miRNA-676-3p靶向调节PFDN1对肾癌增殖和侵袭的影响

Effects of miRNA-676-3p on the proliferation and invasion of renal cancer by targeting PFDN1

目的探讨miRNA-676-3p相对表达量与肾癌患者生存期的关系,及其靶向调节前折叠蛋白1(PFDN1)对肾癌增殖和侵袭的影响。方法选择OncoRank在线软件分析miRNA-676-3p相对表达量与肾癌患者生存率的关系。采用实时荧光定量聚合酶链反应(RT-qPCR)检测miRNA-676-3p在肾癌细胞系中的相对表达量。复苏肾癌CAKI1细胞,以转染miRNA-NC作为对照组,以转染前体miRNA-676-3p作为过表达组。RT-qPCR检测miRNA-676-3p的相对表达量。通过CCK-8法、Transwell实验分别测定两组的细胞吸光度和侵袭数。参考TargetScan Release 8.0网站和双荧光素酶报告基因实验对miRNA-676-3p靶基因进行预测和验证。RT-qPCR及Western blotting法分别检测两组细胞中PFDN1基因和Wnt/β-catenin分子通路蛋白的表达量。计量资料以均数±标准差(±s)表示,两组间比较采用t检验,多组间比较采用单因素方差分析。结果miRNA-676-3p相对表达量高的肾癌患者生存率明显高于miRNA-676-3p相对表达量低的肾癌患者,差异具有统计学意义(P<0.01)。肾癌细胞系中miRNA-676-3p相对表达量明显低于正常肾小管上皮细胞,差异具有统计学意义(P<0.01);CAKI1细胞中miRNA-676-3p相对表达量最低,差异具有统计学意义(P<0.01)。对照组和过表达组miRNA-676-3p相对表达量分别为1.04±0.59和15.90±1.70,过表达组显著高于对照组,差异具有统计学意义(P<0.01)。培养24、48、60、72 h,过表达组的细胞吸光度均低于对照组,差异具有统计学意义(P<0.05)。对照组和过表达组细胞侵袭数分别为(115.90±24.73)、(43.83±21.94)个,过表达组的细胞侵袭数明显低于对照组,差异具有统计学意义(P<0.01)。PFDN1是miRNA-676-3p的下游靶点基因(P<0.01)。过表达组的PFDN1基因相对表达量明显低于对照组,差异具有统计学意义(P<0.01)。过表达组的Wnt/β-catenin分子通路蛋白表达量低于对照组。结论miRNA-676-3p高表达的肾癌患者生存率较高,miRNA-676-3p通过显著下调PFDN1表达来抑制肾癌CAKI1细胞增殖和侵袭,进而抑制肾癌发展。

Objective To explore the relationship between the relative expression of miRNA-676-3p and the survival of renal cancer patients,and its effect on the proliferation and invasion of renal cancer by targeting and regulating prefoldin 1(PFDN1).Methods OncoRank online software was selected to analyze the relationship between the relative expression of miRNA-676-3p and the survival rate of renal cancer patients.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the relative expression of miRNA-676-3p in renal cancer cell lines.Renal carcinoma CAKI1 cells were resuscitated,and the transfected miRNA-NC was used as the control group,and the transfected precursor miRNA-676-3p was used as the overexpression group.The relative expression of miRNA-676-3p was detected by RT-qPCR.The cell absorbance and invasion number of the two groups were measured by CCK-8 and Transwell invasion assays,respectively.The target gene of miRNA-676-3p was predicted and verified by referring to the TargetScan Release 8.0 website and dual-luciferase reporter gene experiment.The expression of PFDN1 gene and Wnt/β-catenin molecular pathway protein in the two groups of cells were determined by RT-qPCR and Western blotting,respectively.Measurement data were expressed as mean±standard deviation(x±s),t-test was used for comparison between two groups,and one-way analysis of variance was used for comparison between multiple groups.Results The survival rate of renal cancer patients with high expression of miRNA-676-3p was significantly higher than that of renal cancer patients with low expression of miRNA-676-3p,the difference was statistically significant(P<0.01).The relative expression of miRNA-676-3p in renal cancer cell lines was significantly lower than that in normal renal tubular epithelial cells,the difference was statistically significant(P<0.01),and the relative expression of miRNA-676-3p in CAKI1 cells was the lowest,the difference was statistically significant(P<0.01).The relative expression levels of miRNA-676-3p in the control and overexpression groups were 1.04±0.59 and 15.90±1.70,respectively,and the overexpression group was significantly higher than the control group,the difference was statistically significant(P<0.01).After 24,48,60,and 72 h of culture,the absorbance of cells in the overexpression group was lower than that in the control group,the difference was statistically significant(P<0.05).The number of invasion cells in the control group and the overexpression group were(115.90±24.73)and(43.83±21.94)cells,respectively,and the number of cell invasion in the overexpression group was significantly lower than that in the control group,the difference was statistically significant(P<0.01).PFDN1 was the downstream target gene of miRNA-676-3p(P<0.01).The relative expression of PFDN1 gene in the overexpression group was significantly lower than that in the control group,the difference was statistically significant(P<0.01).The expression of Wnt/β-catenin molecular pathway proteins in the overexpression group was lower than that in the control group.Conclusions Renal cancer patients with high expression of miRNA-676-3p had a higher survival rate.miRNA-676-3p inhibited the proliferation and invasion of renal cancer CAKI1 cells by significantly down-regulating the expression of PFDN1,thereby inhibiting the development of renal cancer.