miR-122在肝癌细胞TLR4介导的天然免疫中的作用及机制研究

miR-122 is most abundant in liver and extensively involved in hepatitis virus infection and apoptosis of hepatoma cells. Toll-like receptor 4(TLR4) is expressed in a varity of tumors, and TLR4 activation can promote the apoptosis resistance and invasion of tumors. Using RNAhybrid software we found the predicted binding of complementary sequences between miR-122 and TLR4 mRNAs. Over-expression of miR-122 experiment demonstrated that miR-122 could specifically inhibit TLR4 transcription, may be important for the involvment of miR-122 on the host innate immunity mediated by TLR4. Therefore, the subject intends to identify the correlation from three aspects: (1) correlation studies between miR-122 and TLR4 expression, (2) confirmation of the targets of miR-122 in TLR4 mRNA, (3) influence of miR-122 over-expression/down-expression on the expression of natural immune factors of TLR4 downstream and the hepatoma cells characteristics. Based on the results, we aim to confirm that miR-122 might regulate host innate immunity by adjusting the TLR4 expression to participate in the occurrence of hepatoma development process, which reveals a new molecular mechanism of miR-122 on the regulation of innate immunity. This study provides basis not only for research of miRNA and natural immune but also for the study of miRNA and neoplasm occurs.

miR-122是肝细胞中表达最丰富的一类小分子RNA，参与肝炎病毒感染及肝癌细胞凋亡。Toll样受体4（TLR4）能识别肝炎病毒，参与免疫防御及肿瘤的免疫逃逸。我们前期研究发现miR-122可与TLR4靶向结合，且可以抑制TLR4 mRNA的表达，推测其可能参与TLR4介导的天然免疫过程，从而影响肿瘤发生发展。为此，本课题拟进行三方面研究：① miR-122与TLR4表达的相关性研究；② miR-122在TLR4 mRNA上的靶点确认；③ miR-122的沉默与过表达对TLR4下游天然免疫因子表达及肝癌细胞特性的影响。综合分析，明确miR-122通过调节TLR4的表达，从而影响TLR4下游的天然免疫因子的表达及功能，可能参与肝癌的发生发展过程，揭示miR-122调节天然免疫的新分子机制，具有创新性。本研究不仅为研究miRNA与天然免疫的相关性提供依据，更为研究其在肿瘤发生中的作用提供基础。

miR-122是肝细胞中表达最丰富的一类小分子RNA，参与肝炎病毒感染及肝癌细胞凋亡。Toll样受体4（TLR4）能识别肝炎病毒，参与免疫防御及肿瘤的免疫逃逸。本研究发现：①miR-122与TLR4的mRNA及蛋白表达水平在正常肝细胞和肝癌细胞中存在负相关性；②miR-122的沉默可以升高TLR4 mRNA及蛋白的表达，miR-122的过表达可以抑制TLR4 mRNA及蛋白的表达，且miR-122可以影响LPS诱导正常肝细胞中TLR4的表达；③miR-122的沉默与过表达可以影响TLR4下游天然免疫因子的表达及肝癌细胞特性；④通过构建含有报告基因的TLR4野生型和突变型载体，确认miR-122在TLR4 mRNA上的靶点位置（3’ UTR 1603-1609区域）。综上，已明确miR-122通过调节TLR4的表达，从而影响TLR4下游的天然免疫因子的表达及功能，可能参与肝癌的发生发展过程，揭示miR-122调节天然免疫的新分子机制。本研究不仅为研究miRNA与天然免疫的相关性提供依据，更为研究其在肿瘤发生中的作用提供基础。