miR-21和miR-130b靶向性人工设计circRNA抑制肝癌细胞上皮间质转化

mi R-21 and miR-130b Targeted Artificial Modification of circRNA Can Inhibit the EpithelialMesenchymal Transition of Hepatocellular Carcinoma Cells

目的探讨人工改造的CDR1as(M-CDR1as)是否可通过特异性结合miR-21和miR-130b,体外抑制小鼠Hepa1-6肝癌细胞的上皮间质转化(EMT)。方法将CDR1as中miR-7结合位点替换为特异性结合mmu-miR-21或mmu-miR-130b的碱基序列。将小鼠Hepa1-6肝癌细胞分为对照组,空质粒组(NC)和M-CDR1as组。采用RT-qPCR检测Hepa1-6细胞内M-CDR1as的水平及其对靶miR-21和miR-130b水平的影响。利用RT-qPCR和Western blot法检测各组肝癌细胞中EMT相关Vimentin,E-cadherin及N-cadherin的mRNA水平和蛋白表达量。结果仅M-CDR1as组可在阈值以上检测到M-CDR1as。M-CDR1as组miR-21水平较对照组和NC组有所下降(P>0.05),M-CDR1as组miR-130b水平较对照组及NC组均明显下调(P<0.001)。Vimentin和N-cadherin mRN水平较对照组和NC组明显下降(P<0.01),E-cadherin mRNA较对照组和NC组明显升高(P<0.01)。Vimentin和N-cadherin蛋白表达量较对照组和NC组明显下降(P<0.01);而E-cadherin较对照组和NC组表达明显升高(P<0.01)。结论 M-CDR1as可通过结合吸附miR-21和miR-130b体外抑制小鼠Hepa1-6肝癌细胞的EMT。

Objective To explore if the artificial modification of CDR1as(M-CDR1as)can specifically binding miR-21and miR-130b,thereby inhibit the epithelial-mesenchymal transition(EMT)in Hepal-6.Methods The binding site of miR-7in CDR1as was substituted by specific binding sequence of mmu-miR-21or mmu-miR-130b.The mice Hepal-6liver cancer cells were divided into control group,null vector transfection group(NC)and M-CDR1as group.The level of M-CDR1as as well as its effect on miR-21and miR-130b were detected by RT-qPCR.Besides,the expression protein/mRNA level of EMT-related Vimentin,E-cadherin and N-cadherin in liver cancer cells were detected by RT-qPCR and Western blot.Results Only the M-cdr1as group could detect M-cdr1as above the threshold.Mir-21level in the M-cdr1as group was lower than that in the control group and NC group(P>0.05).Besides,the miR-130b level in the M-CDR1as group were significantly decreased than that of control and NC group(P<0.001).The mRNA levels of Vimentin and N-cadherin were significantly lower than those of the control group and NC group(P<0.01).The mRNA of E-cadherin was significantly higher than that of control group and NC group(P<0.01).Both the Vimentin and N-cadherin protein expression levels were significantly lower than those of the control group and NC group(P<0.01).While the expression of E-cadherin was significantly higher than that of the control group and NC group(P<0.01).Conclusion M-cdr1as can inhibit EMT of hepa1-6hepatocellular carcinoma cells of mice in vitro by binding and adsorbing mir-21and mir-130b.