circRNA_1989竞争性结合miR-185-3p调控SCARB1促进HSC活化/肝纤维化的机制研究

The mechanism of occurrence and development of liver fibrosis needs to be deeply elucidated. Previously we detected gene expression profiles in clinical liver fibrosis tissues by using high-throughput technique and verified them by qRT-PCR , and found that: as compared with human normal hepatic tissue, circRNA_1989 and SCARB1 were significantly upregulated, and miR-185-3p was downregulated in liver fibrosis samples; Bioinformatics analysis predicted that there are the binding sites complementary to miR-185-3p both in circRNA_1989 and in SCARB1. CircRNA_1989 is upregulated during primary culture HSC activation; and knockdown of circRNA_1989 increased the expression of miR-185-3p, decreased SCARB1 and collagen；downregulation of SCARB1 inhibited the HSC proliferation and increased the amount of VA in HSC. Therefore, we speculated that circRNA_1989 regulates hepatic fibrosis by competitive binding with miR-185-3p, impairing its inhibitory effect on SCARB1 expression, and then promoting HSC activation/liver fibrosis. In present project, we will clarify the function of circRNA_1989 in hepatic fibrosis and elucidate the roles of circRNA_1989/miR-185-3p/SCARB1 regulation axis by using the molecular biologic and other methods and making in vitro and in vivo functional experiments and other experiments; and analyze the potential value for clinical research/application of related molecules by RNA-scope and other technologies, which will shed a new light on clinical prevention/treatment study on liver fibrosis.

肝纤维化发生发展机制亟待深入阐明。本组前期高通量基因检测、验证发现与人正常肝相比肝纤维化组织circRNA_1989和SCARB1表达上调、miR-185-3p下调；生物信息预测circRNA_1989和SCARB1序列均存在miR-185-3p的结合位点；原代HSC活化后circRNA_1989上调；下调HSC circRNA_1989后miR-185-3p表达升高、SCARB1和胶原下降；下调SCARB1后HSC增殖受抑等。推测circRNA_1989经竞争性结合miR-185-3p、削弱对SCARB1表达的抑制、促HSC活化/肝纤维化。现拟利用分子生物学等手段和体内外功能实验明确circRNA_1989在肝纤维化中的功能、阐明 circRNA_1989/miR-185-3p/SCARB1调控轴在肝纤维化进程中的作用机制；并检测和分析相关分子临床转化价值。为肝纤维化防治研究增添新基础。

肝纤维化是多种慢性肝病的共同病理过程，但目前缺乏针对于肝纤维化诊疗的有效无创性手段，提示有必要对肝纤维化发生发展的分子机制进行深入研究。 . 本课题前期RNAseq测序发现：circRNA_1989 在临床肝纤维化样本中高表达。课题组进一步在22对人肝纤维化组织样本和TGF-β1激活的LX-2细胞中证实circRNA_1989均表达上调；构建Cy3-labeled-circRNA_1989探针，进行荧光原位免疫杂交，发现circRNA_1989在肝纤维化区域高表达。Sanger测序及RnaseR消化实验证明了circRNA_1989的环状结构。后构建了circRNA_1989干扰的LX-2稳定株，CCK-8实验、AnnexinV-APC等及细胞功能实验，证实circRNA_1989通过促进HSC活化/抑制其凋亡而具有促进肝纤维化形成的作用。. 荧光素酶报告实验、AGO2-RIP及RNA pulldown实验证明miR-185-3p与circRNA_1989具有直接结合关系。在TargetScan数据网站，预测到SCARB1、Col1a1等为miR-185-3p靶基因，而我们实验中发现Col1a1在miR-185-3p mimic转染后，下调最明显。故我们后续实验选择Col1a1作为其关键性靶基因进行下游验证，荧光素酶报告实验证实Col1a1是miR-185-3p的直接靶标，实验证实miR-185-3p inhibitor可逆转circRNA_1989敲低的LX-2细胞中Col1a1水平，进一步说明circRNA_1989对Col1a1的调节作用取决于其与miR-185-3p的相互作用。也很好地证明了circRNA_1989/miR-185-3p/Col1a1轴在HSC活化中的调控作用。circRNA_1989可能是肝纤维化的有效的治疗靶标。