雷帕霉素对RAW264.7巨噬细胞10种与细胞自噬相关的miRNAs表达的影响

Effects of rapamycin on expression of ten kinds autophagy-related miRNAs in RAW264. 7 macrophages

目的:为了探讨雷帕霉素对RAW264.7细胞miR-30b、miR-200a和miR-17-5p等10种与细胞自噬相关的miRNAs表达水平的影响,为进一步研究miRNAs调控细胞自噬的机制提供理论依据。方法:雷帕霉素刺激RAW264.7巨噬细胞后,分别在2、4、6、8 h提取细胞small RNA,利用miRNA特异性的茎环引物反转录成cDNA,采用Real-Time PCR检测miR-30b、miR-30c、miR-106a、miR-214、miR-183、miR-200a、miR-376c、miR-17-5p、miR-142-3p、miR-377分子的表达情况。结果:雷帕霉素作用RAW264.7细胞后,miR-17-5p、miR-106在2、4、6 h表达上调(大于2.1倍P<0.05),miR-214在2、8小时表达上调(>2.4倍,P<0.05),miR-30b、miR-30c、miR-183、miR-200a、miR-376c、miR-142-3p在2、6、8 h表达上调(>2.4倍,P<0.05),而miR-183、miR-200a在4 h表达下调(>2.1倍,P<0.05),miR-30b在8小时则是显著性表达下调(大于50倍,P<0.05),miR-377在4 h则是表达上调(>2.5倍,P<0.05),但在2、8 h则是显著表达下调(>50倍,P<0.05)。结论:雷帕霉素刺激RAW264.7巨噬细胞后miR-200a、miR-30b、miR-377、miR-30c、miR-376c、miR-17-5p有显著性变化,说明这些miRNAs可能通过调控某些自噬相关基因在细胞自噬过程中发挥着重要作用。

To detect the influence of rapamycin on the expression of miR-30b,miR-200a and miR-17-5p etc in macrophages and provide the basis to study the regulation of miRNA in autophagy mechanism of macrophages .Methods： Small RNA was extracted at different times after stimulated with rapamycin in cultured RAW 264.7 cells.After using the stem-loop reverse transcription primers to reverse transcribed into cDNA ,the expression of miR-30b ,miR-30c,miR-106a,miR-214,miR-183,miR-200a, miR-376c,miR-17-5p, miR-142-3p, miR-377 was detected by Real-Time PCR.Results： After RAW264.7 cells was treated by rapamycin for 2,4,6 and 8 hours,the expression of miR-17-5p and miR-106 increased （More than 2.1 times,P〈0.05） in 2,4 and 6 hours;miR-214 was up regulated in 2 and 8 hours （More than 2.4 times,P〈0.05）;miR-30b,miR-30c,miR-183,miR-200a,miR-376c and miR-142-3p was up regulated in 2,6 and 8 hours （2.4 times,P〈0.05 ）;while miR-183 and miR-200a was down regulated at 4 hours（More than 2.1 times,P〈0.05）;miR-30b was significantly low expression in 8 hours （more than 50 times,P〈0.05）;miR-377 was up regulated at 4 hours （more than 2.5 times,P〈0.05）,but was significantly down regulated at 2 and 8 hours （More than 50 times,P〈0.05） Conclusion： The expression of miR-200a,miR-30b,miR-377,miR-30c,miR-376c and miR-17-5p is significantly changed after rapamycin stimulated RAW264.7 macrophages,indicated the miRNA may plays an important role in autophagy through the regulation of autophagy-related genes.