Studies On The Roles And Mechanism Of Circulating MiR-429in Predicting The Occurrence And Early Diagnosis Of Hepatocellular Carcinoma

Part I:Studies on the roles and mechanism of circulatingmiR-429in predicting the occurrence and early diagnosis ofhepatocellular carcinomaWe investigated circulating miRNAs signatures of time-series plasma samplescollected from a large cohort including28612healthy and3070chronic HBsAg-positivepersons. Two secreted miRNAs (miR-429and miR-193a-3p) were identified as thepredictive and very early diagnostic biomarkers with the predominant performance beyondtwo world-wide recognized methods, alpha-fetoprotein (AFP) and ultrasound. Theoverexpression and internalization of miR-429in liver tumor initiating cells (T-ICs) andhepatocytes endowed cell self renewal, proliferation, chemoresistance and tumorigenicitythrough directly targeting a tumor suppressor gene RBBP4in vitro and in vivo. In addition,aberrant epigenetic modification upstream of miR-200b/200a/429cluster responsible forexpression manipulation was found in EpCAM+T-ICs and HCC tissues.Conclusion: Those studies provide novel surveillance biomarker and promisingtherapeutic target for HCC. Part II:The nuclear factor High-mobility group box1mediatingthe activation of TLR4signaling in hepatocytes in the earlystage of NAFLD in mice.One of the challenges surrounding nonalcoholic fatty liver disease (NAFLD) is todiscover the mechanisms that underlie the initiation of it. The aim of the present study wasto elucidate the effects of toll-like receptor4(TLR4) signaling in liver parenchymal cellsduring the early stage of NAFLD. Male TLR4-wildtype, TLR4-knockout, TLR2-knockout,MyD88-knockout and TRIF-knockout mice were fed normal diet or high-fat diet (HFD).Liver steatosis, alanine aminotransferase levels, nuclear translocation of NF-κB (p65),macrophage accumulation and neutrophil infiltration were assessed. By using Kupffer celldepletion or bone marrow transplantation, we examined the potential role of kupffer cellsand myeloid infiltrating cells during the initiation of NAFLD. Immunohistochemistry andwestern blotting were implemented to determine the release of High-mobility group box1(HMGB1). The neutral-antibody against HMGB1was used to block the activity of freeHMGB1. Here, we report that the activation of TLR4signaling in hepatocytes,accompanied with the relocation of P65in nucleus was proven playing an important roleduring the initiation of NAFLD. Importantly, HMGB1releasing from hepatocytes inresponse to FFA infusion was firstly reported as the key molecule for the TLR4/MyD88activation and cytokines expression in vitro and in vivo. Treatment with neutralizingantibody to HMGB1protects against FFA-induced TNF-α and IL-6production.Conclusion: Our study supports the notion that TLR4/MyD88signaling in liverparenchymal cells plays a pivotal role during the early progression of HFD-inducedNAFLD, in which free HMGB1served as a positive component mediating TLR4activation.