circRNATUBD1作为miR-146a-5p海绵参与调控TLR4信号通路影响放射性肝病发生的分子机制研究

MiR-146a-5p negatively regulates TLR4 pathway in hepatic stellate cell (HSC) to influence the development of radiation-induced liver disease (RILD), but the upstream regulation mechanism remains unclear. Preliminary study reveals that circRNA TUBD1(circTUBD1，hsa_circ_0044897) with miR-146a-5p binding site is significantly up-regulated and correlated with the changes of TLR4 pathway molecules in irradiated HSC, thus we speculate that circTUBD1 may be involved in the regulation of TLR4 signaling pathway to participate in the development of RILD through competitive binding of miR-146a-5p. This project is intended to use the in-vitro and in-vivo animal experiments in combination with the clinical validation to investigate the mechanism that circTUBD1 serving as the miR-146a-5p sponge involved in competing endogenous RNA regulation pattern to affect the function of HSC, TLR4 signaling pathway, hepatic micro-environment and radiation sensitivity of hepatocyte, by use of RNA antisense purification-quantitative real-time PCR, immuneprecipitation of AGO2, gene overexpression or interference. This project may offer a new perspective to understand the mechanism as well as providing the preventative and therapeutic strategies for RILD.

miR-146a-5p负向调控肝星状细胞(HSC)TLR4通路，影响放射性肝病(RILD)发生发展，但其上游调控机制仍不清楚。前期实验显示，与miR-146a-5p有结合位点的环状RNA TUBD1(circTUBD1，hsa_circ_0044897)在接受照射的HSC中表达明显升高，且其表达变化影响TLR4通路分子的表达，故我们推测circTUBD1可能通过竞争性结合miR-146a-5p参与调控TLR4信号通路，从而影响RILD的发生发展。本项目拟采用体内外实验与临床验证相结合，应用RAP-qRT-PCR、AGO2免疫共沉淀、基因过表达或干扰等实验技术，探讨circTUBD1作为miR-146a-5p海绵，参与竞争性内源RNA调控模式，影响肝星状细胞功能、TLR4信号通路、肝微环境和肝细胞放射敏感性的机制，为RILD发病机制的研究开拓思路、提供防治策略。

辐射诱导的肝损伤（radiation-induced liver disease,RILD）一直是肝脏恶性肿瘤放射治疗后的主要障碍。CircRNA在RILD发展中的功能和分子机制仍然很大程度上未知。本研究旨在探讨circTUBD1在RILD发展中的潜在作用和机制。我们的研究首次揭示了circTUBD1作为miR-146a-5p海绵通过TLR4通路影响LX-2细胞的活力和促炎细胞因子的产生。沉默circTUBD1抑制增殖，促进LX-2细胞凋亡，并显著降低辐射和LPS刺激的LX-2细胞中促炎细胞因子的表达水平，包括IL-1β、IL-6和TNF-α。机制分析表明circTUBD1作为miR-146a-5p海绵，通过调节Toll样受体4(TLR4)、白细胞介素受体相关激酶1(IRAK1)、肿瘤坏死因子相关受体(TRAF6)、核因子-kappaB(pNF-κB)的表达来影响促炎细胞因子的产生。此外，在三维(3D)培养系统和体内模型中，发现circTUBD1的敲低减轻了辐射诱导的早期肝纤维化通过circTUBD1/micro-203a-3p/Smad3反馈回路。敲低circTUBD1不仅降低了作为LX-2细胞活化标志物的α-SMA的表达，而且显着降低了肝纤维化分子（I 型胶原蛋白α1[COL1A1]、胶原蛋白型III alpha1[COL3A1]和结缔组织生长因子[CTGF]）。本研究为缓解RILD进展提供了一种新的治疗策略。