抑制miR-221表达对膀胱癌细胞增殖和凋亡的影响

Effects of inhibition of miR-221 expression on proliferation and apoptosis of bladder cancer cells

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 effects of inhibition of microRNA-221（miR-221） expression on the proliferation and apoptosis of bladder cancer cells. Methods： Has-miR-221 inhibitor and has-miR-221 inhibitor negative control were synthesized, and then transfected into bladder cancer T24 and J82 cells. The transfection efficiency was observed under a fluorescence microscope 5 h after transfection. The expression levels of miR-221 in T24 and J82 cells were detected by real-time fl uorescence quantitative PCR at 24, 48 and 72 h after transfection; the proliferation of T24 and J82 cells was also detected by MTT assay. The expression levels of p53 upregulated modulator of apoptosis（PUMA）, Bax and Bcl-2 mRNAs and proteins in T24 and J82 cells 48 h after transfection were measured by RT-PCR and Western blotting, respectively; the apoptosis of T24 and J82 cells was determined by fl ow cytometry（FCM） and acridine orange（AO）-ethidium bromide（EB） staining. Results： The effi ciencies of has-miR-221 inhibitor transfection into T24 and J82 cells were 80% and 90%, respectively. The expression levels of miR-221 in T24 and J82 cells after transfection with has-miR-221 inhibitor were lower than those in the negative control group and the blank control group（only adding liposome）（P 〈 0.05）. The proliferative inhibition rates of T24 and J82 cells after transfection with has-miR-221 inhibitor were higher than those in the negative control group and the blank control group（P 〈 0.05）, and this effect was in a time-dependent manner. The expression levels of PUMA and Bax mRNAs and proteins in T24 and J82 cells after transfection with has-miR-221 inhibitor were higher than those in the negative control group and the blank control group（P 〈 0.05）, and the expression levels of Bcl-2 mRNA and protein were opposite（P 〈 0.05）. The apoptosis rates of T24 and J82 cells after transfection with has-miR-221 inhibitor were higher than those in the negative control group and the blank control group（P 〈 0.05）. Conclusion： Inhibition of miR-221 expression can suppress the proliferation of bladder cancer cells and induce apoptosis.