Research Of Human Arsenic-resistance Related Gene Function By RNAi Technique

Objective:Arsenic is widely distributed in the environment, its poison is rather strong . Almost all organisms developed strategies to exclude the toxic substance and acquire tolerance. We had found 5 genes which are possible arsenic-resistance related genes from ECV(304) model in the former research. We identified three ATP-binding cassette subfamily member which are up-regulated as target genes in arsenic resistance cells . Firstly, we observed the expression of human arsenic-resistance related genes were inhibited by siRNA. Secondly, We analyze the change of arsenic tolerance around silencing target gene by MTT assay. Finally, we obtained the possible arsenic-resistant gene of human being.Methods:Synthysized siRNA were transfected into ECV(304) cells which were confirmed to resist to arsenic with siPORTTMNeoFXTM.The changes of target gene mRNA and protein levels were determined by semi-quantitative RT-PCR and Western blot.The viability of cells transfected siRNA was determined by MTT assay.Results: ( 1 )ABCA1 Three siRNA specifically targeting ABCA1 mRNA were successfully transfected into ECV(304) cells. Differrent siRNA showed different reduction in ABCA1 expression. At 48h post transfection, the degrees of reduction with siRNA-1, siRNA-2, siRNA-3 were 32.08%士0.746%,55.08%±0.075%,87.09%±0.031% respectively compared with the control(p<0.05). siRNA-3 was the most efficient. Moreover, ECV(304) cells treated with negative control siRNA showed no markable reduction in ABCA1 expression (p>0.05).The results of Western blotting assay indicated ABCA1 protein levels was inhibited after transfection efficiently.MTT assay demonstrated that transfected cells ,comparing with arsenic-resistant cells which were untreated with siRNA , exhibited decreasing in resistance to acute arsenite toxicity. The viable cell rations of transfected cells were significantly reduced in siRNA-3 groups at 48h post transfection.LC50 for acute arsenite exposure in these chronic arsenic-exposed ECV(304) cells was 8.05μΜversus 11.09μΜin control cells.( 2 ) ABCE1 Two siRNA specifically targeting ABCE1 mRNA were successfully transfected into ECV(304) cells. Differrent siRNA showed different reduction in ABCE1 expression. At 48h post transfection, the degrees of reduction with siRNA-1, siRNA-2 were 46.6%士0.339%,73.8%±0.545% respectively compared with the control(p<0.05). siRNA-2 was the most efficient. Moreover, ECV(304) cells treated with negative control siRNA showed no markable reduction in ABCE1 expression (p>0.05).The results of Western blotting assay indicated ABCE1 protein levels was inhibited after transfection efficiently.MTT assay demonstrated that transfected cells still exhibited strong resistance to acute arsenite toxicity, that is to say, ABCE1 does not affect the tolerance of arsenic-resistant cells to arsenic.( 3 ) ABCF1 Three siRNA specifically targeting ABCF1 mRNA were successfully transfected into ECV(304) cells. Differrent siRNA showed different reduction in ABCF1 expression. At 48h post transfection, the degrees of reduction with siRNA-1, siRNA-2, siRNA-3 were 76.73%士0.768%,65.39%±0.699%,52.16%±0.014% respectively compared with the control(p<0.05). siRNA-1 was the most efficient. Moreover, ECV(304) cells treated with negative control siRNA showed no markable reduction in ABCF1 expression (p>0.05).The results of Western blotting assay indicated ABCF1 protein levels was inhibited after transfection efficiently.MTT assay demonstrated that transfected cells still exhibited strong resistance to acute arsenite toxicity. After ABCF1 was knocked down, the arsenic-resistant cells still keep the arsenic resitant activity.Conclusion: siRNA were synthysized and transfected into ECV(304) cells, which could reduce the expression of ABCA1 gene level specially, tolerance of arsenic resistant cells were weakened obviously after ABCA1 were knocked down. So we can identify that ABCA1 gene is mostly a arsenic-resistance of related gene . This study is an important basis for solving the problem of arsenic drugs tolerance in the clinical practice as well as preventing and treating arsenic poisoning.