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.