Research On Screening Of SiRNA Targetting To Silence Expression Of Mas Of NRK

Objective: Through direct transfecting small interferingRNA(siRNA) of the targeted to NRK-49F MAS gene in vitro, toobserve mRNA express inhibition and protein level of the MAS gene inthe NRK-49F, so as to select the effective siRNA，for further researchingthe MAS gene mechanism of action in kidney disease provide theexperimental basis. Methodes:（1）Design and synthesis of siRNA：three pairs of siRNA and a pair of negative-controled siRNA weresynthesized according to siRNA order design philosophy by retrieveingMAS gene sequence in NCBI. The three of them are respectivelysiRNA-1（5’-CCUGACCAGAGCUUUCAAATT-3’，5’-UUUGAAAGCUCUGGUCAGGTT-3’），siRNA-2(5’-GACCAAUCAAAUAUGACAUTT-3’,5’-AUGUCAUAUUUGAUUGGUCTT-3’) and siRNA-3(5’-GCCAUUACUACACAAUCGUTT-3’,5’-ACGAUUGUGUAGUAAUGGCTT-3’)，at the same times，negative control was siRNA (siRNA-neg)（5’-UUCUCCGAACGUGUCACGUTT-3’,5’-ACGUGACACGUUCGGAGAATT-3’）.（2）Cell culture: the NRK-49F cell placed in culturemedium of DMEM/F12which contained10%of fetal calf serum，100U/ml of penicillin and100U/ml of streptomycin at37℃,5%ofcarbon dioxide sterile incubator，1×105/ml of cell suspension were madeand inoculated in six orifice plate for experiment after cells in suspensionculture showing logarithmic growth.(3) Groups: five groups weredivided: group One was MAS siRNA-1which mixedtransfection complexes contained the siRNA sequence one, group twoand three espectively contained the siRNA sequence two and three, group four was negative control which mixed the transfection complexes of thesiRNA sequence negative, the last group just put into hiperfecttransfection reagent, each group had the three replicate wells.(4)Transfection: after12ul of hiperfect transfection reagent mixed nutrientsolution without serum and double antibody into100ul oftransfection complexes, added10nM of final concentration siRNA, thenthe blending incubated at room temperature for5-10minutes. Thetransfection complexes TC was put into six orifice plate, and gentlyshaked to ensure it evenly distributed, transfection efficiency wasmeasured after incubated in the constant temperature box for48houres.(5) Gene expression detect: the mRNA and protein expression of allNRK-49F cell line were detected by Reverse Transcription-PolymeraseChain Reaction and Western Blot.(6) Image process: each group grayvalue were assayed by Quantity One4.4.0.(7) statistical analysis: thespss17.0software was applied for statistical analysis, and the differencesall groups were compared with single factor analysis of variance，theywere statistically significant as P<0.05. Results: MAS gene express aredifferent degree of inhibited by all three of targeted siRNAs. Comparedwith the blank control group, the mRNA expression of NRK-49F cellMAS gene in group siRNA-1, siRNA-2and siRNA-3had significantlydecreased with method of RT-PCR, the ratio of MAS，GAPDH grey valueof each groups were0.5772±0.0220，0.3380±0.0434,0.6164±0.0767, theinhibition rate respectively were26.89％,57.12％,21.73％, there wereno significant decline between the negative control group and blankcontrol group. The Protein expression of specific sequence siRNAtargeted MAS were obviously lower than the blank control group withmethod of Western blot, the inhibition rate respectively were59.47％,67.81％,50.32％. the negative control group also was no evidently difference with the blank control group, who were approximatelyconsistent with the resrult of the methode of RT-PCR. Conclusion：1．ThesiRNA of transfection and synthesis in vitro which target to gene MAScould transfect into the NRK-49F by cationic compounds, and inhibit thegene express.2．The siRNA that could efficiently and specifically silenceMAS of the NRK-49F was screened out.