miR-19a对非小细胞肺癌细胞组织因子的调控作用及细胞增殖的影响

The regulation ef ects of miR-19a on expression of tissue factor and the cell proliferation of NSCLC cells

目的 :探讨微小RNA(microRNA,miRNA,miR)-19a对非小细胞肺癌(non-small cell lung cancer,NSCLC)PC9细胞组织因子(tissue factor,TF)的调控作用及细胞增殖的影响。方法 :将hsa-miR-19a模拟物(hsa-miR-19a-mimic)、hsa-miR-19a抑制物(hsa-miR-19a-inhibitor)、hsa-miR阴性对照(hsa-miR-negative control,hsa-miR-NC)分别瞬时转染PC9细胞后,应用实时荧光定量PCR法检测各组细胞中miR-19a的表达,CCK-8(cell counting kit-8)法检测细胞的增殖情况,FCM法检测细胞周期的变化。利用生物信息学软件预测miR-19a的靶基因,通过荧光素酶报告基因检测系统和蛋白质印迹法对预测的潜在靶基因进行验证。结果 :Hsa-miR-19a-mimic转染组PC9细胞中miR-19a的表达水平明显高于空白对照组(只加脂质体)和阴性对照组(P<0.01),而hsa-miR-19a-inhibitor转染组PC9细胞中miR-19a的表达水平明显低于空白对照组和阴性对照组(P<0.05)。转染hsa-miR-19a-mimic可抑制PC9细胞的增殖,而转染hsa-miR-19a-inhibitor可促进PC9细胞的增殖(P<0.05)。Hsa-miR-19a-mimic转染组PC9细胞被阻滞于G0/G1期,而hsa-miR-19ainhibitor转染组G2/M期细胞所占比例增加(P<0.01)。荧光素酶报告基因检测提示,TF基因受miR-19a负调控。Hsa-miR-19a-mimic转染组TF蛋白的表达水平低于空白对照组和阴性对照组,hsa-miR-19a-inhibitor转染组则相反(P<0.01)。结论 :miR-19a可以调控TF的表达并抑制PC9细胞的体外增殖。

Objective： To investigate the regulation effects of microRNA （miRNA, miR）-19a on expression of tissue factor （TF） and the cell proliferation of non-small cell lung cancer （NSCLC） PC9 cells. Methods： The hsa-miR-1 9a-mimic, hsa-miR-19a-inhibitor and hsa-miR-negative control （hsa-miR-NC） were transiently transfected into PC9 cells, respectively. Then the expression of miR-1 9a was determined by real-time fluorescence quantitative-PCR. The proliferation of PC9 cells transfected with hsa-miR-1 9a- mimic, hsa-miR-19a-inhibitor or hsa-miR-NC was detected by cell counting kit-8 （CCK-8） assay. The cell cycle distribution was analyzed by flow cytometry （FCM）. Bioinformatics software was used to predict downstream target genes of miR-19a. The potential target genes were verified by luciferase reporter assay and Western blotting. Results： The expression level of miR-19a in hsa-miR-19a-mimic-transfected group was higher than those in the blank control group （only adding liposomes） and the negative control group （P 〈 0.01）, while the expression level of miR-19a in hsa-miR-19a-inhibitor-transfected group was opposite （P 〈 0.05）. Hsa-miR-19a-mimic inhibited the proliferation of PC9 cells, while hsa-miR- 19a-inhibitor was opposite （P 〈 0.05）. The cell cycle of PC9 cells was arrested at G0/G1 phase in hsa- miR-19a-mimic-transfected group, and the proportion of cells in G2/M phase in hsa-miR-19a-inhibitor- transfected group was increased （P 〈 0.01）. The result of luciferase reporter assay revealed that miR- 19a negatively regulated the expression of TF gene. The expression level of TF protein in hsa-miR-19a-mimic-transfected group was lower than that in the blank control group or the negative control group, whereas the expression level of TF protein in hsa-miR-19a- inhibitor-transfected group was opposite （P 〈 0.01）. Conclusion： The miR-19a can regulate the expression of TF and inhibit the proliferation of PC9 cells in vitro.