The Construction Of P53 RNA Interference System And The Study Of Its Function

As a very important tumor suppressor gene, p53, which locates in 17pl3.1, plays a significant role in the regulation of cell cycle, the inhibition of cell growth and the induction of tumor cell apoptosis. The absence or mutation of p53 is an important reason for the occurrence of many kinds of tumors. p53 also plays an important role in the blood vessel regulation and the chemotherapeutic sensitivity in tumors. p53 exerts its biological function by activation or inhibition of its downstream genes, but the particular mechanism has not been revealed yet .RNA interference is a process of post-transcriptional gene silencing triggered by double-stranded RNA . It is a gene-inactivating technology that can efficiently identify the function of specific genes. RNAi becomes to be a powerful tool in Functional Genomics.There are many strategies to knockout the specific gene by RNAi in mammal cells, among those strategies, construct a siRNA expression vector is preferable for its stability and permanence. So, in this study, a specific siRNA expression vector which targeted p53 was designed and constructed, and then transfect the vector into the gastric cancer cell line SGC-7901 in order to detect its inhibition efficiency and at the same time, the growth change of cells inhibited p53 expression was observed.Methods1. Design and construct a specific siRNA expression vector which targeted p53 (pSG-p53i), identify the vector by enzyme digestion and sequencing to make sure the exact correction of the construction. An unspecific siRNA expression vector (pSG-p53c) was constructed as a control.2. Transfect pSG-p53i/pSG-p53c vector into the gastric cancer cell line SGG-7901. Select the positive cell clone by antibiotic G418. Detect the inhibition efficiency of pSG-p53i vector by RT-PCR and Western blot.3. Analyse the growth change of the gastric cancer cell line SGC-7901 by Flow Cytometer and cell growth curve.Results1. A specific siRNA expression vector which targeted p53 (pSG-p53i) was constructed. Enzyme digestion and sequencing suggested that thesequence had been inserted into the correct site.2. RT-PCR showed that the level of mRNA which p53 expressed in gastric cancer cell line SGC-7901 with pSG-p53i was 62% lower than the control cell (SGC-7901 with pSG-p53c). And western blot also showed that the level of p53 protein expressed in SGC-7901 with pSG-p53i was lower than the control cell.3. Compare with the cells with pSG-p53c , Flow Cytometer showed that the number of gastric cancer cell with pSG-p53i which in G1 period decreased by 24.2%, while S period cells increased by 81.0% and G2 period cells decreased by 77. 9%;The cell growth curve showed thatthe curve of cells with pSG-p53i had shifted to the left.ConclusionThe results demonstrated that pSG-p53i ,a p53 gene siRNA expression vector, had been successfully constructed. It could lower the level of raRNA and protein of p53 gene, which suggested that pSG-p53i have an effective inhibition in the expression of p53 .The p53 gene siRNA expression vector pSG-p53i speeded the growth of the gastric cancer cell line SGC-7901 , which was the result of the inhibition of p53 expression.