The Research Of Insulin Resistance For FOXO1 Gene Involved In Regulation Of Human Liver Cancer Cell

Objective:Human liver cancer cell line Hep G2 was selected in this study, Hep G2 insulin resistancemodel was established by inducing Hep G2 cell with high concentration of insulin.Glycogenconsumptions of Hep G2 liver cells under different concentrations were analyzed, therefore, the best time and concentration of insulin induction was confirmed. To examine the differences of FOXO1 gene expressions and FOXO1 protein expressions in different grades of concentrations and time. Thus, the outcome of whether FOXO1 gene is plays an major role in the regulation of insulin resistance of liver cancer cel.Method:Human liver cancer cell line Hep G2 was cultured, and cells was subcultured when the growth area was 85% that of the flask disk. The cells was divided into 6 groups according to the density which was 1×105/ml, then the cells was seeded onto the 96-well plates. Serum was removed after the attachment of the cells, and to synchronize for 24 hour. The 6groups of cells were treated with different kinds of concentration of insulin, which were 1×10-5,1×10-6,1×10-7,1×10-8,1×10-9,1×10-10(mol/L),respectively. After the interference for 24 hour, glucose was analyzed by using Glucose oxidase and peroxidase. Cell viability was tested by using MTT method. Then the best experimental concentration was obtained. Then, the cells was divided into 3 groups according to the density which was 1×105/m, the seeded on to the 96-well plates.The cells was synchronized for 24 hour after attachment. Then insulin with the best concentration was used to interfere the procedure, and induced the ells in 24 h,36h and 48 h respectively. Glucose was analyzed by using Glucose oxidase and peroxidase. Cell viability was tested by using MTT method. The best testing time was analyzed and confirmed RT-PCR technique was used to examine the FOXO1 gene expressions in different insulin treatment groups. Western blot was used to analyze the FOXO1 protein expressions, thus to assure the correlation of FOXO1 and insulin resistance. MTT technique was used to test the influence of Pioglitazone to cell adipose. The group that had the least influence was selected, cells in test group was cultured both with and without Pioglitazone for 24 hours.Glucose was analyzed by using Glucose oxidase and peroxidase. RT-PCR technique was used to examine the FOXO1 gene expressions in this group. Western blot was used to analyze the FOXO1 protein expressions in all groups. Thus, proving the correlation of FOXO1 and insulin resistance and clarifying the regulation of insulin resistance of FOXO1 gene I human liver cancer cel s.Result: Hep G2 cell insulin resistance model induced the best conditions for 10-7 mol/L insulin treatment for 36 hours, the optimal concentration of 1.25 induced by insulin resistance reversal model tendency/L hydrochloric acid pyrrole column methadone treatment for 24 hours, cell with insulin resistance occurs Fox O1 gene and protein expression were significantly higher, and insulin resistance reversal occurs, the expression of Fox O1 level is close to the control group.Conclusion: For induction in vitro model of insulin resistance and insulin resistance reversal model, the cellular sensitivity to insulin and negatively correlated with Fox O1 gene expression, prompt Fox O1 likely as insulin sensitization agent of drug screening targets and efficacy evaluation index.