Inducing Of Autophagy And Apoptosis By Mi R-148a Through Hedgehog Pathway In Hepatic Stellate Cells

Chapter 1: Gas1 is a direct target of mi R-148 a in Hepatic stellate cells Objective:Increasing evidence indicates that dysregulated mi RN As significantly contribute to hepatic fibrosis. The purpose of this study was to investigate the effect of mi R-148 a through its potential target, growth arrest-specific gene 1(Gas1) in hepatic stel ate cel s(HSCs) under stress. Methods:LX-2 and T-6 cells were cultured under starvation conditions for 1 h, 2 h and 4 h. The expression levels of mi R-148 a and Gas1 m RNA were detected, mi R-148 a potential target Gas1 was validated using Dual- Luciferase Reporter Assay System, and examined whether Gas1 was the direct target gene of mi R-148 a. Finally, mi R-148 a mimic or inhibitor have been transfected into LX-2 and T-6 cells through Lipofectamine 2000 respectively and detected Gas1 protein and m RNA expression levels by Western blot and q RT-PCR. Results:The mi R-148 a expression level was a sustained, time-dependent upregulation under treatment with Earle’s balanced salt solution in both cell lines. Bioinformatic prediction revealed a putative mi R-148 a binding with the 3’-UTRs of the Gas1 m RN A. Dual- Luciferase Reporter Assay System indicated that mi R-148 a can combine with the 3’-UTR of Gas1, thereby inhibiting the activity of luciferase. HSCs transfected with the mi R-148 a mimic significantly reduced Gas1 protein and m RNA expression levels compared with the negative control group. Conclusions:Starvation induces mi R-148 a upregulation and Gas1 downregulation in a time-dependent manner. Dual- luciferase reporter assays showed that mi R-148 a directly interacts with the 3’-UTRs of Gas1 m RNA with high specificity and Gas1 is a direct functional target of mi R-148 a. Chapter 2: Gas1 inhibits autophagy through Hedgehog pathway in HSCs Objective:Gas1, which encodes a Hedgehog surface binding receptor and facilitates the Hedgehog(HH) signaling pathway and hedgehog signaling pathway puts a damper on autophagy. This study was conducted to explore whether Gas1 could regulate the HH pathway and autophagy in HSCs. Methods:LX-2 and T-6 cells were treated with different concentrations of Gas1 for 48 h(0、0.25μg/μl 、 0.5μg/μl 、 0.75μg/μ l). Western blot, transmission electron micrograph(TEM), Fluorescence microscopy were used to determine the levels of autophagic relative protein expression in HSCs. Meanwhile, HH signaling pathway signatures and autophagy marker were determined after treatment with Gas1 si RN A, Smo agonists and antagonists. Autophagosomes were observed using fluorescence microscopy and TEM. Results:Compared with the negative control group, Gas1 can cause a gradual, dose-dependent reduction in LC3-II and increase in SQSTM1/p62 following by different concentrations of Gas1 treatment(especially for 0.75μ g/μ l). Furthermore, autophagosomes could be easily observed in the cells that were cultured in the EBSS medium but were less prevalent in cells that had been treated with Gas1. and treatment with Cyclopamine decreased Patch1 and Gli1 expression, It is promoted LC3-II accumulation and p62 degradation in both HSCs lines. And treatment with Purmorphamine increased Patch1 and Gli1 expression, but decreased Gas1-induced autophagy.Conclusions:Gas1 can inhibit autophagic activity of LX-2 and T-6 cells by targeting Hedgehog signaling pathway and play the negative regulatory role. O ur present results implicate targeted regulation of Gas1 may become a potential new target for liver fibrosis. Chapter 3: Mi R-148 a induces autophagy, inhibits proliferation and promotes apoptosis in HSCs Objective:Micro RNAs(mi RN As) participate in a variety of biological processes. Mi R-148 a has been found to be downregulated in hepatic fibrosis and human hepatocellular carcinoma. However, the role of mi R-148 a in the autophagy activity of HSCs remains largely unknown. In this study, we describe the epigenetic regulation of mi R-148 a and its impact on autophagy, proliferation and apoptosis in HSCs. Methods:Gain or loss-of- function methods were used to evaluate roles of mi R-148 a in autophagy modulation under starvation. Autophagy was measured by Western blotting, fluorescence microscopy, transmission electron microscopy and Bafilomycin al assay; proliferation and apoptosis that mi R-148 a influence on HSCs were assessed via MTS assay and flow cytometry. Results:Mi R-148 a induces autophagy activation under starvation by promote the conversion of LC3-I/LC3-II, p62 degradation, increase in GFP-LC3 puncta, accumulation of autophagosomes. The modulation of autophagy by mi R-148 a is relies on suppressing target gene Gas1. In the presence of Bafilomycin al, mi R-148 a caused a significant increase in LC3-II and p62 degradation. Meanwhile, overexpressing mi R-148 a resulted in a significantly inhibited proliferation and increased apoptosis, and transected with the mi R-148 a inhibitor showed the opposite effect. Conclusions:Mi R-148 a overexpression induces autophagy in HSCs, Gas1 dramatically blocked mi R-148 a induced autophagy and further indicated that Gas1 is required for inducing autophagy by mi R-148 a.Mi R-148 a inhibits proliferation and promotes apoptosis in HSCs lines.