摘要
目的:研究micro RNA21(miR-21)对人骨髓增生异常综合征转白血病细胞株SKM-1增殖和凋亡的影响。方法:通过构建携带miR-21基因重组慢病毒干扰载体感染SKM-1细胞株,下调miR-21的表达,荧光显微镜下观察感染效率。体外设LV-miR-21干扰组(LV-miR-21-si RNA组)、阴性对照组(negative control组)和空白对照组(normal组)三组。然后采用q RT-PCR的方法检测各组细胞miR-21的相对表达量,通过CCK8法检测细胞增殖情况,Hoest 33258分析各组细胞的凋亡情况,并运用western blot检测凋亡通路中cleaved-caspase-3、Bax关键因子的表达。结果:通过慢病毒介导的miR-21-si RNA下调miR-21的表达后,q RT-PCR检测结果显示LV-miR-21-si RNA组中miR-21的表达量较negative control组、normal组表达量明显降低(P<0.05);CCK8结果显示LV-miR-21-si RNA组细胞增殖能力与negative control组、normal组比较明显下降(P<0.05),Hoechst 33258结果显示LV-miR-21-si RNA组细胞较negative control组、normal组凋亡增加(P<0.05);western blot证实cleaved-caspase-3、Bax在LV-miR-21-si RNA组中的表达明显升高(P<0.05)。结论:miR-21在骨髓增生异常综合征转白血病细胞株SKM-1中高表达,通过下调miR-21的表达可以抑制SKM-1细胞增殖,促进其凋亡;miR-21可能在骨髓增生异常综合征向急性髓系白血病转化进程中起重要作用。
Objective： This study was designed to explore the effects of microRNA21 （miR-21） expression on proliferation and apoptosis in myelodysplastic syndrome cell line SKM-1. Methods： SKM-1 cell line was transfected by recombinant lentiviral interference vector carrying miR-21 gene to down regulate miR-21 expression, and the infection efficiency was observed by fluorescence microscope. We set up LV-miR-21 interference group （LV-miR-21-siRNA group）, negative control group and blank control group （normal group） in vitro. Then the method of qRT-PCR was applied to validate the relative expression of miR-21 in each group. The proliferation of SKM-1 was observed by the technique of CCK8. The apoptosis was detected by Hoest 33258, and the expressions of protein cleaved-caspase-3 and Bax with the application of western blot. Results： We used lentiviral mediated miR-21-siRNA to down regulate miR-21 expression. QRT-PCR showed that the expression ofmiR-21 in the LV-miR-21-siRNA group was significantly lower than that in the negative control group and the normal group （P 〈 0.05）. The results of CCK8 showed that the cell proliferation rate in the LV-miR-21-siRNA group was significantly lower than that of negative control group and the normal group （P 〈 0.05）. The results of Hoechst 33258 showed that the apoptotic rate in LV-miR-21-siRNA group was significantly higher than that in the negative control group and the normal group （P 〈 0.05）. Western blot showed that to down regulate of miR-21 resulted in up-regulation of cleaved-caspase-3 and Bax in the LV-miR-21-siRNA group （P 〈 0.05）. Conclusions： miR-21 was highly expressed in SKM-1 cell lines of leukemia transformed from myelodysplastic syndrome. To down regulate miR-21 expression could inhibit proliferation and promote apoptosis of SKM-1 cells. MiR-21 may play an important role in the progression of myelodysplastic syndromes to acute myeloid leukemia.
出处
《现代生物医学进展》
CAS
2017年第34期6657-6661,6683,共6页
Progress in Modern Biomedicine
基金
陕西省自然科学基础研究计划项目(2013JM4016)
