The Effect And Mechanism Of MicroRNA-34A In Nonalcoholic Fatty Liver Disease

Background and aim:With the improvement of people’s living standard, the prevalence of nonalcoholic fatty liver disease (NAFLD) increased in recent years. NAFLD has become one of the most common chronic liver diseases. The mechanism of NAFLD is not yet fully understood. Recent studies have found that miRNA is closely related with liver diseases. We previously found that the expression of miR-34a were significantly up-regulated in the livers of two animal models of NAFLD, which prompted that miR-34a plays an important role in the development of NAFLD. Our aim of this study was to explore and analyze the target of miR-34a and to reveal miR-34a-specific regulatory mechanisms. In this study, qPCR and western blot were used to validate the expression of miR-34a in NAFLD models both in vitro and in vivo, and RNAi technology was used to explore the biological function of miR-34a in NAFLD. Further, the predicted target of miR-34a was verified.Methods:1. In L02cells, we use free fatty acids (FFAs) to induce NAFLD model in vitro. And we construct NAFLD animal models by feeding C57BL/6mice with high fat diet (HFD). qPCR was used to detect the expression of miR-34a in L02cells and in the livers of C57BL/6mice.2. Through transfecting miR-34a inhibitor in vitro or mouse tail vein injection of miR-34a knockdown lentivirus vector approach, we set out to explore the functional mechanism. Oil red O staining, HE staining and biochemical analysis were used to explore the effect of miR-34a inhibition on the degree of steatosis and inflammation.3. We used an online database to predict the target of miR-34a. It turned out PPARa. Then we used the method of western blot to validate the protein expression levels of PPARa, next we adopted a luciferase reporter system to identify the sequence of target of miR-34a. Western blot and qPCR were used to observe the expression of PPARa downstream genes which were involved in lipid metabolism.4. We used PPARa siRNA to inhibit the expression of PPARa in vitro to explore the influence of the degree of steatosis of liver cells. Western blot and qPCR were used to observe the expression of PPARa downstream genes which were involved in lipid metabolism. Results:1. The expressions of miR-34a were significantly increased in NAFLD models of L02cells and mice livers.2. Through transfecting miR-34a inhibitor in vitro or mouse tail vein injection of miR-34a knockdown lentivirus vector to inhibit the expression of miR-34a, we found that the droplets of lipid and the contents of triglycerides in L02cells and liver tissues were significantly decreased. Furthermore, liver weight/body weight ratios and serum levels of AST were significantly decreased in miR-34a inhibitor groups of mice.3. Using online databases to predict, we found that PPARa maybe the target of miR-34a. The expressions of PPARa were up-regulated after inhibiting the expression of miR-34a in L02cells and C57BL/6mice livers. Further, we confirmed that PPARa was the directly target of miR-34a by luciferase reporter system analysis. MiR-34a interacted with the essential binding sequence located in3’-UTR of PPARa. The expressions of downstream of PPARa which were involved in lipid metabolism were changed. On the other hand, the energy metabolism pathway of AMPK was activated in miR-34a inhibitor group.4. By inhibiting the expression of PPARa though transfecting PPARa siRNA in vitro, we found that the lipid droplets and the contents of triglyceride were significantly increased in L02cells and HepG2cells. The expressions of downstream of PPARa which were involved in lipid metabolism were changed.Conclusions:The expression of miR-34a was closely related to NAFLD, and up-regulation of miR-34a was involved in lipid metabolism of NAFLD. MiR-34a may become a potential therapeutic target in NAFLD by targeting PPARa.