摘要
目的:研究microRNA-15a(miR-15a)在多发性骨髓瘤(MM)细胞生长中的作用及可能机制。方法:慢病毒颗粒感染MM细胞株U266和RPMI8226,流式细胞术(FCM)分选获得稳定转染MM细胞株。CCK-8法检测miR-15a高表达前后MM细胞的增殖情况;AO/EB染色、Hoechst 33258荧光染色法及FCM检测miR-15a高表达前后MM细胞凋亡的情况;FCM检测miR-15a高表达前后MM细胞周期的情况;real-time PCR方法检测miR-15a高表达前后MM细胞miR-15a、BMI-1及BCL-2 mRNA的表达;Western blot方法检测miR-15a高表达前后MM细胞BMI-1蛋白表达。结果:获得高表达miR-15a的MM稳定转染细胞株。CCK-8结果显示miR-15a高表达可抑制M M细胞(U266和RPM I8226)的增殖;AO/EB、Hoechst 33258染色和FCM结果显示,miR-15a高表达可显著诱导MM细胞(U266和RPM I8226)的凋亡,U266和RPM I8226细胞高表达组与对照组细胞凋亡率分别为:90.52%vs37.08%,59.40%vs 44.17%;同时,miR-15a高表达可诱导MM细胞(U266和RPM I8226)G1期阻滞,G1期细胞分别为(41.50±0.64)%、(45.31±0.77)%。real-time PCR结果显示,在高表达miR-15a抑制骨髓瘤细胞生长的过程中BCL-2 mRNA表达显著降低,BMI-1 mRNA表达则没有改变,但是Western blot结果却显示BMI-1蛋白表达出现显著降低。结论:miR-15a高表达通过诱导骨髓瘤细胞周期阻滞和凋亡抑制骨髓瘤细胞生长,其机制涉及miR-15a在转录后水平对BMI-1、BCL-2基因的负调控。
Objective：To explore the effect and possible mechanisms of miR-15a on growth of multiple myeloma （MM） cells. Methods：MM cell lines （U266 and RPMI8226） were transfected by lentiviral particles. MM stable cell lines were selected and collected by flow cytometry （FCM）. Proliferation of MM cells before and after miR-15a high expression was detected by CCK-8 method. Apoptosis of MM cells before and after miR-15a high expression was detected by AO/EB dying, Hoechst 33258 dying and FCM, respectively. Cell cycle of MM cells before and after miR- 15a high expression was detected by FCM. The expressions of miR-15a, BMI-1 and BCL-2 mRNA of MM cells before and after miR-15a high expression were detected by real-time PCR. The expressions of BMI-1 protein of MM cells before and after miR-15a high expression were detected by Western blot. Results：MM stable cell lines with miR-15a high expression was acquired. CCK-8 result showed that high expression of miR-15a could inhibit growth of MM cells （U266 and RPMI8226）. AO/EB dying, Hoechst 33258 dying and FCM testing results showed that high expression of miR-15a could significantly induce apoptosis of MM cells （U266 and RPMI8226 ）. The apoptosis rates of U266 and RPMI8226 cells in high expression group and control group were 90. 52% vs 37.08% and 59.40% vs 44. 17%, respectively. Meanwhile, FCM testing results showed that high expression of miR-15a could induce G1 arrest of MM cells（ U266 and RPMI8226 ）, which proportion of G1 phase were 41.50% ± 0.64%, 45.31%± 0.77%, respectively. Real-time PCR results showed that during the growth inhibition process of MM cells caused by miR-15a high expression, the expression of BCL-2 mRNA decreased, but there was no significant changes in the expression of BMI-1 mRNA, while the expression of BMI-1 protein decreased significantly. Conclusion： High expression of miR-15a can induce cell cycle arrest and apoptosis of MM cells, then inhibit their growth. The mechanisms may be related with the negative regulation of BMI-1 and BCL-2 genes in post-transcription level caused by miR-15a.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2015年第3期706-712,共7页
Journal of Experimental Hematology
基金
国家和福建省临床重点专科建设项目资助
国家自然科学基金青年科学基金资助项目(81201872)
福建省自然科学基金资助项目(2013J01308)
福建医科大学教授学术发展基金(JS11005)
