长链非编码RNA RP11-1212A22.4通过靶向miRNA-483-5p对食管癌细胞株细胞活力和侵袭能力的影响

Effects of long non-coding RNA RP11-1212A22.4 on the cell viability and invasive ability of ovarian cancer cell lines through targeting miRNA-483-5p

目的探讨长链非编码RNA RP11-1212A22.4通过靶向miRNA-483-5p(miR-483-5p)对食管癌细胞株细胞活力和侵袭能力的影响。方法采用GEPIA在线数据库分析RP11-1212A22.4在食管癌组织中的表达情况。采用实时荧光定量聚合酶链反应(qRT-PCR)检测RP11-1212A22.4在人食管癌细胞株EC9706、KYSE30、TE-13、Eca109和正常食管黏膜上皮细胞株HET-1A中的表达情况。将RP11-1212A22.4相对表达量最低的细胞株EC9706分为RP11-1212A22.4组(转染pcDNA-RP11-1212A22.4质粒)和对照组(转染pcDNA-NC质粒)。采用四甲基偶氮唑盐(MTT)法分析EC9706细胞活力,采用Transwell法检测EC9706细胞侵袭能力。StarBase数据库预测和双荧光素酶报告基因实验验证RP11-1212A22.4和miR-483-5p的靶向关系。qRT-PCR检测两组EC9706细胞中miR-483-5p的相对表达量。蛋白质印迹法检测两组EC9706细胞中细胞周期蛋白依赖性激酶6(CDK6)、基质金属蛋白酶2(MMP-2)、细胞周期蛋白依赖性激酶4(CDK4)、基质金属蛋白酶9(MMP-9)的表达情况。结果GEPIA在线数据库中,食管癌组织中RP11-1212A22.4相对表达量较癌旁组织降低,差异有统计学意义(P<0.001)。RP11-1212A22.4在食管癌细胞株EC9706、KYSE30、TE-13、Eca109和正常食管黏膜上皮细胞株HET-1A中的相对表达量分别为0.11±0.08、0.32±0.09、0.72±0.09、0.59±0.13和0.97±0.12,差异有统计学意义(F=40.42,P<0.001)。RP11-1212A22.4组和对照组EC9706细胞中RP11-1212A22.4相对表达量分别为11.9±2.4和1.0±0.3,差异有统计学意义(t=8.89,P<0.001)。接种第2天起,RP11-1212A22.4组EC9706细胞活力较对照组均降低(均P<0.05)。RP11-1212A22.4组侵袭细胞数为(48±12)个,低于对照组的(106±22)个(t=4.63,P<0.001)。StarBase数据库预测和双荧光素酶报告基因实验证实RP11-1212A22.4靶向结合miR-483-5p。RP11-1212A22.4组miR-483-5p相对表达量为0.24±0.11,低于对照组的1.02±0.23(t=5.98,P=0.001)。RP11-1212A22.4组CDK6、MMP-2、CDK4、MMP-9蛋白表达均较对照组降低。结论RP11-1212A22.4在食管癌组织和细胞株中均呈低表达,其通过靶向结合miR-483-5p抑制食管癌细胞的活力和侵袭能力。

Objective To investigate the effects of long non-coding RNA(lncRNA)RP11-1212A22.4 on the cell viability and invasive ability of esophageal cancer cell lines by targeting miRNA-483-5p(miR-483-5p).Methods The expression of RP11-1212A22.4 in esophageal cancer tissues was analyzed by using GEPIA online database.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of RP11-1212A22.4 in human esophageal cancer cell lines EC9706,KYSE30,TE-13,Eca109 and normal esophageal epithelial cell line HET-1A.The lowest expression level of EC9706 cell line in RP11-1212A22.4 was divided into RP11-1212A22.4 group(transfected with pcDNA-RP11-1212A22.4 plasmid)and the control group(transfected with pcDNA-NC plasmid).The cell viability of EC9706 cell was analyzed by using methyl thiazolyl tetrazolium(MTT)method,and the invasion ability of EC9706 cell was detected by using Transwell assay.The targeting relationship between RP11-1212A22.4 and miR-483-5p was verified by using StarBase database prediction and dual luciferase reporter assay.The relative expression level of miR-483-5p of EC9706 cell in two groups was detected by using qRT-PCR.Western blot was used to detect the expressions of cyclin-dependent kinase 6(CDK6),matrix metalloproteinase 2(MMP-2),cyclin-dependent kinase 4(CDK4),and matrix metalloproteinase 9(MMP-9)proteins in two groups.Results In GEPIA online database,compared with adjacent tissues,the relative expression level of RP11-1212A22.4 in esophageal cancer tissues was decreased,and the difference was statistically significant(P<0.001).The relative expression levels of RP11-1212A22.4 in esophageal cancer cell lines EC9706,KYSE30,TE-13,Eca109 and normal esophageal mucosal epithelial cell line HET-1A were 0.11±0.08,0.32±0.09,0.72±0.09,0.59±0.13 and 0.97±0.12,and the difference was statistically significant(F=40.42,P<0.001).The relative expression levels of RP11-1212A22.4 in EC9706 cells of RP11-1212A22.4 group and the control group were 11.9±2.4 and 1.0±0.3,respectively,and the difference was statistically significant(t=8.89,P<0.001).Compared with the control group,the cell viability of EC9706 cell in RP11-1212A22.4 group was decreased(all P<0.05).The number of invasive cells in RP11-1212A22.4 group was lower than that in the control group(48±12 vs.106±22,t=4.63,P<0.001).StarBase database prediction and dual luciferase reporter assay both showed that RP11-1212A22.4 targeted miR-483-5p.The relative expression level of miR-483-5p in RP11-1212A22.4 group was lower than that in the control group(0.24±0.11 vs.1.02±0.23,t=5.98,P=0.001).Compared with the control group,the expressions of CDK6,MMP-2,CDK4 and MMP-9 proteins in the RP11-1212A22.4 group were decreased.Conclusions RP11-1212A22.4 is lowly expressed in esophageal cancer tissues and cell lines,and it inhibits the cell viability and invasive ability of esophageal cancer cells by targeting miR-483-5p.