长链非编码RNA锌指蛋白反义链1靶向微小RNA-512-3p抑制肾癌细胞侵袭和转移

Long noncoding RNA zinc finger protein antisense chain 1 targeting micro-512-3p inhibits the invasion and metastasis of renal cell carcinoma cells

目的探讨长链非编码RNA锌指蛋白反义链1(lncRNAZFAS)靶向调控微小RNA-512-3p(miR-512-3p)表达及对透明细胞肾细胞癌(ccRCC)侵袭和迁移的影响。方法收集2019年2月至2020年4月我院泌尿外科接受肾癌根治术的ccRCC患者30例,获取肾癌及癌旁组织标本。采用实时定量反转录聚合酶链反应(RT-qPCR)检测30个ccRCC癌肿组织和30个邻近非肿瘤正常组织中lncRNAZFAS和miR-512-3p和可能靶向miRNAs的表达水平。分别通过细胞计数盒(CCK-8)测定、细胞克隆试验、Transwell迁移和侵袭测定测定敲低lncRNAZFAS1对细胞增殖、细胞克隆、迁移和侵袭的影响。lncRNAZFAS1和miR-512-3p的相关性通过生物信息学软件RAIDv2.0和AnnoLnc预测进行分析。lncRNAZFAS1和miR-512-3p之间的相互作用通过荧光素酶报告基因检测验证。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,进一步两两比较采用SNK-q检验。结果RT-qPCR结果显示lncRNAZFAS1在ccRCC癌肿组织表达显著高于邻近非肿瘤组织(1.96±0.17比1.00±0.01,t=2.691,P<0.01),miR-512-3p在ccRCC癌肿组织表达显著低于邻近非肿瘤组织(0.67±0.06比1.00±0.01,t=1.319,P<0.05)。CCK-8结果显示,si-lncRNAZFAS1组细胞活性均低于空白组及si-NC组(0.43±0.09比0.96±0.08比0.98±0.05,F=3.759,P<0.05);细胞克隆试验结果显示,si-lncRNAZFAS1组肾癌细胞的克隆形成数显著低于空白组及si-NC组(52.4±8.2比100.2±5.4比99.6±4.3,F=7.924,P<0.05);Transwell侵袭和迁移结果显示,si-lncRNAZFAS1组穿越细胞数低于空白组及si-NC组(132.2±11.4比179.6±16.8比175.2±19.8,F=3.817,P<0.05),差异有统计学意义。双荧光素酶报告基因实验证实miR-512-3p被确定为ZFAS1的靶标。结论lncRNAZFAS1靶向miR-512-3p抑制ccRCC的生长和转移,lncRNA ZFAS1可能成为ccRCC的新治疗靶点。

Objective To investigate long noncoding RNA zinc finger protein antisense chain 1(lncRNA ZFAS1)targeted regulation of micro RNA-512-3p(miR-512-3p)expression and its effect on the invasion and migration of clear cell renal cell carcinoma(ccRCC).Methods From February 2019 to April 2020,we collected a total of 30 patients with ccRCC who underwent radical nephrectomy to obtain re-nal cancer and adjacent tissue samples.The expression level of ZFAS1 and miR-512-3p in 30 ccRCC and 30 adjacent non-tumor tissues were detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The effect of knockdown of ZFAS1 on cell proliferation,migration and invasion was measured by CCK-8 assay,Cell cloning,Transwell migration and invasion assays,respectively.The correlation of ZFAS1 and miR-512-3p was analyzed by bioinformatics RAID v2.O and AnnoLnc.Interactions between ZFAS1 and miR-512-3p were verified by luciferase reporter assay.Independent sample t-test was used for comparison between the two groups.The one-way ANOVA was used for comparison between multiple groups,and SNK-q test was used for further pairwise comparison.Results RT-qPCR results showed that the expression of IncRNA ZFAS1 in ccRCC cancer tissues was significantly higher than that in adjacent non-tumor tissues(1.96±0.17 vs.1.00±0.01,t=2.691,P<0.01),and that of miR-512-3p in ccRCC tissues was significantly lower than that in adjacent non-tumor tissues(0.67±0.06 vs.1.00±0.01,t=1.319,P<0.05).CCK-8 assay results showed that the cell activity of si-lncRNA ZFAS1 group was lower than that of blank group and si-NC group(0.43±0.09 vs.0.96±0.08 vs.0.98±0.05,F=3.759,P<0.05).The results of cell cloning assay showed that the clone formation number of ccRCC cells in si-lncRNA ZFAS1 group was significantly less than that in blank group and si-NC group(52.4±8.2 vs.100.2±5.4 vs.99.6±4.3,F=7.924,P<0.05).The results of Transwell invasion and migration assays showed that the number of crossing cells in si-lncRNA ZFAS1 group was less than that in blank group and si-NC group,with statistical significance(132.2±11.4 vs.179.6±16.8 vs.175.2±19.8,F=3.817,P<0.05).Dual luciferase reporter assay confirmed that miR-512-3p was identified as the target of ZFAS1.Conclusion Knockdown of IncRNA ZFAS1 inhibits the growth and metastasis of ccRCC by targeting miR-512-3p,which may represent that IncRNA ZFAS1 is one of the new therapeutic targets or biomarkers of ccRCC.