致瘤性转化过程中HRG的转录调控及其抑肝癌效应的分子机制研究

Based on previous research, the expression of HRG was significantly lower in HCC tissues by immunohistochemical and Western blot analysis. HRG overexpressed Huh7 and MHCC-97H hepatoma cell line showed decreases in cell proliferation, colony forming ability and orthotopic tumor formation, meanwhile an increase in cell apoptosis. HRG could competitively bind to HS with bFGF and inhibit ERK1/2 phosphorylation in FGF-ERK1/2 signaling pathway. We explored HRG function during HCC tumorigenesis in WB F344 hepatic oval-like cells. The expression of HRG decreased gradually in neoplastic transformation, which suggested HRG may act as a potential tumor suppressor and an essential factor in neoplastic transformation. We design this project to clarify the inhibitory effect of HRG in neoplastic transformation, by HRG knockout in WB-F344 with Crispr-Cas9 and neoplastic transformed by MNNG. The transcriptional regulation of HRG will be analyzed by EMSA, Chip-qPCR, luciferase reporter assay, and pyrosequencing. Proliferation, apoptosis, invasion and other behavior changes in cells will be monitored in HRG overexpressed and knock-downed transformed WB-F344 cells. RNA sequencing will be used to analyze the transcriptome level changes correspondent to HRG up- and down- regulation. Validation of the HRG regulatory network and its underlying mechanism will be performed through western blot, immunofluorescence, and qPCR.

课题组前期研究发现，富组氨酸糖蛋白（HRG）在肝癌组织中低表达，HRG过表达能抑制Huh7、MHCC-97H的细胞增殖、软琼脂克隆形成、原位瘤生长、促进细胞凋亡；HRG与bFGF竞争性结合硫酸乙酰肝素抑制FGF-ERK1/2通路的磷酸化；用WB-F344细胞探索HRG在肝癌发生中的作用，发现其在细胞致瘤性转化中表达下降，提示其可能是抑癌因子，在致瘤性转化中发挥重要作用。本研究拟用Crispr-Cas9敲除WB-F344的HRG基因，亚硝基胍诱导致瘤性转化，确认其在致瘤性转化中的抑癌效果；EMSA、Chip-qPCR、双荧光素酶报告法、焦磷酸测序等解析HRG的表达调控特点及方式；在转化的癌细胞中过表达/敲除HRG，检测细胞增殖、凋亡、侵袭等细胞行为学变化；RNA测序检测HRG改变引起的转录组变化，分析HRG相关调控网络，用免疫印迹、细胞免疫荧光、qPCR等技术进行验证，阐明其调控机制。

课题组前期研究发现，富组氨酸糖蛋白（HRG）在肝癌组织中低表达，HRG过表达能抑制Huh7、MHCC-97H的细胞增殖、软琼脂克隆形成、原位瘤生长、促进细胞凋亡；HRG与bFGF竞争性结合硫酸乙酰肝素抑制FGF-ERK1/2通路的磷酸化；用WB-F344细胞探索HRG在肝癌发生中的作用，发现其在细胞致瘤性转化中表达下降。本研究通过CRISPR-Cas9敲除WB-F344的HRG基因，亚硝基胍诱导致瘤性转化，验证HRG为肿瘤抑制因子；EMSA、Chip-qPCR、双荧光素酶报告法、焦磷酸测序等解析HRG的核心转录区域，调控HRG表达的转录因子，及甲基化修饰对HRG表达调控的影响；细胞功能试验表明HRG表达变化后可影响细胞增殖、凋亡、侵袭等细胞行为学行为；通过RNA-seq解析HRG调控网络，其中表达97个基因表达上调，192个基因表达下调。选取差异表达基因RAD52、MCM6进行验证及进一步研究，RAD52、MCM6进在肝癌具有潜在的临床诊断意义。本研究为深入理解HRG在肝癌发生中的作用及其机制，为临床诊断及寻找药物治疗靶点提供了新的理论依据。