Effect Of PNPLA3 Gene I148M Polymorphism On Cell Cycle Of Huh - 7 Cell Line

Objective:To overexpression of PNPLA3 wild type and I148 M variant in Huh-7 cells and investigate its cell cycle affected by I148 M polymorphism of PNPLA3 gene and the possible mechanisms. Methods: The wild type and I148 M variant of PNPLA3 gene sequences were dispersed using a double digestion method and integrated into the vector plasmids respectively. The vector plasmids with the specific target gene were purified and amplified. These plasmids and the viral packaging plasmids were used to co-transfect the 283 T cells to obtain the lentivirus vectors containing the specific genes we needed. Subsequently Huh-7 cells we cultured were infected by lentivirus vectors. After screening we constructed the Huh-7 cells overexpressing PNPLA3 wild type and I148 M variant respectively. Huh-7 cells with zero load plasmids were used as matched control, Flow cytometry was conducted to detect the cell cycles of these 3 type of Huh-7 cells. Western blot and fluorescence quantitative PCR were applied to investigate the expression of regulatory factors, Cyclin D1 and p53. The data were analyzed by SPSS 17.0 Microsoft and expressed with “mean±standard deviation”. The comparison between two groups was statistically treated by Independent-Samples T Test or χ2 Test. The difference was not statistically significant unless P<0.05. Results:(1) Plasmids were successfully constructed: positive clones were DNA sequenced and the sequencing results showed plasmids carried the target gene sequence we needed.(2) We successfully constructed the lentirus vectors containing target plasmids with virus titer up to 1×10^8 PFU/ml: inverted fluorescence microscope showed bright EGFP expression in 293 T cells followed by puromycin screening.(3) Lentirus vectors successfully infected Huh-7 cells: inverted fluorescence microscope showed the rate of EGFP expression was over 95% in Huh-7 cells followed by puromycin screening. The expression of PNPLA3 protein in wild type group and variant type group were higher than in the control group. This result can also be seen in fluorescence quantitative PCR test(variant type group 166.65±117.25, wild type group 1149.55±278.17, control group 1.02±0.23; P<0.05).(4) PNPLA3 gene I148 M polymorphism in Huh-7 cells showed the disorder of Cell Cycle: cell cycle phase distribution was presented by the proportion of cells in each phases(%), compared with the control group, the cell cycle phase distribution(G1 phase 59.27±0.15, G2/M phase 24.23±0.31, S phases 16.50±0.26) had no differences in wild type group(G1 phase 58.53±0.35, G2/M phase 24.87±0.60, S phases 16.60±0.26; P<0.05). While between variant type group and wild type group, G1 phase was significantly decreased(variant type group G phase138.37±0.21, P<0.05), S phase and G2/M phase were increased(variant type group S phase 27.47±0.35, P<0.05; G2/M phase 34.17±0.15, P<0.05), respectively.(5) The expression of cell cycle regulating protein p53 in variant type group was increased while Cyclin D1 had no difference: compared with control group, the relative expression of P53 m RNA in variant type group was significantly upregulated(control group 1.06±0.41, variant type group 6.54±0.34; P<0.05) and there was no statistical significance in wild type group(1.66±0.30,P>0.05); Cyclin D1 expression showed no statistical significance in any of these three groups(control group 1.00±0.10, wild type group 1.06±0.03, variant type group,1.11±0.04; P>0.05). Conclusions: I148 M polymorphism of PNPLA3 gene promoted Huh-7 cells growth via the increasing DNA duplication and cell cycles of Huh-7 cells. And we founded that upregulating the expression of P53 participated in this process.