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微小RNA 125b(miR-125b)在许多恶性肿瘤的增殖、分化和凋亡等过程中具有很重要的作用,但miR-125b是否涉及肝癌的上皮-间质转换过程(EMT)还有待进一步研究。本研究通过构建过表达miR-125b的肝癌稳转细胞株,初步检测miR-125b对于肝癌的EMT过程和相关的TGF-β信号通路的影响,以及对于肝癌细胞凋亡的影响。以慢病毒载体p HRS-1cla-EGFP构建过表达miR-125b的载体质粒(p HRS-1cla-miR125b-CMV-EGFP),并对上述载体进行NheⅠ、XbaⅠ双酶切和测序鉴定,鉴定正确后,在293T细胞中进行慢病毒包装,浓缩病毒后,对MHCC97-H进行慢病毒感染并采用流式分选GFP阳性的细胞。实时定量PCR检测表明肝癌细胞稳转株MHCC97-H-PHRSmiR-125b-EGFP的miR-125b表达量是空载体转染组的6倍。Western印迹检测发现,与空载体对照组相比,MHCC97-H-PHRS-miR-125b-EGFP细胞中间质细胞标志α-SMA表达显著下调,上皮细胞标志E-cadherin表达显著上调,同样的,用Western印迹检测也发现MHCC97-H-PHRS-miR-125b-EGFP细胞中TGF-β信号通路关键下游分子Smad2和Smad4的表达显著下调,细胞凋亡检测结果表明,与对照组相比,过表达miR-125b的稳转株凋亡率增加到19.66%,加入TGF-β1后,过表达miR-125b的稳转株凋亡率进一步增加到74.7%。同样的,在体内治疗实验中,我们采用商品化的体内核酸转染试剂,在皮下肿瘤组织中过表达miR-125b mimics,结果表明miR-125b的过表达与肿瘤组织的凋亡成正相关性(r=0.83463,P<0.01),且免疫组化结果也表明,miR-125b过表达后,E-cadherin表达显著上调,α-SMA及Smad2和Smad4的表达显著下调。上述结果表明,我们成功构建了过表达miR-125b的肝癌细胞稳转株,并成功建立了肿瘤组织中过表达miR-125b mimics的动物模型,在体内外均观察到过表达miR-125b后对肝癌细胞EMT过程的抑制作用和对细胞凋亡的促进作用。相关研究结果加深了我们对miR-125b在肝癌中抑制肝癌发展作用机制的理解,及其作为潜在的治疗肝癌的新靶点的重要性。
MicroRNA 125b (miR-125b) plays an important role in the proliferation, differentiation and apoptosis of many malignant tumors. Whether miR-125b is involved in epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma remains to be further studied. In this study, miR-125b-overexpressing hepatocellular carcinoma cell line was constructed to detect the effect of miR-125b on the EMT process and TGF-β signaling pathway in hepatocellular carcinoma and its effect on hepatocellular carcinoma cell apoptosis. The vector (HRS-1cla-miR125b-CMV-EGFP) overexpressing miR-125b was constructed with the lentiviral vector p HRS-1cla-EGFP. The vector was identified by Nhe I and Xba I digestion and sequencing. , Lentiviral packaging in 293T cells, and after concentration of the virus, Lentiviral infection of MHCC97-H was performed and GFP positive cells were sorted by flow cytometry. Real-time quantitative PCR showed that the expression level of miR-125b in MHCC97-H-PHRSmiR-125b-EGFP hepatocellular carcinoma cells was 6-fold higher than that in the vector-transfected group. Western blotting showed that the expression of α-SMA in MHCC97-H-PHRS-miR-125b-EGFP cells was significantly down-regulated and the expression of E-cadherin in epithelial cells was significantly up-regulated compared with the control group. Similarly, The expression of Smad2 and Smad4, a key downstream molecule of TGF-β signaling pathway, was significantly down-regulated in MHCC97-H-PHRS-miR-125b-EGFP cells by Western blot. The results of apoptosis assay showed that compared with the control group, The apoptosis rate of miR-125b stable metastasis increased to 19.66%. After addition of TGF-β1, the apoptosis rate of stable transgenic cells over-expressing miR-125b further increased to 74.7%. Similarly, in in vivo treatment experiments, we overexpress miR-125b mimics in subcutaneous tumor tissues using commercial in vivo nucleic acid transfection reagents, indicating that miR-125b overexpression correlates positively with tumor tissue apoptosis ( r = 0.83463, P <0.01). The results of immunohistochemistry also showed that the expression of E-cadherin was up-regulated and the expressions of α-SMA, Smad2 and Smad4 were down-regulated when miR-125b was overexpressed. The above results indicate that we successfully constructed stable hepatoma cells overexpressing miR-125b and successfully established an animal model of overexpression of miR-125b mimics in tumor tissue. The overexpression of miR-125b was observed in vivo and in vitro Inhibition of EMT in hepatocellular carcinoma cells and its role in promoting apoptosis. Our findings reinforce our understanding of the mechanisms by which miR-125b inhibits the development of hepatocellular carcinoma in hepatocellular carcinoma and its potential as a potential new target for hepatocellular carcinoma.