lncRNA-MALAT1通过靶向下调miR-570-3p促进胃癌细胞增殖  

lncRNA-MALAT1 targets and downregulates miR-570-3p expression to promote proliferation of gastric cancer cells

作　　者：侯婧瑛[1] 凌辉 金小岩[3] 罗信[2] 李楚强[2] 王凌云[2] HOU Jing-ying;LING Hui;JIN Xiao-yan;LUO Xin;LI Chu-qiang;WANG Ling-yun(Department of Emergency, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120 , China;Department of Gastroenterology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120 , China;The General Department, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120 , China)

机构地区：[1]中山大学孙逸仙纪念医院急诊科,广东广州510120 [2]中山大学孙逸仙纪念医院消化内科,广东广州510120 [3]中山大学孙逸仙纪念医院综合科,广东广州510120

出　　处：《中国病理生理杂志》2018年第12期2145-2152,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81700242);广东省科技计划项目(No.2017A020215176);广东省医学科研基金资助项目(No.A2016264,No.A2017001);广州市科技计划项目(No.201704020121).

摘　　要：目的:探讨长链非编码RNA MALAT1(lncRNA-MALAT1)是否可通过靶向下调微小RNA-570-3p(miR-570-3p)促进胃癌细胞增殖。方法:将体外培养的人胃癌细胞株SGC7901分为3组:空白对照组、si-MALAT1组和si-MALAT1 NC组,其中空白对照组为单纯的SGC7901细胞株,si-MALAT1组和si-MALAT1 NC组分别转染lncRNA-MALAT1 siRNA及其阴性对照。MTS法检测各组细胞的增殖情况。RT-qPCR检测单纯的SGC7901细胞株培养不同时点的miR-570-3p及不同组别lncRNA-MALAT1和miR-570-3p的表达情况。通过生物信息学软件RegRNA预测获取lncRNA-MALAT1与miR-570-3p潜在的互补结合位点。将MALAT1及其突变体克隆到萤光素酶载体psiCHECK-2中,构建MALAT1野生型和突变型质粒,并采用酶切和测序方法鉴定psi CHECK-2-MALAT1载体是否构建成功。将MALAT1野生型和突变型质粒分别与miR-570-3p模拟物、miR-570-3p抑制剂、miR-570-3p模拟物阴性对照、miR-570-3p抑制剂阴性对照在293T细胞中共转染,收集细胞后通过双萤光素酶报告系统检测不同组别的萤光素酶活性,从而对lncRNA-MALAT1与miR-570-3p的靶向调节关系进行验证。结果:与空白对照组和si-MALAT1NC组相比较,si-MALAT1组在不同时点(24、48和72 h)的A490值显著降低(P <0. 01)。在单纯的SGC7901细胞株中,随着时间的推移,miR-570-3p的表达量逐渐下降(P <0. 05)。si-MALAT1组lncRNA-MALAT1的表达水平显著降低,而miR-570-3p表达显著升高(P <0. 01)。双萤光素酶报告基因检测显示,与miR-570-3p模拟物阴性对照组相比,miR-570-3p模拟物组MALAT1野生型报告基因的萤光素酶活性显著降低(P <0. 01),而miR-570-3p抑制剂组MALAT1野生型报告基因的萤光素酶活性较miR-570-3p模拟物组明显增高(P <0. 01); miR-570-3p模拟物、miR-570-3p抑制剂、miR-570-3p模拟物阴性对照及miR-570-3p抑制剂阴性对照对MALAT1突变型的表达均无明显影响。结论:lncRNA MALAT1能够通过靶向结合并下调miR-570-3p促进胃癌细胞增殖。AIM:To investigate whether long non-coding RNA MALAT1(lncRNA-MALAT1)targets and down-regulates microRNA-570-3p(miR-570-3p)expression to further promote the proliferation of gastric cancer cells.METHODS:Gastric cancer cell line SGC7901was cultured in vitro and divided into3groups:blank control,si-MALAT1and si-MALAT1NC.The si-MALAT1and si-MALAT1NC groups were transfected with MALAT1siRNA and its negative control,respectively.The cell proliferation was evaluated by MTS assay.The expression of miR-570-3p was detected at different time points in the pure SGC7901gastric cancer cell line,and the expression of lncRNA-MALAT1and miR-570-3p in different groups was detected by RT-qPCR.The potential complementary binding sites of lncRNA-MALAT1and miR-570-3p were predicted by RegRNA.The MALAT1gene and its mutant fragment were cloned into luciferase reporter vector psiCHECK-2.Restriction enzyme analysis and sequencing were used to identify whether the recombinant plasmids carrying MALAT1or MALAT1-Mut were successfully constructed.miR-570-3p mimic,miR-570-3p inhibitor,miR-570-3p mimic negative control and miR-570-3p inhibitor negative control were co-transfected into the293T cells with the luciferase repor-ters containing MALAT1or MALAT1-Mut.Dual-luciferase reporter assay was performed to detect luciferase activity in different groups in order to verify the relationship between lncRNA-MALAT1and miR-570-3p.RESULTS:Compared with blank control group and si-MALAT1NC group,the A490value in si-MALAT1group was significantly decreased(P<0.01).The expression of miR-570-3p presented an obvious declining trend over time.The expression of lncRNA-MALAT1in si-MALAT1group was remarkably decreased,whereas the expression of miR-570-3p was obviously increased.The dual-luciferase reporter assay indicated that the MALAT1reporter luciferase activity decreased significantly in miR-570-3p mimic group compared with mimic negative control(P<0.01),and the luciferase activity of MALAT1reporter was obviously up-regulated in miR-570-3p inhibitor group compa

关 键 词：长链非编码RNAMALAT1 微小RNA-570-3p 胃癌 细胞增殖 

分 类 号：R735.2[医药卫生—肿瘤] R363.2[医药卫生—临床医学]