针对DNMT3多靶点siRNA的构建及对肾细胞癌786-0细胞的影响

Establishment and evaluation the effection of DNMT3 multi-targeting siRNA on RCC786-0 cell line

目的:设计、验证针对DNA甲基化转移酶3家族同源保守区域的多靶点siRNA,体内、外评价同时沉默DNMT3对肾细胞癌增殖、侵袭能力的影响。方法:基于DNMT3A/3B的同源保守区域,设计制作针对二者同源区域的2对siRNA序列(si DNMT3A/B-1及si DNMT3A/B-2),以Western印迹法验证沉默效率。转染肾细胞癌786-0细胞系后,分别以体外、体内实验验证其对于肾细胞癌增殖、侵袭能力的影响。结果:针对DNMT3A/B同源保守区siRNA序列,能够显著降低DNMT3A及3B的蛋白表达,并可以显著降低786-0细胞划痕迁移、增殖、侵袭能力,其侵袭、增殖关键基因(Ki-67、Vimentin、VEGF、CD31、MMP2)表达显著下调。体内研究证实,敲除组小鼠皮下瘤生长速度显著降低伴总体生存率显著延长(P<0.05),且皮下瘤内侵袭、增殖关键基因表达显著下调(P<0.05)。结论:基于DNMT3同源保守区域可获得多靶点有效siRNA;靶向下调肾细胞癌786-0细胞系DNMT3A及3B表达能够显著降低细胞增殖、侵袭能力;靶向DNMT3是肾细胞癌治疗的潜在靶点。

Objective：Designing and synthesis the multi - targeting siRNA based on the homology region of DN-MT3A and 3B. Evaluating the effect of DNMT3A/ B knockout on cell progression and proliferation in RCC cell line. Methods：Two multi - targeting siRNA were designed and synthesized based on the homology region of DNMT3A and 3B. DNMT3A and 3B expression was detected by Western blot after transfection for 48 hours. The in vitro potency of invasion,proliferation,immigration and related gene expression were analyzed by wound healing assay,MTT assay,in-vasion assay,migration assay and real - time PCR assay. The in vivo proliferation and survival ability were detected by xenograft tumor model. Results：Transfection of siDNMT3A/ B - 1 and siDNMT3A/ B - 1 in 786 - 0 cell significant knockdown the expression of DNMT3A and 3B at the same time. The silencing of DNMT3A and 3B significantly sup-press the potency and expression of invasion,migration and progression related genes（ Ki - 67,Vimentin,VEGF, CD31,MMP2）by in vitro and in vivo assays,and also prolong xenograft mice survival rate. Conclusion：The multi -targeting siRNA suppressing the cell proliferation and progression abilities. DNMT3A/ B targeting therapy maybe the novel approach for renal cell carcinoma treatment.