Rna Interference By Cervical Cancer Hela Survivin Expression And Biological Effect Of Study

Cervical carcinoma having been one of the common theriomas in female in recently years, it was induced by human papillomavirus (HPV). Two hundred thousands patients were killed by this disease every year in the world, and the new cases are more than one hundred and thirty thousands. Thus, researches associated with prevention and therapy cervical carcinoma became urgent in medicine.RNA interference (RNAi) is a new field in the researches of cancer therapy, which induces a specific gene silencing after the transcription by a double strands RNA homogenous to the target gene. The double strands RNA can degradated by a specific nuclease and produced the interference RNA (siRNA), which complement fixes with the homogenous gene, and the target RNA were degradated by the specific enzyme induce the gene silencing.Survivin is a new member of the family of apoptosis-inhibit proteins, it associated with survive of the cell with cancer specific and only expresses in the caner tissue. Survivin can induce apoptosis through the endogenous and exogenous channels. Through these two channels, cytochromec C was released from the mitochondria, this is the key step in cell apoptosis. The released cytochromec C can combine with the apoptosis associated factor1(Apaf-1) with the existing of dATP and form a polymer, which combine with the caspase-9form an apoptotic body. The active caspase-9can active other caspases such as caspase-3, and induce the apoptosis. Survivin can also react with caspase direct by depressing the active of Caspase-3and Caspasc-7, or indirect inhibit the Caspase by P21.Aim:This study aimed to use the RNAi construct siRNA recombinant plasmid aim at the mRNA in survivin in human, and transfect the plasmids to Hela cell in cervical carcinoma to analyze the cytobiological effects, such as the cells grow form, expression of survivin protein and mRNA in survivin, apoptosis of cells and the activation of caspase-3. Thus to give basis of protection, therapy and surveillance in healing of caner in clinical.Method:A short hairpin DNA sequence of survivin was synthesised and cloned to the expression RNAi vector pcdna6.2. The pcdna6.2-siRNA(+) and pcdna6.2-siRNA (-) recombination vectors were constructed. An pcdna6.2vector as blank control. The recombination vectors were identified by sequencing. Then the vectors were transfected into the Hela cell mediate by liposome. The total RNA and protein were extracted and real time PCR and western blot were used to analyze the expression of mRNA and protein. The flow cytometry was used to discover the apoptosis of Hela cell after the survivin having been down regulated, and the Braford method were used to detect the concentration of Caspase3.Results:1. The eukaryotic-expressed RNAi vector aim at survivin has been constructed, which can influence the growth of Hela cell and inhibit the expression of sruvivin mRNA and protein after having been transfected into Hela cell. The interface effect of relative expression of Survivin mRNA in the pcDNA6.2-GW/EmGFP-siR (+) group was optimal at the48hours point after transfection.2. The results of real time PCR and western blot revealed that the mRNA and protein of survivin decreased dramaticlly after the pcdna6.2-siRNA(+) vector has been transfected into Hela cell.3. The proliferation ability of Hela cell has been inhibited obviously after the survivin RNAi vector has been transfected.4. The result of flow cytometry test revealed the proportion of apoptosis in experiment group and control group were11.44%and4.05%respectively. The change of concentration of Caspase-3revealed that gene silencing of survivin can increase its activation.Conclusions:We have constructed an RNAi vector aim at survivin and revealed it can down regulate the survivin in Hela cell. This study also revealed that gene silencing of survivin can inhibit the abilities of proliferation and transference in vitro, and also can accelerated the apoptosis of Hela cell. Survivin has a potential foreground in the protection and therapy of caner as an evaluating indicator in therapy or a target of gene therapy. This study has laid the groundwork for effect of survivin in the therapy of cervical carcinoma by using the RNAi.