长链非编码RNA肝癌高表达转录本促进人骨髓间充质干细胞向肝样细胞的分化

Long-chain non-coding RNA highly up-regulated in liver cancer promotes the differentiation of human bone marrow mesenchymal stem cells into hepatocytes

背景:骨髓间充质干细胞在体外可以被诱导分化为肝样细胞,但是细胞功能不成熟。研究表明,长链非编码RNA可以影响间充质干细胞的分化,但是关于长链非编码RNA肝癌高表达转录本是否能够参与骨髓间充质干细胞向肝细胞分化还未有报道。目的:探讨长链非编码RNA肝癌高表达转录本在人骨髓间充质干细胞向肝样细胞分化中的作用。方法:体外添加细胞因子诱导人骨髓间充质干细胞(购自上海中科院细胞库)向肝样细胞分化,诱导4周后倒置显微镜观察细胞的形态学变化,免疫荧光技术检测肝细胞特异性蛋白的表达,qRT-PCR检测分化过程中肝癌高表达转录本的表达;然后通过慢病毒转染构建稳定表达肝癌高表达转录本的人骨髓间充质干细胞株,以转染阴性对照病毒作为对照组,两组同时添加细胞因子进行肝向细胞诱导分化,qRT-PCR、Western blot检测过表达组与对照组肝细胞特异性基因、蛋白表达的差异。结果与结论:①人骨髓间充质干细胞在体外添加细胞因子下可以成功被诱导分化为肝样细胞,在分化过程中肝癌高表达转录本的表达逐渐增高;②过表达肝癌高表达转录本后促进了人骨髓间充质干细胞诱导分化后肝细胞特异性基因及蛋白的表达;③结果表明,肝癌高表达转录本在人骨髓间充质干细胞向肝样细胞分化中发挥着至关重要的作用,将为细胞治疗肝脏疾病奠定坚实的理论基础。

BACKGROUND:Human bone marrow mesenchymal stem cells can be induced to differentiate into hapatocytes without mature function.Long-chain non-coding RNA(lncRNA)plays an important part in the differentiation of mesenchymal stem cells.To date,there is still no report on whether the lncRNA highly up-regulated in liver cancer(HULC)is involved in the differentiation of bone marrow mesenchymal stem cells into hepatocytes.OBJECTIVE:To investigate the mechanism of lncRNA HULC in the differentiation of human bone marrow mesenchymal stem cells into hepatocyte-like cells.METHODS:Human bone marrow mesenchymal stem cells were purchased from the Cell Bank of Chinese Academy of Sciences,Shanghai,China and were cultured in the hepatic differentiation medium containing cytokines in vitro.After 4 weeks of induction,cell morphology was observed under inverted microscope.Hepatocyte specific proteins were analyzed by immunofluorescence.The expression of lncRNA HULC was examined by qRT-PCR.Bone marrow mesenchymal cell lines stably expressing lncRNA HULC were then produced through lentiviral transfection.Bone marrow mesenchymal stem cells transfected with negative lentivirus were used as controls.Two groups of cells were concurrently induced with the hepatic differentiation medium containing cytokines.The expressions of hepatocyte specific genes and proteins were detected and compared between the two groups by qRT-PCR and western blot,respectively.RESULTS AND CONCLUSION:Human bone marrow mesenchymal stem cells could be successfully induced into hepatocytes in vitro through the addition of cytokines.The expression of lncRNA HULC was gradually increased during the differentiation.Overexpression of lncRNA HULC caused a marked increase in the expression of hepatocyte specific genes and proteins.To conclude,lncRNA HULC plays an important role in the differentiation of human bone marrow mesenchymal stem cells into hepatocytes.It will provide a theoretical basis for the application of cell therapy in liver diseases.