MicroRNA-10b对人低转移肺癌细胞株95-C增殖和侵袭的作用及其机制的研究

Effect and mechanism of miRNA-10b on proliferation and invasion of low metastasis of lung cancer cell line 95-C

目的微小RNA(microRNA,miRNA)-10b被证实可促进多种肿瘤细胞的生长及增强其侵袭能力。文中旨在研究miRNA-10b对人低转移肺癌细胞95-C的增殖及侵袭力的影响及其作用机制。方法用脂质体法将miRNA-10b真核表达质粒瞬时转染入95-C,实验设置空白对照组、阴性对照组和miRNA-10b质粒转染组,实时定量荧光PCR(Real-Time PCR,RT-PCR,)检测各组细胞miRNA-10b的表达及KLF4mRNA的表达,细胞增殖实验检测各组细胞的增殖情况,Transwell实验检测各组细胞侵袭能力,Western blot检测各组细胞KLF4蛋白的表达。结果转染后48h,RT-PCR检测,miRNA-10b质粒转染组miRNA-10b表达(1.61±0.12)较空白对照组(1.01±0.08)、阴性对照组(0.86±0.07)明显上调(P<0.05),阴性对照组与空白对照组差异无统计学意义(P>0.05)。生长曲线反映miRNA-10b质粒转染组细胞生长速度明显高于空白对照组和阴性对照组,差异有统计学意义(P<0.05),空白对照组和阴性对照组比较,差异无统计学意义(P>0.05);Transwell结果显示,miRNA-10b质粒转染组[(188.0±15.1)/高倍显微镜]较空白对照组[(151.0±11.3)/高倍显微镜]、阴性对照组[(136.0±10.8)/高倍显微镜]有更多的细胞迁移到Transwell膜的另一侧(P<0.05),空白对照组与阴性对照组差异无统计学意义(P>0.05);转染后48 h,miRNA-10b质粒组KLF4蛋白表达(0.86±0.06)较空白对照组(1.48±0.05)和阴性对照组(1.54±0.08)降低(P<0.05);各组细胞KLF4 mRNA表达差异无统计学意义(P>0.05)。结论 miRNA-10b可能过下调KLF4蛋白的表达促进95-C细胞的增殖及侵袭能力。

Objective MiRNA-10b is an important member of the MiRNA family , which has been proven that miRNA-10b can promote the growth and invasion of a variety of tumor cells .The aim of this study was to to investigate the effect and mechanism of miR-NA-10b on proliferation and invasion of low metastasis of lung cancer cell line （95-C）. Methods The recombinant of miRNA-10b was transfected into 95-C by lipofectin method .The experiment set up 3 groups：blank control group , negative control group and miRNA-10b expression plasmid transfected group .MiRNA-10b expression level and KLF4mRNA expression level were detected by real-time fluores-cence quantitative PCR （ RTFQ-PCR） .The cell proliferation was detected by cell proliferation assay .The invasive ability of cell was de-tected by Transwell experiment .The expression of KLF4 protein was assessed by Western blot . Results At 48 hours after transfection, compared with blank control group （1.01 ±0.08） and negative control group （0.86 ±0.07）, the miRNA-10b expression level in miRNA-10b expression plasmid transfected group （1.61 ±0.12） increased significantly （P〈0.05） and there was no statistical difference between blank control group and negative control group （P〉0.05）.From the growth curve, the cell proliferation rate was obviously increased in miRNA-10b expression plasmid transfected group （[188.0 ±15.1]/HP） compared with the other two groups （[151.0 ±11.3]/HP）, （[136.0 ±10.8]/HP） （P 〈0.05） and there was no statistical difference between these two groups （ P 〉0.05 ）.Transwell result showed more cells transferred to the other side of Transwell compared with the other two groups （ P 〈0.05 ） and there was no statistical difference between these two groups （P 〉0.05）.The expression of KLF4 protein decreased in miRNA-10b expression plasmid transfected group compared with the other two groups （ P〈0.05）.KLF4mRNA expression decreased, but the difference had no statistical significance （P〉0.05）. Conclusion MiRNA-10b might promote the pro-liferation and invasion of 95-C through down-regulation of KLF4 protein expression .