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目的 :探讨抑制微RNA(microRNA,miRNA,miR)-221表达对膀胱癌细胞增殖和凋亡的影响。方法 :将化学合成的miR-221抑制片段has-miR-221 inhibitor和miR-221抑制阴性无意义片段(阴性对照组)(has-miR-221inhibitor negative control)转染至膀胱癌T24和J82细胞中,转染5 h后,在荧光显微镜下观察转染效率;实时荧光定量PCR法检测转染48 h后,T24和J82细胞中miR-221的表达情况;MTT法检测转染24、48和72 h后,T24和J82细胞的增殖情况;RT-PCR和蛋白质印迹法检测转染48 h后,T24和J82细胞中p53上调凋亡调控因子(p53 upregulated modulator of apoptosis,PUMA)、Bax和Bcl-2 mRNA及蛋白的表达;FCM法和吖啶橙(acridine orange,AO)-溴化乙锭(ethidium bromide,EB)染色检测转染48 h后,T24和J82细胞的凋亡情况。结果 :Has-miR-221 inhibitor转染至T24和J82细胞的效率分别为80%和90%。Has-miR-221 inhibitor转染后,T24和J82细胞中miR-221的表达水平明显低于阴性对照组和空白对照组(只加入转染试剂)(P<0.05);转染后T24和J82细胞的增殖抑制率高于阴性对照组和空白对照组(P<0.05),且呈时间依赖性;转染组PUMA和Bax mRNA及蛋白的表达水平明显高于阴性对照组和空白对照组(P<0.05),Bcl-2 mRNA及蛋白的表达水平明显低于阴性对照组和空白对照组(P<0.05);has-miR-221 inhibitor转染组T24和J82细胞的凋亡率明显高于阴性对照组和空白对照组(P<0.05)。结论 :抑制miR-221的表达可抑制膀胱癌细胞的增殖,并促进其凋亡。
Objective: To investigate the effect of inhibiting the expression of microRNA (miRNA, miR) -221 on the proliferation and apoptosis of bladder cancer cells. METHODS: The transfected bladder cancer T24 and J82 cells were transfected with the chemically synthesized miR-221 inhibitor has-miR-221 inhibitor and the miR-221 negative suppressor fragment (negative control) (negative control) , Transfection 5 h after transfection efficiency was observed under a fluorescence microscope; real-time fluorescent quantitative PCR 48 h after transfection, T24 and J82 cells miR-221 expression; MTT assay transfected 24,48 and 72 h, the proliferation of T24 and J82 cells; the expression of p53 upregulated modulator of apoptosis (PUMA), Bax and Bcl-2 in T24 and J82 cells were detected by RT-PCR and Western blot 48 h after transfection; 2 mRNA and protein were detected by FCM and acridine orange (AO) - ethidium bromide (EB) staining. The apoptosis of T24 and J82 cells was detected 48 h after transfection. Results: The efficiencies of Has-miR-221 inhibitor transfected T24 and J82 cells were 80% and 90%, respectively. The expression of miR-221 in T24 and J82 cells after transfected with Has-miR-221 inhibitor was significantly lower than that in the negative control group and blank control group (only transfected with transfection reagent) (P <0.05) The inhibitory rate of proliferation of cells was higher than that of the negative control group and the blank control group (P <0.05), and the time-dependent manner. The expression of PUMA and Bax mRNA and protein in the transfection group was significantly higher than that in the negative control group and the blank control group <0.05). The expression of Bcl-2 mRNA and protein was significantly lower than that of negative control group and blank control group (P <0.05). The apoptosis rate of T24 and J82 cells transfected with has-miR-221 inhibitor was significantly higher than that of negative Control group and blank control group (P <0.05). Conclusion: Inhibition of miR-221 expression can inhibit bladder cancer cell proliferation and promote its apoptosis.