Effect Of SiRNA Interference On The Expression Of EIF5A2 And Invasion Of Human Gallbladder Carcinoma Cells In Vitro

BACKGROUND:Gallbladder cancer(GC)is a common primary malignant tumor of biliary system in clinical practice.Because of its high degree of malignancy and rapid growth rate of cancer cells,the prognosis of patients suffering from this disease is very poor.And because gallbladder cancer is often very likely to appear before the patient is diagnosed with cancer cell metastasis,so that patients receive surgical resection of the treatment,easy to relapse.And the 5-year survival rate of hand patients after surgical resection was also lower than that of other cancers.In addition,gallbladder cancer cells are also less sensitive to chemotherapeutic drugs,while some radiation therapeutic maneuvers are also less effective in the adjuvant treatment of gallbladder cancer disease.Therefore,there is an urgent clinical need to study and develop new treatments for gallbladder cancer,and gene therapy in the clinical treatment of the gallbladder has become the focus of scientists.Among them,siRNA uses the antisense nucleotide strand of the target gene in cancer cells to achieve that the mRNA of the target gene cannot be translated into protein through the effect of base complementation,achieving the silencing of the expression of the target gene at the epigenetic level.At present,this strategy has become a common strategy for the study of cancer gene therapy,of which the cancer-related gene EIF5A2 gene has been studied in other types of cancer,while the study on the EIF5A2 gene of gallbladder cancer has rarely been reported.Objectives:Transfecting gallbladder carcinoma GBC-SD cell line with siRNA small interference fragment,thereby interfering with EIF5A2 gene,and using western blot and PCR technology to detect the knockdown efficiency.The effects of EIF5A2 gene on the proliferation,migration and invasion of human gallbladder cancer cell lines were investigated by means of Cell Counting Kit-8,Tranwell migration and other experimental techniques.Thus laying a theoretical foundation for the treatment of gallbladder carcinoma.Methods:1.After the design and synthesis of siRNA,it was transiently transferred into gallbladder cancer cells by liposome mediated way.After 24 hours and 48 hours of culture,the silencing effect of siRNA on eif5a2 gene of gallbladder cancer cells at RNA level and protein level was verified by Q-PCR and Western blot experiments respectively,so as to screen out the appropriate siRNA.2.SiRNA was screened out and transited into gallbladder cancer cells by liposome mediated method.After 24 hours of transfection,CCK8 method was used to explore the change of proliferation ability of gallbladder cancer cells in vitro.3.The qualified siRNA was screened out and transited into gallbladder cancer cells by liposome mediated method.After 24 hours of transfection,Transwell method was used to explore the migration and invasion of gallbladder cancer cells on Transwell plate.Results:1.The mean relative expression of EIF5A2 gene in gallbladder carcinoma cells transfected with siRNA was 0.673±0.132,while the mean relative expression of EIF5A2 gene in gallbladder carcinoma cells not transfected with siRNA was 4.771 ±0.514.There was a significant statistical difference in the relative expression of EIF5A2 gene in the transfected group(P<0.05).In addition,the band of the transfected group was significantly weaker than that of the untransfected group in the Western blot test.These results indicated that the siRNA designed in this experiment,NM 020390 siRNA 112,had a significant silencing effect on the EIF5A2 gene of gallbladder cancer cells at the RNA and protein levels.2.the proliferation ability of gallbladder cancer cells was detected by CKK8.There was no significant statistical difference in the A450 absorbance values of the transfected group at 0 h and 12 h after transfection with siRNA,with P values of 0.732 and 0.172,respectively.However,after 24 h,36 h and 48 h of transfection,the A450 absorbance values of the transfected group were significantly different,P<0.05.These results indicated that the proliferation ability of gallbladder cancer cells in the transfected group was inhibited.3.in transwell plates to detect the migration of gallbladder cancer cells.The mean number of three chambers of gallbladder cancer cells in the transfected group was 91.67,while the mean number of three chambers in the untransfected group was 162.31.Statistical analysis showed a significant mathematical statistical difference(0.004).In the invasion assay of gallbladder cancer cells detected by transwell plates.The mean number of three chambers of gallbladder cancer cells in the transfected group was 61.58,while the mean number of three chambers of gallbladder cancer cells in the untransfected group reached 140.12,and statistical analysis showed that there was a significant mathematical statistical difference between the two groups(0.007).Conclusions:1.In this study,we verified that siRNA-NM 020390_siRNA112 had a significant silencing effect on the EIF5A2 gene in gallbladder cancer cells at the RNA and protein levels by Q-PCR validation and Western blot assay.2.by targeting and silencing the expression of EIF5A2 gene in gallbladder cancer cells,it was able to inhibit the proliferation of gallbladder cancer cells.3.After targeted silencing of EIF5A2 gene in gallbladder carcinoma cells,the migration and invasion of gallbladder carcinoma cells were detected by Transwell assay.The results showed that the migration and invasion of gallbladder carcinoma cells were significantly inhibited after silencing the expression of EIF5A2 gene in gallbladder carcinoma cells.