The Study Of The Therapy To Colon Cancer With Replication-Competent Adenovirus Highly Expressing Shrna Of Survivin

ObjectiveTo Construct the conditionally replicating adenovirus vector Ad-delE1b55KD-shRNA/Survivin-EGFP which can transfect to HT-29 effectually and selectively and containing shRNA to human Survivin gene. And investigate the effect of the vector silencing on expression of mRNA and protein of Survivin gene in HT-29 cells, on propagation and apoptosis of colon carcinoma cell lines HT-29, and on Survivin gene in colon carcinoma cell lines HT-29 lastingly.MethodInserted EGFP gene and the shRNA gene to human Survivin mRNA into pAd-delE1b55KD, then we have obtained the plasmid pAd-delE1b55KD-shRNA/Survivin-EGFP. The plasmid and pBHGE3 were cotransfected into HEK-293 cell for obtaining the conditionally replicating adenovirus vector Ad-delE1b55KD-shRNA/Survivin-EGFP which containing shRNA to human Survivin mRNA. Then the DNA of the adenovirus vector was extracted and identified. We transfected Ad-delE1b55KD-shRNA/Survivin-EGFP to HT-29 (control is replication defective adenovirus and liposome vector which was contained the same shRNA as Ad-delE1b55KD-shRNA/Survivin-EGFP, the 3th control is Ad-delE1b55KD/Survivin-EGFP ). The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot. The propagation of colon carcinoma cell lines HT-29 were detected by MTT and the apoptosis of HT-29 cells after transfection were evaluated by AO/EB dyeing and Annexin V-PE/7-AAD dyeing. At last, we transfected Ad-delE1b55KD-shRNA/Survivin-EGFP to HT-29 (control is replication defective adenovirus and liposome vector which was contained the same shRNA as Ad-delE1b55KD-shRNA/Survivin-EGFP, the 3th control is Ad-delE1b55KD/Survivin-EGFP). The expression of EGFP,Survivin mRNA and Survivin protein in HT-29 were detected at the 1st day,7th day,14th day and 28th day after transfection.ResultEGFP gene and the shRNA gene to human Survivin mRNA was inserted into conditionally replicating adenovirus vector successfully, which was proved by sequencing. The transfection efficiency to HT-29 of Ad-delE1b55KD-shRNA/Survivin-EGFP was significant higher than replication defective adenovirus and liposome vector(P<0.01). After transfection of Ad-delE1b55KD-shRNA/Survivin-EGFP, mRNA and protein of Survivin gene in HT-29 cells were obviously reduced. The death rate and apoptosis rate of HT-29 cells were obviously more increased than Ad-delE1b55KD/Survivin-EGFP, replication defective adenovirus and liposome vector which was contained the same shRNA. The expression of EGFP,the inhibition of Survivin mRNA and Survivin protein in HT-29 were high in each groups at the 7th day after transfection, among the total, the effect of Ad-delE1b55KD-shRNA/Survivin-EGFP group was most high; at the 14th day, the effect of Ad-delE1b55KD/Survivin-EGFP, replication defective adenovirus group and liposome vector group were decrease obviously, and it was still high in Ad-delE1b55KD-shRNA/Survivin-EGFP group; the effect of Ad-delE1b55KD-shRNA/Survivin-EGFP group was disappeared at the 28th day, and it was still high in Ad-delE1b55KD- shRNA/Survivin-EGFP group like before(P<0.01).ConclusionA conditionally replicating adenovirus vector was constructed and identified successfully. It could transfect to HT-29 and silence Survivin effectually and selectively. The effect of RNAi-mediated Survivin gene with Ad-delE1b55KD-shRNA/Survivin-EGFP silencing was higher than replication defective adenovirus and liposome vector ; the effect of Ad-delE1b55KD-shRNA/Survivin-EGFP on propagation and apoptosis were higher than Ad-delE1b55KD/Survivin-EGFP, replication defective adenovirus and liposome vector. RNAi-mediated Survivin gene with Conditionally replicating adenovirus can silence Survivin gene in colon carcinoma cell lines HT-29 lastingly.