microRNA-185下调AKT1信号通路对淋巴母细胞淋巴瘤细胞增殖和凋亡的影响及相关的分子机制的研究

Effect of microRNA-185 down-regulation of AKT1 signaling pathway on proliferation and apoptosis of lymphoblastoid lymphoma cells and its related molecular mechanism

目的探讨miR-185对淋巴母细胞淋巴瘤(T-LBL)细胞增殖和致肿瘤性的影响及相关的分子机制。方法 q PCR检测人T-LBL组织和正常的胸腺组织中miR-185表达水平。miR-185模拟物及NC-miRNA转染Jurkat细胞,MTT法测定细胞增殖情况,q PCR和MTT法分别检测miR-185表达水平和细胞增殖情况,细胞流失术检测细胞凋亡。生物信息学软件Target Scan预测miR-185的靶作用位点,并通过q PCR、Western blot和荧光素酶活性实验证实miR-185的靶作用位点。慢病毒介导的miR-185过表达Jurkat细胞分别通过皮下注射的方式注射入裸鼠左右侧,皮下注射后4 d、8 d、12 d、16 d、20 d、24 d、28 d、32 d分别测量肿瘤体积,以探讨miR-185对体内肿瘤生长的影响。结果与人正常胸腺组织相比,T-LBL组织中miR-185表达显著下调(P<0.05);MTT检测细胞增殖结果表明,与NCmiRNA转染组相比,miR-185转染组Jurkat细胞增殖速率显著下降(P<0.05),且转染miR-185后,Jurkat细胞凋亡比例显著升高(P<0.05);Target Scan生物学软件预测表明AKT1为miR-185潜在的靶基因,实验结果进一步证实AKT1的3'非翻译序列(3'UTR)是miR-185的直接靶点;裸鼠移植瘤试验结果表明,miR-185能抑制体内肿瘤生长。结论miR-185通过靶向作用于AKT1基因抑制Jurkat细胞增殖,其可能为T-LBL的临床治疗提供新的靶标。

Objective To investigate the effect and mechanism of miR - 185 negative regulation of AKT1 gene expression in T - lympho-blastic lymphoma （T - LBL）. Methods Expression of miR - 185 in human T - lymphoblastic lymphoma （T - LBL） tissues and normal thymus were detected by qPCR. miR - 185 mimics and NC - miRNA were transfected into Jurkat cells, then MTT assay was used to detect Jurkat cells proliferation, qPCR was used to detect the expression of miR - 185 and flowcytometry was used to determine cells apoptosis. TargetScan was used to predict the target site of miR - 185 , and double luciferase reporter assay, qPCR, western blot and site - directed mutagenesis were used to further confirm the target site of miR - 185. miR - 185 - positive cells or non - miR - 185 - positive cells were directly injected subcutaneously into the ？ anks of BALB/c nude mice, tumor volume was determined by caliper measurement at4d,8d,12d,16d,20d,24d,28d,32d after injection. Results miR - 185 was significantly down - regulated in T - LBL tissues compared with human normal thymus tissues （ P〈0.05）. MTT results showed that, compared with NC - miRNA transfection group, Jurkat cells proliferation rate in miR - 185 transfected group decreased significantly （ P 〈 0. 05） , and the apoptosis of Jurkat cells increased significantly （ P〈0.05） after miR - 185 transfection. Bioinformatics pre-diction results showed that, 3′- UTR sequences of AKT1 may be a miR - 185 target site, and results further confirmed that the 3′- UTR sequence of AKT1 gene is a direct target site of miR - 185. Conclusion MiR - 185 can inhibit Jurkat cells proliferation and tumorigenicity by targeting AKT1 signaling pathway, and this study may provide a new target for clinical treatment of T - LBL.