Circ-CL5A1受TDP-43调控并抑制肝细胞癌转移的研究

Circ-COL5A1 is Regulated by TDP-43 and Inhibit HCC Metastasis

目的:探索Circ-COL5A1的生物学功能、调控机制和作用机制,进而为HCC转移的干预提供候选分子并进一步了解HCC转移。方法:通过前期工作基础选定目标分子Circ-COL5A1。通过慢病毒转染在HCC细胞系中过表达Circ-COL5A1,进而通过划痕愈合实验、transwell实验观察Circ-COL5A1的生物学功能。通过生物信息学分析、表达干扰实验和RNA免疫共沉淀(RIP)实验探究目标分子的调控机制。通过western blot技术、实时定量PCR(qRT-PCR)技术对目标分子的下游作用机制进行初步探索。结果:Circ-COL5A1在肝癌干细胞中表达下调,而且Circ-COL5A1过表达的HCC细胞系侵袭和迁移能力减弱。在Circ-COL5A1生物学合成过程中,RNA结合蛋白TDP-43可以富集其线性前体,并在环化结构形成后解离。Circ-COL5A1还可以降低其亲本基因V型胶原蛋白α1链(COL5A1)的蛋白质表达水平,这可能会影响多个信号通路进而干预HCC的转移过程。结论:内源性的Circ-COL5A1可以抑制HCC的转移能力,可以为阻断HCC转移提供候选分子。TDP-43的促进环状RNA形成提示RNA结合蛋白是环状RNA生物学合成过程中的重要调控因子。Circ-COL5A1可以通过转录后调控抑制其亲本基因COL5A1的表达。

Objective:To explore the biological function,mechanism and regulation factor of circ-COL5A1,providing candidate molecules for the intervention and further understanding of HCC metastasis.Methods:The target molecule Circ-COL5A1 were selected based on the preliminary work.The biological function of circ-COL5A1 is verified through transwell assay and wound healing assay.Bioinformatic analysis,gene expression interference and RNA immunoprecipitation(RIP)experiments were employed to explore the regulation mechanism of circ-COL5A1.Western blot and quantitative real-time PCR(qRT-PCR)were employed to explore the downstream pathway of circ-COL5A1.Result:The expression of Circ-COL5A1 is down-regulated in HCC cancer stem cells.And Circ-COL5A1 overexpressed inhibit the invasion and migration ability in Huh7 and HCC-LM3 cell lines.The result of RIP experiments showed that RNA-binding protein(RBP)TDP-43 can enrich the linear precursor and dissociate after the formation of the circular structure.Circ-COL5A1 can also reduce the protein expression of its maternal gene COL51,which may affect multiple signaling pathways and thus interfere with the metastasis process of HCC.Conclusion:Endogenous Circ-COL5A1 can inhibit the metastasis ability of HCC cell lines,and may provide candidate molecules for blocking HCC metastasis.TDP-43 promotes the biogenesis of Circ-COL5A1,suggesting that RBPs are important regulators in the process of circRNA biogenesis.Circ-COL5A1 inhibits protein expression of its maternal genes COL5A1 through post-transcription regulation.