Effects Of Macrophage Migration Inhibitory Factor Expression Inhibition On Proliferation And Apoptosis And The Expressions Of Bc1-2 And P53 Protein In Human Gastric Cancer Cell Line BGC-823

Objectives:To observe the effects which inhibition of macrophage migration inhibitory factor on proliferation and apoptosis and expression of Bcl-2 and p53 protein in human gastric cancer cell line BGC-823 in vitro,and to further explore the potential pathogenesis of MIF in the human gastric carcinogenesis,so as to provide the experimental foundation to targeting MIF for gastric cancer therapy in clinic.Methods:Randomized control study and human gastric cancer cell line BGC-823 were used for the experiments.There was blank control group,50μg/ml HA(MIF inhibitors,high molecular weight hyaluronate)group,100μg/ml HA group,200μg/ml HA group,400μg/ml HA group and 100μg/ml 5-Fu group,and the experiments begin to process after the cell line was treated 48h by different concentration of drugs.The IC₅₀ of HA to human gastric cancer cell line BGC-g23 was the final concentration which was added into the cells for 24h,48h and 72h,respectively.The methods is following:(1)MTT assay was used to determine the effects on the inhibition of the growth of human gastric cancer cell line BGC-823 by the drugs;(2)Flow cytometry and AO-EB fluorescent staining were used to determine the effects on the apoptosis of BGC-823 cell by the drugs;(3)Flow cytometry was used to assay the effects on the cell cycle of BGC-823 cells by the drugs;(4)Semi-quantitative RT-PCR was used to detect the effects on the expression of MIF mRNA in BGC-823 cells by the drugs;(5)Western Blot was used to detect the effects on the expression of the protein of MIF and apoptotic related gene Bcl-2 and p53 in BGC-823 cells by the drugs. Results:(1) The result of MTT assay showed that the growth of human gastric cancer cells BGC-823 was inhibited by HA with concentration from 50μg/ml to 400μg/ml, and the effect increased with higher concentration or longer time.The minimum effective concentration of HA was 100μg/ml,and the role of inhibition was the most highest in positive control group.(2) The results of apoptosis on the cell cycle of BGC-823 cells were determined between AO-EB fluorescent stainining and flow cytometry were coincident:compared with the blank control group,apoptosis of HA group was significantly increased.The minimum effective concentration of HA was 100μg/ml,and the rates of apoptosisand rupture of membrane and the positive cells were increased with higher concentration.The rates of apoptosis and rupture of membrane in positive control group were the most highest.(3) The result of flow cytometry showed that HA could block off BGC-823 cells in stage of G₀/G₁,and the role was significantly increased compared with the blank control group.The minimum effective concentration of HA was 100μg/ml,and the role increased with higher concentration,and the positive control group were the most highest.(4) The result of semi-quantitative RT-PCR showed that the relative expression level of MIF mRNA in BGC-823 cells was decreased with higher concentration and longer time, and the role was significantly decreased compared with the blank control group.The minimum effective concentration of HA was 100μg/ml,and the positive control group was the most lowest level of MIF expression.(5)The result of Western Blot showed that the relative expression levels of MIF and Bcl-2 protein in BGC-823 cells were decreased with higher concentration or longer time,but the relative expression level of p53 protein was increased with higher concentration or longer time,and the role was significantly decreased/increased compared with the blank control group. The minimum effective concentration of HA was 100μg/ml,and the relative expression levels of MIF and Bcl-2 protein in BGC-823 cells in positive control group were the most lowest,but the relative expression level of p53 protein in positive control group was the most highest.Conclusions:(1) MIF can be expressed in human gastric cancer cells BGC-823,and HA can decrease the expression of MIF mRNA and protein in human gastric cancer cells BGC-823.(2) The inhibition of MIF in human gastric cancer cell line BGC-823 can up-regulate the expression of p53 protein and down-regulate the expression of Bcl-2 protein.(3) The inhibition of MIF in human gastric cancer cell line BGC-823 can inhibit the growth of the cells,and the cells can be block off in stage of G₀/G₁, and the roles decrease BGC-823 cell proliferation and induce its apoptosis.