4种共表达microRNA协同调节大鼠肝细胞增殖和肝再生的分子机制研究

Rat liver regeneration (LR) involves cell proliferation and apoptosis, and is regulated by microRNA.Our previous study showed that miR-182 promoted rat hepatocyte proliferation and LR, and the trend of the relative expression of miR-125a, miR-183, miR-199a and miR-429 in rat LR were in accordance with that of miR-182, and the five miRNAs share common target genes. Therefore, we speculated that they might synergistically regulate rat primary cultured hepatocyte proliferation and rat LR. To verify the speculation, we will firstly study the role of the mimic and inhibitor of four microRNAs in rat primary cultured hepatocyte. And then we will up-regulate and down-regulate the expression of four microRNAs in vitro and in vivo by gene addition and RNA interference, and detect the effect of these interventions on the expression changes of the target genes, and on the expression changes of genes and proteins related to hepatocytes proliferation and apoptosis, and on hepatocytes proliferation and apotosis and liver regeneration. Finally, we will parse the ways and means of four microRNAs in regulating the hepatocytes proliferation and LR by network principle and method, which may provide a theoretical basis for revealing the mechanism of rat LR and establishing the prevention and treatment strategies of liver diseases.

研究表明，大鼠肝再生涉及细胞增殖和凋亡，受microRNA等调节。本课题组研究发现，大鼠肝再生中 miR-125a、miR-183、miR-199a和miR-429等4种microRNAs与促进大鼠肝细胞增殖和肝再生的miR-182表达趋势一致，且它们有共同的靶基因，因此推测，它们可能通过协同方式调控肝细胞增殖和肝再生。为验证上述推测，本项目拟首先检验它们的模拟物和抑制物对体外培养的大鼠（原代）肝细胞增殖的调节作用。然后从体外和体内两个方面、通过基因添加和干涉等方法上调和下调4种microRNA表达，检测这些干预对靶基因、对肝细胞增殖/凋亡相关基因和蛋白表达、对肝细胞增殖/凋亡、对再生肝生长等的影响，用网络原理和方法解析它们调控肝细胞增殖和肝再生的途径和方式，为揭示肝再生机制、建立治疗和预防肝病方法奠定基础。

肝脏是机体的重要解毒器官，在受到物理、化学、生物等因素损伤后，通过肝再生促进肝细胞的增殖来补偿损伤的肝组织和恢复肝脏的生理功能。miRNA是一类长度21-24nt的小分子RNAs，成熟的miRNA通过与靶mRNA互补配对结合引起后者降解和/或抑制后者翻译，起到转录后调控蛋白表达的作用。miRNA调控多种生物学过程，包括细胞分化、增殖、凋亡、代谢等，但对肝再生及肝细胞增殖的调控机制尚不完全清楚。为此，本项目拟进一步研究4种共表达microRNA协同调节大鼠肝细胞增殖和肝再生的分子机制，本研究首先进行了四种miRNA mimic 和inhibitor的合成，然后用MTT法检测miR-429、miR-199a、miR-125a和miR-183四种miRNA对大鼠肝细胞BRL-3A活性的影响，用流式细胞术检测四种miRNA对大鼠肝细胞BRL-3A周期的影响，结果发现：miR-429和miR-125a抑制BRL-3A细胞的增殖，miR-183和miR-199a促进BRL-3A的细胞的增殖；用FITC/PI双染法检测细胞凋亡，结果表明： miR-429和m iR-125a具有显著促进BRL-3A细胞凋亡的作用， miR-183和miR-199a 具有抑制BRL-3A细胞凋亡的作用；利用qRT-PCR和western blot方法分析靶基因下游共同的信号分子在其共同过表达和下调表达后的大鼠肝细胞BRL-3A中的表达。miRNA-429和miR-125a可能通过JUN/MYC/BCL2/CCND1信号传导途径协同调控大鼠肝细胞BRL-3A的增殖。本研究为miRNA协同调控肝细胞增殖作用奠定了基础。