The Biological Effect And Mechanism Of PPAR? V227A Polymorphism On The Hepatocyte

Background and aim:Peroxisome proliferator-activated receptor alpha(PPARa), known as a member of nuclear receptor super-family, plays a critical role in the pathogenesis of nonalcoholic fatty liver disease(NAFLD). Our previous study showed that the V227A polymorphism in the PPARa gene was shown to have different distributions in the patients with fatty liver and the healthy subjects. The level of weight, body mass index, hip circumference, waist circumference, waist-hip ratio, percentage of body fat, abdominal wall fat thickness in subjects with V227A variant were significantly lower than those in V227 wide type. PPARa V227A polymorphism may be involved in the mechanism of NAFLD and obesity. The aim of this study was to investigate the biological effects and mechanism of genomic variations in PPARa V227A on hepatocyte. Methods:The fusion PCR cloning method was used for generation of the fragment of PPARa gene with V227 or 227A variation. The fusion PCR products were cloned into multiple clone sites of eukaryotic expression vector pEGFP-N1 and then the recombinant plasmid of pEGFP-Nl-PPAR?V227 and pEGFP-N1-PPAR?227A was constructed. After confirmed by DNA sequencing, the recombinant plasmid of pEGFP-N1-PPAR?V227, pEGFP-N1-PPAR?227A and the empty eukaryotic expression vector pEGFP-N1 were transfected into human liver cell line L02 respectively by liposome transfection and got their stable expression in cells. The positive clones were screened by G418 and the expression of fusion protein PPARa V227A-EGFP was identified by immunofluorescence analysis and Western blot analysis. Then the L02 cells were divided into 4 groups:cells transfected with PPARa V227 (wild-type), cells transfected with PPARa 227A (mutation), cells transfected with empty vectors and L02 cells without treatment. Each group was further subdivided into intervention group (with PPARa agonist WY14643) and control group (no intervention). The effects of PPARa V227A gene variations on cell growth and proliferation were examined by MTT method. The effect of PPARa V227A gene variations on cell apoptosis was analyzed by flow cytometry. The examination of protein expression levels was performed by Western blot analysis.Results:(1) The recombinant eukaryotic expression vectors pEGFP-N1-PPAR?V227 and pEGFP-N1-PPAR?227A were successfully constructed.(2) L02 cells were transfected with the recombinant eukaryotic expression vectors and got their stable expression of fusion protien PPARa V227-EGFP and PPARa 227A-EGFP.(3) The MTT assay showed that cellular proliferative activities and cell viability in the mutation group were significantly higher compared with the wild-type group(P<0.01).(4) The result of flow cytometry following Annexin V/PI staining showed that the apoptosis rate in the mutation group was lower compared with that in the wild-type group(P<0.05).(5) The expressions of PPARa, RXRa were not different between the wide-type group and mutation group. The expressions of PPARa and RXRa could be induced by PPARa agonist WY14643.(6) The expression of phospho-p44/42MAPK (Thr202/Tyr204) was higher in the mutation group than that in the wide-type group. And the expression of phospho-NF-KB-p65 (Ser536) was lower in the mutation group than that in the wide-type group.Conclusions:(1) The recombinant eukaryotic expression vectors pEGFP-N1-PPAR?V227 and pEGFP-N1-PPARa 227A were successfully constructed.(2) L02 cells were transfected with the recombinant eukaryotic expression vectors and the stable expressions of fusion protien PPAR?V227-EGFP and PPARa 227A-EGFP were obtained.(3) The cellular proliferative activity and cell viability in the mutation group were higher compared with those in the wild-type group, and the apoptosis rate in the mutation group was lower compared with that in the wild-type group.(4) PPAR?227A may increase the expressions of phospho-p44/42MAPK (Thr202/Tyr204) and decrease the expressions of phospho-NF-?B-p65 (Ser536), which probably contributed to the higher cell viability and lower apoptosis rate in the mutation group. Genomic variation with PPAR?227A may benefit L02 cell line.This study provides vital evidence for the function of V227A variation in PPAR?gene, however, the specific mechanisms of how these two variants work are poorly understood, which needs further research and exploration.