lncRNA CDK5RAP3通过miR-223-3p调节胃癌细胞的增殖和侵袭

lncRNA CDK5RAP3 regulates the proliferation and invasion of gastric cancer cells via miR-223-3p

目的探究长链非编码RNA(lncRNA)CDK5RAP3在胃癌组织中的表达及对胃癌细胞增殖和侵袭的调节作用。方法通过TCGA数据库分析CDK5RAP3在胃癌组织和癌旁组织中的表达差异。通过转染pcDNA3.1-CDK5RAP3质粒至Hs-746T细胞,构建过表达CDK5RAP3胃癌细胞株(CDK5RAP3组),转染pcDNA3.1质粒至Hs-746T细胞(对照组)。通过实时荧光定量PCR(qRT-PCR)检测两组细胞中CDK5RAP3表达量的变化。CCK-8法和Transwell实验分别检测过表达CDK5RAP3对Hs-746T细胞增殖、侵袭的影响。通过starBase v2.0数据库预测CDK5RAP3和miR-223-3p的结合位点。双荧光素酶报告基因实验验证CDK5RAP3和miR-223-3p的直接结合。qRT-PCR检测各组Hs-746T细胞中miR-223-3p表达水平。Western blotting检测各组Hs-746T细胞中增殖蛋白和侵袭蛋白的表达水平。实验数据采用SPSS 17.0软件进行分析,符合正态分布的计量资料以均数±标准差(±s)表示,两组间比较采用t检验,多组均数间比较采用单因素方差分析。结果与癌旁组织相比,胃癌组织中CDK5RAP3表达水平明显降低(P<0.01)。对照组和CDK5RAP3组Hs-746T细胞中CDK5RAP3表达分别为(1.08±0.77)和(10.63±2.14),差异有统计学意义(P<0.01)。上调CDK5RAP3显著降低Hs-746T细胞的增殖活性(P<0.05)。对照组和CDK5RAP3组侵袭细胞数分别为(137.80±28.72)个和(57.76±24.95)个,差异有统计学意义(P<0.01)。CDK5RAP3能够直接结合miR-223-3p(P<0.01)。对照组和CDK5RAP3组Hs-746T细胞中miR-223-3p表达分别为(6.22±1.20)和(1.01±0.98),差异有统计学意义(P<0.01)。与对照组相比,上调CDK5RAP3显著减少增殖蛋白和侵袭蛋白的表达水平。结论胃癌组织中CDK5RAP3呈现低表达,CDK5RAP3通过靶向miR-223-3p抑制胃癌Hs-746T细胞的增殖和侵袭。

Objective To explore the expression of long non-coding RNA(lncRNA)CDK5RAP3 in gastric cancer tissue and its regulatory effect on gastric cancer cell proliferation and invasion.Methods The expression differences of CDK5RAP3 in gastric cancer tissues and adjacent tissues were analyzed by TCGA database.By transfecting the pcDNA3.1-CDK5RAP3 plasmid into Hs-746T cells,a gastric cancer cell line overexpressing CDK5RAP3(CDK5RAP3 group)was constructed,and the pcDNA3.1 plasmid was transfected into Hs-746T cells as a control group.The changes of CDK5RAP3 expression in the two groups of cells were detected by real-time quantitative PCR(qRT-PCR).The effects of overexpression of CDK5RAP3 on the proliferation and invasion of Hs-746T cells were detected by CCK-8 assay and Transwell assay,respectively.The binding sites of CDK5RAP3 and miR-223-3p were predicted by the starBase v2.0 database.The direct binding of CDK5RAP3 and miR-223-3p was verified by dual-luciferase reporter gene experiment.The expression levels of miR-223-3p in Hs-746T cells in each group were detected by qRT-PCR.Western blot was used to detect the expression levels of proliferation proteins and invasion proteins in Hs-746T cells in each group.The experimental data were analyzed by SPSS 17.0 software,and the measurement data conforming to the normal distribution were expressed as Mean±SD.The t-test was used to compare between two groups,and the one-way analysis of variance was used to compare the means of multiple groups.Results Compared with adjacent tissues,the expression level of CDK5RAP3 in gastric cancer tissues was significantly lower(P<0.01).The expressions of CDK5RAP3 in Hs-746T cells in the control group and CDK5RAP3 group were(1.08±0.77)and(10.63±2.14),respectively,and the difference was statistically significant(P<0.01).Up-regulation of CDK5RAP3 significantly decreased the proliferation activity of Hs-746T cells(P<0.05).The number of invasive cells in the control group and CDK5RAP3 group were(137.80±28.72)and(57.76±24.95),respectively,and the difference was statistically significant(P<0.01).CDK5RAP3 could directly bind miR-223-3p(P<0.01).The expression of miR-223-3p in Hs-746T cells in control group and CDK5RAP3 group were(6.22±1.20)and(1.01±0.98),respectively,and the difference was statistically significant(P<0.01).Compared with the control group,up-regulation of CDK5RAP3 significantly reduced the expression levels of proliferation and invasive proteins.Conclusion The expression of CDK5RAP3 is low in gastric cancer tissue,and CDK5RAP3 inhibits the proliferation and invasion of gastric cancer Hs-746T cells by targeting miR-223-3p.