Yan Gangli, Li Binbin, Xin Xuan, Xu Midie, Ji Guoqing, Yu Hongyu
Department of Pathology, Changzheng Hospital, The Second Military Medical University, Shanghai, China (mainland).
Department of Pathology, Changzheng Hospital,the Second Military Medical University, Shanghai, China (mainland).
Med Sci Monit. 2015 Oct 6;21:3008-15. doi: 10.12659/MSM.894000.
The incidence of liver fibrosis remains high due to the lack of effective therapies. Our previous work found that microRNA (miR)-34a expression was increased, while acy1-CoA synthetase long-chain family member1 (ACSL1) was decreased, in a dimethylnitrosamine (DNS)-induced hepatic fibrosis rat model. We hypothesized that miR-34a may play a role in the process of hepatic fibrosis by targeting ACSL1.
From days 2 to 14, cultured primary hepatic stellate cells (HSCs) underwent cell morphology, immunocytochemical staining, and quantitative reverse transcription PCR (RT-qPCR) for alpha smooth muscle actin (a-SMA), desmin, rno-miR-34a, and ACSL1 expression. Wild-type and mutant luciferase reporter plasmids were constructed according to the predicted miR-34a binding site on the 3'-untranslated region (UTR) of the ACSL1 mRNA and then transfected into HEK293 cells. rno-miR-34a was silenced in HSCs to confirm that rno-miR-34a negatively regulates ACSL1 expression. mRNA and protein expression of α-SMA, type I collagen, and desmin were assayed in miR-34a-silenced HSCs.
HSCs were deemed quiescent during the first 3 days and activated after 10 days. rno-miR-34a expression increased, and ACSL1 expression decreased, from day 2 to 7 to 14. rno-miR-34a was shown to specifically bind to the 3'-UTR of ACSL1. miR-34a-silenced HSCs showed higher ACSL1and lower α-SMA, type I collagen, and desmin expression than that of matching negative controls and non-transfected cells.
miR-34a appears to play an important role in the process of liver fibrosis by targeting ACSL1 and may show promise as a therapeutic molecular target for hepatic fibrosis.
由于缺乏有效的治疗方法，肝纤维化的发病率仍然很高。我们之前的研究发现，在二甲基亚硝胺（DNS）诱导的肝纤维化大鼠模型中，微小RNA（miR）-34a表达增加，而酰基辅酶A合成酶长链家族成员1（ACSL1）表达降低。我们推测miR-34a可能通过靶向ACSL1在肝纤维化过程中发挥作用。
在第2天至第14天，对培养的原代肝星状细胞（HSCs）进行细胞形态学、免疫细胞化学染色，并对α平滑肌肌动蛋白（α-SMA）、结蛋白、rno-miR-34a和ACSL1表达进行定量逆转录PCR（RT-qPCR）。根据预测的miR-34a与ACSL1 mRNA 3'-非翻译区（UTR）的结合位点构建野生型和突变型荧光素酶报告质粒，然后转染到HEK293细胞中。在HSCs中沉默rno-miR-34a以证实rno-miR-34a负向调节ACSL1表达。检测miR-34a沉默的HSCs中α-SMA、I型胶原蛋白和结蛋白的mRNA和蛋白表达。
HSCs在最初3天被认为是静止的，10天后被激活。从第2天到第7天再到第14天，rno-miR-34a表达增加，ACSL1表达降低。rno-miR-34a被证明特异性结合ACSL1的3'-UTR。与匹配的阴性对照和未转染细胞相比，miR-34a沉默的HSCs显示出更高的ACSL1表达和更低的α-SMA、I型胶原蛋白和结蛋白表达。
miR-34a似乎通过靶向ACSL1在肝纤维化过程中发挥重要作用，并且可能有望成为肝纤维化的治疗分子靶点。
