长链非编码RNA LINC01224对肾癌细胞增殖和转移影响机制探讨

Long-chain non-coding RNA LINC01224 regulates proliferation and metastasis of renal cancer cells by regulating miR-542-3p

目的长链非编码RNA(long-chain non-coding RNA,lncRNA)被发现在肾癌发生和发展调控方面具有重要作用。本研究观察lncRNA LINC01224在肾癌组织和细胞株中表达,探讨LINC01224对肾癌细胞增殖和转移影响及作用机制。方法实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,qRT-PCR)检测肾癌组织14例和肾癌细胞株(Caki-1、ACHN、786-O、OS-RC-2和A498)中LINC01224表达。将载有LINC01224编码序列的慢病毒载体(Lenti-LINC01224)感染LINC01224表达量最低的肾癌细胞株作为观察组,以载有绿色荧光基因的慢病毒(Lenti-GFP)为对照组。细胞增殖实验和Transwell迁移实验检测感染后细胞增殖和迁移能力。生物信息学技术预测LINC01224的下游基因。qRT-PCR检测感染后细胞中LINC01224和下游基因mRNA表达。蛋白质印迹法检测下游蛋白表达。结果癌旁组织和肾癌组织LINC01224表达量分别为4.27±0.42和1.94±0.23,差异有统计学意义,t=4.826,P<0.001。人正常肾小管上皮细胞HK-2和人肾癌细胞株Caki-1、ACHN、786-O、OS-RC-2和A498中LINC01224表达量分别为6.95±0.73、2.74±0.38、4.73±0.48、4.76±0.28、1.04±0.16和3.09±0.35,差异有统计学意义,F=27.530,P<0.001。观察组细胞增殖能力从第3天开始降低,t=6.104,P=0.004。对照组和观察组OS-RC-2细胞迁移数分别为(191.50±31.15)和(71.12±16.52)个,差异有统计学意义,t=3.415,P=0.014。LINC01224可结合miR-542-3p,miR-542-3p可结合MFN2mRNA。与对照组比较,观察组OS-RC-2细胞测定指标表达量差异有统计学意义的包括:LINC01224表达量分别为1.03±0.15和7.93±0.80,t=8.511,P<0.001;miR-542-3p表达量分别为1.03±0.13和0.32±0.04,t=5.005,P=0.002;MFN2mRNA表达量分别为1.03±0.15和4.32±0.37,t=8.155,P<0.001。结论 LINC01224在肾癌中表达减少,可抑制肾癌细胞的增殖和迁移能力,其机制可能是抑制miR-542-3p的表达,减少miR-542-3p表达对MFN2基因抑制作用。

OBJECTIVE Long-chain non-coding RNA(lncRNA)has been found to play an important role in the regulation and development of renal cell carcinoma.This study investigated the expression of lncRNA LINC01224 in renal cell carcinoma and cell lines,and explored the effect of LINC01224 on proliferation and metastasis of renal cell carcinoma and its mechanism.METHODS Real-time quantitative PCR(qRT-PCR)was used to detect the expression of LINC01224 in renal cancer tissues and renal cancer cell lines.The lentiviral vector carrying the LINC01224 coding sequence(LentiLINC01224)was infected into the kidney cancer cell line with the lowest expression of LINC01224 and used as an experimental group,and the lentivirus carrying the green fluorescent gene(Lenti-GFP)was used as a control group.The cell proliferation assay and Transwell migration assay were used to detect the proliferation and migration ability of cells after infection.Bioinformatics technology predicts downstream genes for LINC01224.qRT-PCR was used to detect the expression of LINC01224 and downstream gene mRNA in infected cells.Western blotting was used to detect the expression of downstream gene protein.RESULTS The expression levels of LINC01224 in adjacent tissues and renal cancer tissues were 4.27±0.42 and 1.94±0.23,respectively.Compared with adjacent tissues,the expression of LINC01224 in renal cell carcinoma was decreased(t=4.826,P<0.001).The expression levels of LINC01224 in human kidney cancer cell lines Caki-1,ACHN,786-O,OS-RC-2 and A498 were 2.74±0.38,4.73±0.48,4.76±0.28,1.04±0.16 and 3.09±0.35,respectively.The expression level of LINC01224 in human normal tubular epithelial cells HK-2 was 6.95±0.73.Compared with HK-2 cells,the expression of LINC01224 was decreased in renal cancer cell lines(F=27.530,P<0.001).The proliferative capacity of the experimental group cells decreased from day 3(t=6.104,P=0.004).The migration of OS-RC-2 cells in the control group and the experimental group were(191.50±31.15)and(71.12±16.52),respectively.Compared with the control group,the migration ability of the OS-RC-2 cells in the experimental group was reduced(t=3.415,P=0.014).LINC01224 binds to miR-542-3 p and miR-542-3 p binds to mitochondrial fusion protein 2(MFN2)mRNA.The expression levels of LINC01224 in OS-RC-2 cells of the control group and the experimental group were 1.03±0.15 and7.93±0.80 respectively(t=8.511,P<0.001).In the control group and the experimental group,miR-542-combined in OS-RC-2 cells.The expression levels of 3 p were 1.03±0.13 and 0.32±0.04,respectively(t=5.005,P=0.002).The expression levels of MFN2 mRNA in OS-RC-2 cells of control group and experimental group were 1.03±0.15 and 4.32±0.37,respectively(t=8.155,P<0.001).The expression of MFN2 protein was increased in the experimental group.CONCLUSIONS The expression of LINC01224 in renal cell carcinoma is significantly reduced,which can significantly inhibit the proliferation and migration of renal cancer cells.The mechanism may be to inhibit the expression of miR-542-3 p and reduce the inhibitory effect of miR-542-3 p expression on MFN2 gene.