长链非编码RNA锌指蛋白667反义RNA1通过下调微小RNA-296表达抑制胶质瘤细胞U87MG侵袭、转移

Long non-coding RNA zinc finger protein 667-antisense RNA 1 inhibits the invasion and metastasis of U87MG cells by down-regulating the expression of microRNA-296

目的:观察长链非编码RNA(lncRNA)锌指蛋白667反义RNA1(ZNF667-AS1)下调微小RNA(miRNA,miR)-296对胶质瘤细胞U87MG侵袭、转移的影响。方法:双荧光素酶鉴定ZNF667-AS1与miR-296的靶向关系。实验分为对照组、空载体质粒组、sh-ZNF667-AS1组、sh-ZNF667-AS1+阴性对照组、sh-ZNF667-AS1+miR-296 mimic组。空载体质粒组转染pEGFPN1空载体,sh-ZNF667-AS1组转染pEGFPN1-sh-ZNF667-AS1,质粒转染成功后,在sh-ZNF667-AS1组基础上,sh-ZNF667-AS1+阴性对照组转染miR-296 mimic NC,sh-ZNF667-AS1+miR-296 mimic组转染miR-296 mimic。实时定量反转录聚合酶链反应(RT-qPCR)检测细胞中ZNF667-AS1、miR-296水平;细胞计数试剂盒(CCK-8)检测细胞增殖;Transwell检测细胞侵袭和迁移;蛋白质免疫印迹实验检测细胞中迁移侵袭抑制蛋白(MIIP)、高迁移率族蛋白1(HMGB1)蛋白水平。多组间比较行单因素方差分析,进一步两两比较行SNK-q法。结果:生物信息学软件(Starbase)分析显示ZNF667-AS1序列上存在miR-296潜在结合位点,且经荧光素酶实验验证,miR-296与ZNF667-AS1呈正相关(P<0.05)。0 h各组细胞吸光度(A450)值差异无统计学意义(F=1.071,P>0.05),12、24、36、48 h各组细胞A450值比较,差异有统计学意义(F=17.238、93.997、48.271、60.646,P<0.05)。细胞处理48 h,对照组、空载体质粒组、sh-ZNF667-AS1组、sh-ZNF667-AS1+阴性对照组、sh-ZNF667-AS1+miR-296 mimic组中ZNF667-AS1水平分别为1.01±0.12、0.98±0.14、0.44±0.09、0.46±0.07、0.76±0.12;miR-296水平分别为1.01±0.14、1.02±0.13、0.39±0.06、0.42±0.05、0.72±0.09;侵袭细胞数量分别为(83.25±6.05)、(84.37±8.76)、(39.49±4.15)、(42.15±7.42)、(63.18±7.13)个;迁移细胞数量分别为(126.60±8.79)、(124.49±9.12)、(76.16±6.48)、(79.44±12.15)、(118.18±6.86)个,且差异有统计学意义(F=36.498、55.130、58.771、47.314,P<0.05)。结论:降低ZNF667-AS1表达可下调miR-296表达抑制胶质瘤细胞U87MG侵袭、转移,而过表达miR-296可逆转上述过程。

Objective To observe the effect of long non-coding RNA(lncRNA)zinc finger protein 667-antisense RNA 1(ZNF667-AS1)down-regulating microRNA(miRNA,miR)-296 on the invasion and metastasis of glioma cell U87MG.Methods The target relationship between ZNF667-AS1 and miR-296 was identified by double luciferase.The experiment was divided into three groups:control group,empty vector plasmid group,sh-ZNF667-AS1 group,sh-ZNF667-AS1+negative control group,sh-ZNF667-AS1+miR-296 mimic group.The empty vector of pegfpn1 was transfected in the empty-vector plasmid group,the sh-ZNF667-AS1 group was transfected with pEGFPN1-sh-ZNF667-AS1,after the plasmid was successfully transfected,on the basis of sh-ZNF667-AS1 group,miR-296 mimic NC was transfected into sh-ZNF667-AS1+negative control group,and miR-296 mimic was transfected into sh-ZNF667-AS1+miR-296 mimic group.The levels of ZNF667-AS1 and miR-296 were detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR);cell counting kit-8(CCK-8)was used to detect cell proliferation;Transwell was used to detect cell invasion and migration;in addition,the levels of migration and invasion inhibitor protein(MIIP)and high mobility group protein 1(HMGB1)were detected by Western blotting.Results The analysis of bioinformatics software(Starbase)showed that there was a potential binding site of miR-296 in ZNF667-AS1 sequence,and the positive correlation between miR-296 and ZNF667-AS1 was confirmed by luciferase experiment(P<0.05).There was no significant difference in A450 between the three groups at 0 h(F=1.071,P>0.05),but there was a significant difference in A450 between the groups at 12,24,36,48 h(F=17.238,93.997,48.271,60.646,P<0.05).Cells were treated for 48h,the levels of ZNF667-AS1 were 1.01±0.12,0.98±0.14,0.44±0.09,0.46±0.07,0.76±0.12 in control group,empty vector plasmid group,sh-ZNF667-AS1 group,sh-ZNF667-AS1+negative control group and sh-ZNF667-AS1+miR-296 mimic group,respectively;the levels of miR-296 were 1.01±0.14,1.02±0.13,0.39±0.06,0.42±0.05,0.72±0.09,respectively;the number of invasive cells were(83.25±6.05),(84.37±8.76),(39.49±4.15),(42.15±7.42),(63.18±7.13)cells,respectively;the number of migration cells were(126.60±8.79),(124.49±9.12),(76.16±6.48),(79.44±12.15),(118.18±6.86)cells,respectively,and the difference was statistically significant(F=36.498,55.130,58.771,47.314,P<0.05).Conclusion Decrease of ZNF667-AS1 can down-regulate the expression of miR-296 and inhibit the invasion and metastasis of U87MG,and over-expression of miR-296 can reverse the above process.