Constructing RNAi Vector And Silencing MT P53 Gene In PLC/PRF/5 Cancer Cells By Using RNA Interference

P53 protein was firstly discovered in 1979. P53 gene can code P53 protein that is a nuclear phosphoprotein with cancer-inhibiting properties. Thus it is tumor suppressor gene. Change or loss of wild type (WT) p53 function has close relation to most of various human tumors.The majority of p53 gene mutations are missense mutations. Minority of mutant proteins can retain wild-type activity, and most of MT p53 have changed their properties. They can lose normal function, or can inhibit the activity of WT p53 or inactivate WT p53 at the same time, and some of them can acquire new properties to promote carcinogenesis.Gene therapy of p53 becomes the focus of researches because p53 gene playes an important role in cell growth. There are two main researches: gene replacement approach of WT p53 gene and block approach of MT p53 gene. The fundamental strategy of gene replacement approach is replaced MT p53 by WT p53. In primary hepatocellular carcinoma, however, high expression of WT p53 existed, suggested that the function of WT p53 was inhibited. Thus, the proach can't be used in patients with high expression of WT p53. Block approach of MT p53 gene is to inhibit expression of hurtful genes orout-of-control genes, such as by using antisense RNA.RNA interference (RNAi) is that double-stranded RNA (dsRNA) molecules of 21-23 nt (short interfering RNAs, siRNAs) can effectively and specifically silence expression of targeted genes through sequence-specific cleavage of the cognate RNA transcript, and then can improve mRNA degradation, as a result, can induce the process that cells show lost of expression of targeted genes. So RNAi is an ideal tool of gene silencing therapy and a widely used method to analyze gene function because of its high specificity and effectivity. At present, mounting evidence suggests RNAi can be used in gene therapy to treat tumors and virus infection in mammalian cells. But using RNAi in vivo is limited by absence of vectors of high effectivity and lower toxicity.The targeted gene of RNAi in this experiment is mutant p53 gene which codes mutant P53 protein with R249S.The reasons lie in that mutant p53 protein with R249S is harmful gain-of-function mutant protein, has played an important role in the carcinogenesis and devolopment of hepatocellular carcinoma, and has specific regional distribution, such as in South Africa and some areas of China.The first aim of our study was to construct expression vector of RNA interference, and identified the right vector by using known siRNA to silence the expression of WT p53 in HepG2 cells. The second aim was to design the oligonucleotides with small hairpin structure which target was mutant p53 gene, which codes mutant P53 protein with R249S, to silence MT p53 gene in PLC/PRF/5 cells by using RNAi vector we constructed, and to observe the effect of MT p53 with R249S mutant on the cell cycle and cell growth of hepatocellular caicinoma cells.Firstly, we constructed expression vector of RNAi, and then used known oligonucleotides with small hairpin structure that targeted WT p53 to identify the RNAi vector. The oligonucleotides with small hairpin structure were annealed, and then formed dsDNA with corresponded enzyme cleave sites. RNAi vector was digested by the same enzymes and then formed adhering ends. The dsDNA with small hairpin structure that targeted WT p53 was cloned into the expression vector of RNAi vector to construct RNAi vector that targeted WT p53.After identified by digesting by enzymes, the right vectors were stably transfected into HepG2 cells, and obtained the stably transfected cell strain. In order to prove the effect of RNAi, the P53 protein was immediately detected by western blot. We used the more sensitive method that named chemistry luminescence because of short half time of WT p53. MTT method and FCM analysis were applied to measure cell growth curve and cell cycle to observe effect of mutant p53 on tumor cells growth.The results of western blot showed that the level of WT P53 protein markedly decreased compared with the control group, suggested that the expression of WT p53 gene was inhibited. Thus, we had constructed RNAi vector successfully. And we found no obvious difference in cell growth curve and cell cycle between RNAi group and control group.According to the principle of siRNA design, we designed the oligonucleotides with small hairpin structure which target was mutant p53 gene that codes mutant P53 protein with R249S.The corresponded dsDNA was formed and then inserted into RNA interference vectors. After identified by digesting by enzymes, the right vectors were stably transfected into PLC/PRF/5 cells, which express purified mutant P53 protein (codon 249...