摘要
【目的】探讨上调microRNA-99b(miR-99b)表达对宫颈癌SiHa细胞增殖和凋亡的作用及可能的机制。【方法】宫颈癌SiHa细胞分正常对照组、阴性对照组和miR-99b调控组。利用siPORT NeoFX TransfectionAgent,阴性对照组细胞转染Cy3dye labeled PremiR Negative Control,miR-99b调控组细胞转染Pre-miR-hsa-miR-99b miRNA Precursor。实时定量PCR方法检测miR-99b表达,四甲基偶氮唑蓝(MTT)法检测细胞增殖能力,流式细胞术检测细胞凋亡率,Western blot检测靶基因细胞分裂周期25A(CDC25A)蛋白表达。【结果】实时定量PCR结果显示,miR-99b调控组细胞,miR-99b表达增加107.23倍。正常对照组、阴性对照组和miR-99b调控组细胞增殖率分别为(3.26±0.44)%、(3.65±0.47)%和(2.24±0.45)%,细胞凋亡率分别为(8.12±1.54)%、(8.75±1.67)%和(14.26±1.70)%,细胞CDC25A蛋白表达分别为0.50±0.06、0.59±0.05和0.31±0.05。与正常对照组和阴性对照组比较,miR-99b调控组细胞增殖率、凋亡率和CDC25A蛋白表达差异均有统计学意义(P<0.05)。【结论】上调miR-99b表达可通过降低靶基因CDC25A表达,抑制细胞增殖,诱导细胞凋亡,miR-99b可能成为宫颈癌治疗的靶基因。
[Objective] To investigate the effects of microRNA-99b （miR-99b） up-regulation on proliferation and apoptosis in SiHa ceils and its possible mechanism. [Methods] The SiHa cells were divided into normal control, negative control, and miR-99b modulated groups. Cy3 dye labeled PremiR Negative Control was transfected into negative control cells, and Pre-miR-hsa-miR-99b miRNA Precursor was transfected into miR-99b modulated cells with siPORT NeoFX transfection agent. The miR-99b expression was detected by real-time PCR. The cellular growth activity was assayed by MTT assay, and the apoptosis was tested by flow cytometry. The protein expression of CDC25A was measured by Western blot analysis. [Results] The miR-99b expression in miR-99b modulated cells increased 107.23 fold. The cellular growth ratio of normal control group was 3.26% ± 0.44%, of negative control group 3.65% ± 0.47%, of miR-99b modulated group 2.24% ± 0.45%. The apoptosis ratio of normal control group was 8.12% ± 1.54%, of negative control group 8.75% ± 1.67%, of miR-99b modulated group 14.26% ± 1.70%. The CDC25A protein expression of normal control group was 0.50 ± 0.06, of negative control group 0.59 ± 0.05, of miR-99b modulated group 0.31 ± 0.05. The cellular growth ratio, apoptosis ratio, and CDC25A protein expression had significant difference between miR-99b modulated group and two control groups （P 〈 0.05）. [Conclusions] The miR-99b up-regulation could suppress the cellular proliferation and induce apoptosis in SiHa cells by decreased target genes CDC25A expression. The miR-99b may be a target gene for cervical carcinoma treatment.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2008年第6期670-675,共6页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金面上项目(30672221)
广东省自然科学基金博士启动项目(7301386)
