过表达微小RNA miR-125b抑制肝癌细胞上皮-间质转换及促进细胞凋亡

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pHRS-1cla EGFP 构建过表达miR-125b的载体质粒(pHRS-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-PHRS-miR-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。

Overexpression of MiR-125b Inhibits Epithelial-Mesenchymal Transition and Promote Cell Apoptosis in Hepatocellular Carcinoma

MircroRNA-125b(miR-125b) has an important role in processes of malignant neoplasm, including cell proliferation, differentiation, and apoptosis. However, less is known for the involvement of miR-125b in epithelial-mesenchymal transformation(EMT) in hepatocellular carcinoma(HCC). This study aims to exploit the effects of miR-125b overexpression on EMT regulated by TGF-β signaling pathway. A miR-125b overexpression vector pHRS-1cla-miR125b-CMV-EGFP was constructed into pHRS-1cla-EGFP lentivirus vector by double digestion with NheI and XbaI. MHCC97-H cells were infected with viruses; and GFP-positive cells were collected from a flow cytometry sorting. HCC cell stably transfected with MHCC97-H-PHRS-miR-125b-EGFP was established. The qRT-PCR results showed that the miR-125b expression stable cells was nearly 6 fold than that of the control. Western blot showed that the expression of mesenchymal cell marker α-SMA of MHCC97-H-PHRS-miR-125b-EGFP was significantly down-regulated; while the expression of epithelial cell marker E-cadherin was upregulated. The activity of TGF-β signaling pathway was decreased with down-regulated expression of Smad2 and Smad4. In apoptosis assay, a 19.66% increase in apoptosis rate was detected in MHCC97-H-PHRS-miR-125b-EGFP cells. An additional increase to 74.7% was shown after TGF-β1 treatment. In vivo experiment results showed that transfection of miR-125b mimics in mice subcutaneous tumor tissues with a positive correlation between miR-125b expression and apoptosis rate of tumor tissues(r=0.83463，P<0.01). Immunohistochemistry showed that E-cadherin was significantly up-regulated; while α-SMA, Smad2 and Smad4 were down-regulated.