摘要
目的探讨miR-202对肺癌A549细胞增殖和凋亡的调控作用及其相关机制。方法培养肺腺癌A549细胞和肺正常上皮BEAS-2B细胞,利用Real time-PCR检测miR-202在上述细胞中的表达水平;合成并将miR-202 minic和miR-NC转染进入A549细胞后,应用MTT检测12、24、48 h时细胞的抑制率,使用流式细胞术检测转染后48 h细胞的凋亡情况,利用western blot检测GLI-2蛋白的表达情况;构建野生型和突变型GLI-2 3'UTR插入p MIR-REPORTTMluciferase vector载体,并将其与pRL-TK质粒共转染进入A549细胞,之后分别将等量的pre-miR-202和miR-NC再转染进入A549细胞,利用双荧光素酶报告基因检测试剂盒检测萤火虫和海肾荧光素酶活性。结果 miR-202在A549细胞中的相对表达量显著低于在BEAS-2B细胞中的表达量(P<0.01);miR-202 minic组在12、24、48和72 h的细胞抑制率显著高于对照组和miR-NC组(P<0.01);miR-NC组在72 h时细胞抑制率显著高于对照组(P<0.01)。此外,miR-202 minic组细胞凋亡率显著高于miR-NC和对照组(P<0.01),并且miR-NC组的细胞凋亡率显著高于对照组(P<0.01)。荧光素酶报告基因结果说明GLI-2是miR-202的靶基因。miR-202minc转染A549细胞后可显著降低GLI-2蛋白的表达,而miR-NC组与对照组相比较,GLI-2蛋白表达无显著差异。结论 miR-202通过下游靶基因GLI-2调控肺癌A549细胞的增殖和凋亡,其可作为治疗肺癌的潜在靶点。
Objective To explore the mechanism and effect of miR-202 regulation the proliferation and apoptosis of A549 cells. Method A549 cells and lung epithelial BEAS-2B cells were cultured,and then miR-202 level of A549 and BEAS-2B cells was analyzed by realtime-PCR. The sequence of miR-202 and miR-NC was synthesized. They were transfected into A549 cells,the rate of proliferation inhibition was detected by MTT at 12,24,and 48 h,cellular apoptosis rate of A549 cells and the GLI-2 protein expression was measured by FACS and western blot at 48 h,respectively. The wild-type and mutation of GLI-2 3＇UTR was inserted into the plasmid of p MIR-REPORTTMluciferase vector,which was cotransfected into A549 with pRL-TK plasmid. premiR-202 and miR-NC was transfected into these cells,which had been transfected p MIR-REPORTTMluciferase vector and pRL-TK plasmid. Firefly and Renilla reniformis luciferase activities were measured 48 h later. Result the expression of miR-202 was lower in A549 cells than in BEAS-2B cells（ P〈0. 01）. The cellular inhibition rate was higher in miR-202 minic group than in miR-NC and control group at 12,24,48,and 72 h（ P0.01）,and the cellular inhibition rate was significant higher in miR-NC group than control group（ P0.01）. Moreover,the apoptosis of A549 cells was higher in miR-202 minic group than in miR-NC and control group at 72 h（ P0.01）. Luciferase assay showed GLI-2 was a direct target gene of miR-202. Up-regulation of miR-202 was significant decreased the expression of GLI-2 while miR-202 minic was transfected into A549 cells. GLI-2 protein expression was no significant difference between miR-NC group and control. Conclusion miR-202 regulated the proliferation and apoptosis of A549 cells through directed mediated target gene GLI-2,and it would as a novel target for lung cancer treatment.
出处
《中华肺部疾病杂志（电子版）》
CAS
2016年第3期252-257,共6页
Chinese Journal of Lung Diseases(Electronic Edition)
基金
国家自然科学基金资助项目(30801366)
