微小RNA-590-5p靶向CCNG2对神经胶质瘤细胞增殖、侵袭和干细胞特性的影响

Effects of microRNA-590-5p targeting CCNG2 on proliferation,invasion and stem cell characteristics of glioma cells

目的探讨微小RNA(miRNA,miR)-590靶向CCNG2对神经胶质瘤细胞增殖、侵袭和干细胞特性的影响。方法选取2017年3月至2021年3月南阳市中心医院收集的59例神经胶质瘤和癌旁组织作为研究对象,采用荧光定量聚合酶链反应(PCR)分析分析癌旁组织和肺癌组织miR-590表达水平。神经胶质瘤细胞U251分为miRNA对照组、miR-590组和miR-590 KD组,并采用转染试剂转染对照miRNA、miR-590-5p模拟物、miR-590-5p抑制剂,48 h后检测。采用细胞计数试剂盒(CCK-8)和克隆形成实验分析两组细胞增殖能力;采用Transwell实验分析两组细胞的侵袭能力。采用成球实验和流式细胞术分析两组细胞的干细胞特性;采用生物信息学和双荧光素酶报告基因分析miR-590靶基因。采用蛋白质印迹法(Western blot)分析细胞和肿瘤组织靶基因的表达水平。组间比较采用t检验。结果癌旁组织中miR-590-5p表达水平(1.03±0.15)明显低于神经胶质瘤组织(2.25±0.27),差异有统计学意义(t=30.640,P<0.05)。对照组细胞48 h吸光度值(2.01±0.15)明显高于miR-590-5p KD组(1.37±0.11),差异有统计学意义(t=30.640,P<0.05)。克隆形成实验结果显示,对照组细胞克隆形成数量[(81.33±10.78)个]明显高于miR-590-5p KD组[(36.67±6.71)个],差异有统计学意义(t=8.614,P<0.05)。对照组细胞侵袭数量[(122.17±15.13)个]明显高于miR-590-5p KD组[(60.17±7.83)个],差异有统计学意义(t=8.913,P<0.05)。对照组细胞SP亚群比例[(3.67±1.04)个]明显高于miR-590-5p KD组[(0.71±0.32)个],差异有统计学意义(t=6.669,P<0.05)。对照组细胞成球数量[(32.33±8.09)个]明显高于miR-590-5p KD组[(13.33±2.16)个],差异有统计学意义(t=5.557,P<0.05)。CCNG2是miR-590-5p的靶基因。对照组细胞CCNG2蛋白表达水平(0.89±0.08)明显低于miR-590-5p KD组(2.32±0.30),差异有统计学意义(t=11.130,P<0.05)。结论miR-590靶向CCNG2调节神经胶质细胞的增殖、侵袭和干细胞特性等细胞生物学行为。

Objective To investigate the effects of microRNA(miRNA,miR)-590 targeting CCNG2 on the proliferation,invasion and stem cell characteristics of glioma cells.Methods 59 cases of glioma and adjacent tissues collected in our hospital from March 2017 to March 2021 were selected as research objects,and the expression level of miR-590 in adjacent tissues and lung cancer tissues was analyzed by fluorescence quantitative polymerase chain reaction(PCR)analysis.Glioma U251 cells were divided into miRNA control group,miR-590 group and miR-590 KD group.Control miRNA,miR-590-5p analog and miR-590-5p inhibitor were transfected with transfection reagent and detected 48 hours later.cell counting kit-8(CCK-8)and clone formation assay were used to analyze the proliferation ability of the two groups of cells;Transwell experiment was used to analyze the invasive ability of the two groups of cells.The stem cell characteristics of the two groups of cells were analyzed by adjacent test and flow cytometry;Bioinformatics and double luciferase reporter genes were used to analyze miR-590 target genes.Western blotting was used to analyze the expression level of target genes in cells and tumor tissues.The measurement data between groups were compared by t-test.Results The expression level of miR-590-5p in the adjacent tissues of cancer(1.03±0.15)was significantly lower than that in the glioma tissues(2.25±0.27,t=30.640,P<0.05).The 48h absorbance value of cells in the control group(2.01±0.15)was significantly higher than that in the miR-590-5p KD group(1.37±0.11,t=30.640,P<0.05).The results of clone formation experiment showed that the number of cell clones in the control group[(81.33±10.78)cells]was significantly higher than that in the miR-590-5p KD group[(36.67±6.71)cells,t=8.614,P<0.05].The number of cell invasion in the control group[(122.17±15.13)cells]was significantly higher than that in the miR-590-5p KD group[(60.17±7.83)cells,t=8.913,P<0.05].The proportion of SP subsets in the control group[(3.67±1.04)cells]was significantly higher than that in the miR-590-5p KD group[(0.71±0.32)cells,t=6.669,P<0.05].The number of cells in the control group[(32.33±8.09)cells]was significantly higher than that in the miR-590-5p KD group[(13.33±2.16)cells,t=5.557,P<0.05].CCNG2 is the target gene of miR-590-5p.The expression level of CCNG2 protein in the control group(0.89±0.08)was significantly lower than that in the miR-590-5p KD group(2.32±0.30,t=11.130,P<0.05).Conclusion The miR-590 targeting CCNG2 regulates the proliferation,invasion and stem cell characteristics of glial cells.