miR-208a促进非小细胞肺癌A549细胞增殖、侵袭和迁移的功能和机制研究  

Study on the function and mechanism of miR-208a in promoting the proliferation,invasion and migration of non-small cell lung cancer A549 cells

作　　者：李新玲 吴振国[1] 张立海[1] 朱颀峰[1] LI Xin-ling;WU Zhen-guo;ZHANG Li-hai;ZHU Qi-feng(Department of Respiratory and Critical Care Medicine,the First Hospital of Hebei Medical University,Shijiazhuang 050031,China)

机构地区：[1]河北医科大学第一医院呼吸与危重症医学科,050031

出　　处：《天津医药》2021年第3期242-247,I0002,共7页Tianjin Medical Journal

摘　　要：目的探讨miR-208a对非小细胞肺癌A549细胞增殖、侵袭和迁移的影响及其机制。方法(1)分别用miR-208a mimic、NC mimic、miR-208a inhibitor和NC inhibitor转染A549细胞。采用荧光定量PCR(qPCR)检测A549细胞中miR-208a过表达和干扰效率;采用CCK-8实验、Transwell实验和划痕实验检测miR-208a对A549细胞增殖、侵袭和迁移的影响。利用生物信息学网站PicTar、miRanda、Targetscan和miRDB预测与miR-208a结合的下游靶基因;构建蛋白酪氨酸磷酸酶受体G(PTPRG)的3′UTR野生型和突变型双荧光素报告酶基因载体,和miR-208a共转染到HEK-293T细胞后观察荧光素酶活性变化。(2)取对数生长期的A549细胞,设置阴性对照组(si-NC组)、PTPRG敲低组(si-PTPRG组)和PTPRG敲低组+miR-208a干扰组(si-PTPRG+miR-208a inhibitor组),采用CCK-8、Transwell实验和划痕实验检测PTPRG对A549增殖、侵袭和迁移的影响,qPCR和Western blot检测3组细胞PTPRG的mRNA和蛋白表达变化。结果(1)miR-208a mimic组miR-208a基因表达水平、细胞增殖、细胞侵袭和细胞迁移率均高于NC mimic组(P<0.05);miR-208a inhibitor组的miR-208a基因表达水平、细胞增殖、细胞侵袭和细胞迁移率均低于NC inhibitor组(P<0.05)。生物信息学分析显示PTPRG是miR-208a的靶基因,双荧光素报告酶实验结果显示miR-208a过表达能够降低PTPRG 3′UTR野生型的荧光素酶活性,而敲低miR-208a能够增加PTPRG 3′UTR野生型的荧光素酶活性,miR-208a能够与PTPRG的3′UTR序列结合。(2)与si-NC组相比,si-PTPRG组细胞增殖、细胞侵袭和细胞迁移能力明显增高,PTPRG基因和蛋白表达水平下降(P<0.05);而与si-PTPRG组相比,si-PTPRG+miR-208a inhibitor组细胞增殖、细胞侵袭和细胞迁移能力下降,PTPRG基因和蛋白表达水平升高(P<0.05)。结论miR-208a可能通过靶向调控PTPRG来促进A549细胞的增殖、侵袭和迁移。Objective To investigate the functional role and mechanism of miR-208a on proliferation,invasion and migration of non-small cell lung cancer(NSCLC)A549 cells.Methods(1)A549 cells were transfected with miR-208a mimic,NC mimic,miR-208a inhibitor and NC inhibitor,respectively,and the overexpression and interference efficiency of miR-208a were detected by quantitative polymerase chain reaction(qPCR).CCK-8,Transwell and wound healing assays were used to evaluate the effects of miR-208a on proliferation,invasion and migration of A549 cells.Bioinformatics websites such as miRDB,PicTar,Targetscan and miRanda were used to predict the downstream target genes of miR-208a.PTPRG 3′UTR wild-type and mutant double luciferase reporter gene vectors were constructed and co-transfected with miR-208a into HEK-293T cells to observe the changes of luciferase activity.(2)A549 cells in logarithmic phase were taken,and negative control group(si-NC group),PTPRG knockdown group(si-PTPRG group),PTPRG knockdown group+miR-208a interference group(si-PTPRG+miR-208a inhibitor group)were set up.CCK-8,Transwell test and scratch test were used to detect the effects of PTPRG on proliferation,invasion and migration of A549 cells.qPCR and Western blot assays were performed to detect the expression levels of PTPRG mRNA and protein.Results There were increased levels of miR-208a mRNA expression,cell proliferation,migration and invasion in miR-208a mimic group compared with those of NC mimic group(P<0.05).By contrast,miR-208a inhibition exerted the opposite effects.Bioinformatics analysis showed that PTPRG was the target gene of miR-208a,and the overexpression of miR-208a could reduce the luciferase activity of PTPRG 3′UTR wild-type,and miR-208a inhibitor could increase the luciferase activity of PTPRG 3′UTR wild-type,indicating that miR-208a could bind to the 3′UTR sequence of PTPRG.Further studies showed that compared with si-NC group,the proliferation,invasion and migration of cells were significantly increased in si-PTPRG group,and the expres

关 键 词：癌 非小细胞肺 A549细胞 受体样蛋白质酪氨酸磷酸酶类 微RNAS 细胞增殖 细胞运动 miR-208a 

分 类 号：R734.2[医药卫生—肿瘤] R349.5[医药卫生—临床医学]