTR of IRX5.
作者
代龙光
朱丽英
沈婕
钱雯
张競之
朱金凤
许永劼
刘歆蕾
许雯
朱科静
张令
潘卫
李兴
DAI Long-guang;ZHU Li-ying;SHEN Jie;QIAN Wen;ZHANG Jing-zhi;ZHU Jin-feng;XU Yong-jie;LIU Xin-lei;XU Wen;ZHU Ke-jing;ZHANG Ling;PAN Wei;LI Xing(Department of Medical Laboratory,Guizhou Medical University,Guiyang 550004,China;Department of Hematology and Body Fluids,Clinical Laboratory Center,the Affiliated Hospital of Guizhou Medical University;The Affiliated Hospital of Guizhou Medical University,Guizhou Prenatal Diagnosis Center;Guizhou University of Traditional Chinese Medicine)
出处
《天津医药》
CAS
北大核心
2020年第5期358-363,共6页
Tianjin Medical Journal
基金
贵州省科技厅项目(黔科合基础[2016]1123)
贵州省科技厅学术新苗项目(黔科合平台人才[2017]5718)
贵州省教育厅青年科技人才成长项目(黔教合KY[2018]176)
贵州省高等学校大学生创新创业训练计划项目(20195200893)
贵州医科大学附属医院博士启动基金(I-2019-04)。
