Production of Cyclin D۱ specific siRNAs by double strand processing for gene therapy of esophageal squamous cell carcinoma

Background: RNAi (RNA interference) is a new strategy in gene therapy and biotechnology which provides new promises in the treatment of different diseases such as cancer and viral diseases. CCND۱ which is a key gene in cell cycle is amplified and over expressed in esophageal cancer. The objective of this study was production and siRNAs for CCND۱, the key gene in cell cycle. Materials and Methods: We used dsRNA digestion method by using recombinant human dicer enzyme to cleave in vitro transcribed dsRNA into a pool of ۲۲bp siRNA. Total RNA was extracted and cDNA was produced using RT-PCR. T۷ promoter was added to both ends of the DNA template by PCR. RNA was produced from both strands of the DNA using T۷ RNA polymerase. After annealing both strands, dsRNA was prepared. Finally siRNA pool was produced by dicer treatment. Results: RNA extraction yield from HN۵ cell line was ۱۴.۶۹ µg/۱۰۶cell. The results from beta actin control gene confirmed the cDNA integrity. After optimization, T۷ promoter adding was confirmed using gel electrophoresis and DNA sequencing. After optimization dsRNA yield was improved. The best incubation condition was ۱۸h. Each microgram of dsRNA yielded ۰.۵ µg siRNA. Conclusion: dsRNA digestion method includes several steps which the product of each step is used as the precursor for the next step. So optimization and increasing the specificity and product yield should be in the most important goals of the study, because the yield of each step has a direct relationship with the final product yield which is siRNA. Optimizing and increasing the yield, dsRNA digestion method could be a rapid, available and profitable method for siRNA generation, providing large amounts of siRNA.

Keywords:

dsRNA digestion , siRNA , RNAi , dsRNA , esophageal cancer , CCND۱