عدم كارايي shRNA اختصاصي بر عليه ژن E1A دركاهش بيان پايدار در سلول هاي HEK 293

Inability of specific shRNA for stable reduction of E1A gene expression in HEK293 cell line

Objective: E1A oncoprotein of adenovirus type 5 is a regulatory factor which controls transcription of other adenovirus genes. This protein promotes both viral genom replication and host cell transformation by altering the function of certain important cellular proteins such as p21 and Rb. The aim of the present study was to constantly reduce the expression of E1A gene in HEK 293 cell line by RNAi technique in order to analyse the effects of this suppression on these cells. Materials and Methods: The U6 promoter and shRNA regions from the control and E1A specific siRNA coding plasmid as pSP-81 and pSP81-E1A were subcloned into pcDNA3.1. Then these constructs were transfered into the HEK 293 cancerous cells using lipofection method and successfully transfected cell colonies were selected based on neomycin antibiotic resistance. Changes in E1A gene expression were analysed by RT-PCR technique after selection process. Results: Final analysis showed no obvious difference in E1A gene expression level in the suppresed and control groups, upon transfection with the constructed plasmids. In order to examine the possible influence of cloning procedure on the function of U6 promoter, cells were transfected with Dr. Hackerʹs original plasmids, but no inhibition of E1A gene expression was observed again. The results of sequencing revealed existence of a mutation in the siRNA target region for the E1A gene sequence. Conclusion: These results illustrated that no considerable suppression has been occurred by repeating Dr. Hackerʹs expriment, even with application of very effective lipofection method. To examine the sequence of the E1A gene, the PCR product of the 13s region of the gene was sequenced. Sequencing revealed existence of a point mutaion in the siRNA target region. It seems that observed impaired interference could be attributed to this mutation in the E1A gene of the studied cells.