Study On The Mechanism Of PNPLA3 Gene I148M Polymorphism In Nonalcoholic Fatty Liver Fibrosis

Objective:To explore the influence of PNPLA3 I148M gene polymorphism in the expression of hedgehog signaling pathway at animal and cell levels and reveal the potential mechanism between PNPLA3 I148M gene polymorphism and nonalcoholic fatty liver fibrosis.Methods:The three transgenic mouse models, homozygous wild-type (PNPLA3+/+), heterozygous (PNPLA3148M/+), homozygous mutant (PNPLA3148M/M), were constructed by micro injection method. All the mice were feeding with normal rodent chow and water ad libitum for 16 weeks. Blood samples were taken from mice heart after ether anesthesia to detect serum TG, TC, ALT, AST levels. Western blot and fluorescence quantitative PCR were applied to investigate the expression of Hh signaling pathways and genes related to cell proliferation and apoptosis in mice liver tissue. In cell experiments, sequences of PNPLA3 I148M wild type and mutant gene and slow virus vector were synthesised using double digestion method and integrated into the vector plasmids respectively. After amplification and purification, these plasmids were used to transfect the 293T cells to obtain lentivirus vectors containing different genotypes of PNPLA3 gene. Subsequently, we used the lentivirus vectors to infect the HSC-T6 cells we cultured.Then the HSC-T6 cells overexpressing PNPLA3 I148M wild type and mutant gene were constructed after screening by puromycin. On this basis, we tested expression of Hh signaling pathways and cell proliferation and apoptosis related gene, respectively. The validation of the protein levels were used by western blot. The data were expressed with "mean±standard deviation" and analyzed by SPSS 17.0.Independent-Samples T Test was used to test the comparison between two groups. P＜0.05 were considered as statistically significance.Results:(1) Compared to wild-type littermates (PNPLA3+/+), serum alanine aminotransferase (ALT) level was significantly higher in PNPLA3148M/M mice than the wide type animals. (2) Hepatic mRNA expression levels of mouse shh, patched, smo, gli-1 were significantly increased in the PNPLA3148M/M and PNPLA3148M/+ transgenic mice compared with PNPLA3+/+controls. (3) The mRNA expression levels of bcl-2, PDGF and TGFβ1 were significantly higher in the PNPLA3 mutant gene mice carriers than these wide-type transgenic mice. (4) Plasmids were successfully constructed and infected HSC-T6 cells. Fluorescence microscope showed that the rate of EGFP expression was over 85%. After screening by puromycin, the rate of EGFP expression was over 95%. PNPLA3 protein levels were higher in wild type and mutant group, compared to empty vector control cells, which proven HSC - T6 cells overexpressing of PNPLA3 I148M wild type and mutant genes were constructed successfully. (5) The mRNA expression levels of shh, patched, smo, gli-1 gene were higher in HSC-T6 cells of PNPLA3 I148M mutations than wild-type cells group and the difference is statistically significant. (6) Compared to PNPLA3 I148M wild-type HSC-T6 cells, mRNA expression levels of bcl-2, PDGF and TGFβ1 were up-regulated obviously in PNPLA3 I148M mutant HSC-T6 cells and the difference has statistically significance.Conclusions:These results indicated PNPLA3 I148M variant activating the Hh signal pathway may one of the pathogenetic mechanisms that facilitate the transformation of simple steatosis into fibrosis.