Dicer, a large multidomain ribonuclease, is responsible for processing double-stranded RNAs (dsRNAs) to 20-bp-long small interfering RNAs (siRNAs), which act as effectors during RNA interference (RNAi).OBJECTIVE:
To observe the efficacy of siRNA cocktails generated by recombinant human Dicer on the down-regulation of epidermal growth factor receptor (EGFR) expression in human glioma cells.DESIGN, TIME AND SETTING:
The following in vitro experiment was performed at the Department of Neurosurgery, Tianjin Medical University General Hospital and Laboratory of Neuro-Oncology, Tianjin Neurological Institute.MATERIALS:
Mini-RNA isolation kit, human placenta complimentary DNA (cDNA) was produced by Tiangen Biotech (Beijing, China), human glioblastoma U251-MG cells were produced by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.METHODS:
A PCR product from the human EGFR, which corresponded to the tyrosine kinase domain of the 3′-end fragment, was used as the T7-promotor for in vitro transcription. siRNA cocktails were generated by in vitro dicing of double stranded RNA. A total of 500, 250 and 125 μ g siRNA cocktails were transiently transfected into U251 glioma cells through the use of the GeneSilencer.MAIN OUTCOME MEASURE:
Expression of EGFR was detected by real-time PCR.RESULTS:
The total PCR product of the human EGFR, corresponding to the tyrosine kinase domain, is approximately 680 bp in length. The PCR transcriptants included GCC leader sequences and a T7 promoter sequence, with a fragment of EGFR cDNA at the center. The T7 promoter was prepared for in vitro transcription of dsRNA. After dicing for 24 hours, the 21-nt siRNA cocktails were verified by 4% agarose gel. The difference between threshold cycle of a sample assay and threshold cycle of the corresponding endogenous reference (Δ Ct) among parental U251 cells and cells transfected with different doses of siRNA cocktails were determined to be 3.06, 7.35, and 10.31 cycles, respectively. These results indicated effective silencing of EGFR expression by siRNA cocktails through transient transfection.CONCLUSION:
The siRNA cocktails significantly suppressed exogenous expression of EGFR in U251 glioblastoma cells in a dose-dependent manner, thereby providing a more promising RNAi methodology for glioma therapy.