摘要
目的检测雷帕霉素诱导细胞后4种miRNAs的表达情况,并进一步研究miR-144与Beclin-1基因的靶向调控作用。方法对SKOV-3细胞分别进行雷帕霉素(50μg/L,反应2 h)及3-甲基腺嘌呤(10 nmol/L、反应12 h)处理,RT-q PCR检测各组细胞miR-17、miR-20a、miR-144及miR-155的表达;Western blot检测雷帕霉素组Beclin-1蛋白的表达;双荧光素酶报告系统、Western blot及RT-q PCR,验证miR-144与Beclin-1之间的靶向调控关系。结果与正常对照组相比,雷帕霉素组SKOV-3细胞的miR-17、miR-144及miR-155的表达水平显著上调(P<0.05);3-MA组SKOV-3细胞的miR-17、miR-20a及miR-144的表达水平显著下调(P<0.05);雷帕霉素组Beclin-1的蛋白表达明显低于正常细胞组(P<0.05)。miR-144可靶向作用Beclin-1的3'非翻译区(3'UTR),且抑制其表达;miR-144能明显抑制Beclin-1蛋白及mRNA的表达。结论 miR-144可靶向抑制Beclin-1基因的表达,并参与调控SKOV-3细胞自噬的过程。
Objective To detect the influence of rapamycin on the expression of 4 kinds of miRNAs and the effect cell autophagy.To study the relationship of miR-144 and Beclin-1 gene. Methods SKOV-3 cells were treated with 50 ng,/mL rapamycin 2 hours and 10 nmoL/L 3-methyl adenine 12 hours, the expression of miR-17, miR-20a, miR- 144 and miR-155 was detected by RT-qPCR in SKOV-3 cell of different groups, the protein expression of Beclin-1 was detected by Western blot.The targeting effect of miR- 144 on Beclin- 1 gene was verified by the dual-luciferase reporter assay,Western blot and RT-qPCR. Results The expression of miR- 17, miR- 144 and miR- 155 were in- creased compared with NC groups in rapamycin group （P〈0.05） ; miR-17, miR-20a and miR-144 were down reg- ulated compared with NC group in 3-MA group（P〈0.05 ） ;the protein of Beclin- 1 was down expression compared with NC group in rapamyein group.miR-144 could suppress Beclin-1 expression by targeting the specific 3＇ untrans- lated region sequence of Beclin- 1 gene. Conclusions miR- 144 can inhibit the autophagy-related gene Beclin- 1 ex- pression and regulate the autophagy process in SKOV-3 cells.
出处
《基础医学与临床》
CSCD
2017年第7期1021-1025,共5页
Basic and Clinical Medicine
