The Research Of Cervical Carcinoma Cell HIF-1 Gene Expression Inhibited By RNA

ObjectiveTo utilize RNAi technology,apply siRNAs specific targeting of the HIF-1 gene were used to treat on human cervical carcinoma cell SiHa which express HIF-1,to evaluate the special inhibitory effect of siRNA,provide experiment ground to the feasibility of use RNAi technology to cure cervical carcinoma.Methods1.Construct four siRNAs of HIF-1 gene H1,H2,H3,H4 separately and Negative Control siRNA,postive control(Human GAPDH) siRNA,identify no mistake then transfect human cervical carcinoma cell SiHa by liposome.Apply siRNA which fluorescently-labeled with GFP,optimize transfection condition,measure transfection efficiency.2.Use siRNA which target GAPDH as postive control,detect the GAPDH mRNA's expression of cells in the normoxia and hypoxia cultural condition at 48h after transfection separately.3.Detect the expression of mRNA and protein of the target gene in the normoxia or hypoxia cultural condition by RT-PCR and Western Blotting,in order to screen the most effective siRNA.4.The most effective siRNA was transfected into SiHa separately,detect the mRNA and protein's expression of the target gene in the normoxia or hypoxia cultural condition at 24h,48h,72h and 96h after transfection,use the method of RT-PCR and Western Blotting,in order to investigate the time-efficiency and special inhibitory effect of siRNA.Results1.Fluorescence inverted microscope picture display that,the fluorescence is the most strong when the final concentration of siRNA is 30nM in the normoxia or hypoxia cultural condition,illustrate that more siRNA enter cells at this time,transfection efficiency is the highest,exceed 80%.2.Useβ-actin as reference,β-actin mRNA's expression ofβ-actin group has no marked change in the normoxia or hypoxia cultural condition,GAPDH mRNA's expression of GA-GA group all decrease obviously than blank-GA group and NC-GA group(p＜0.05).3.In this experiment,the result of RT-PCR and Western Blotting after transfection cue that,the different siRNA show different inhibitory efficiency,in four siRNAs specific targeting of the HIF-1 gene,the most descent of expression of target gene caused by H2siRNA in the two conditions(P＜0.05).According to our experiment's result,select H2siRNA to carry out the next experiment.4.Use H2siRNA transfect SiHa,the effect of H2siRNA is special and efficient,the expression level of target gene reduce respectively in 24h,48h,72h,96h after transfection in the normoxia or hypoxia cultural condition,especially in 48h (P＜0.05).The inhibitory action sustaines at least 96h in the two conditions, whereas the other correspond non-target gene level have no change at each time points.ConclusionHIF-1siRNA inhibit SiHa's growth by inhibit HIF-1mRNA and protein level's expression.This experiment offer evidence for further illuminate HIF-1's function and HIF-1 gene therapy's target regulation.RNAi is hope to become a new gene therapy method of human cervical carcinoma.