ABSTRACT
Objective To investigate the radiosensitivity of silencing N-Ras by RNA interference in hepatoma carcinoma cell MHCC-97.Methods N-Ras RNA interference (RNAi) vector was constructed by using pcDNA 6.2-GW/EmGFP-mir plamid.The RNAi effect was detected by RT-PCR,Western bolt,immunohistochemisty and MTT method.Survival curve for each cell line were obtained by measuring the clone forming abilities of irradiated cell populations.Results After silencing the N-Ras by RNAi,The expression level of N-Ras mRNA,N-Ras protein,immunohistochemisty were decreased 96.9% ±0.159%(t=40.377,P＜0.05),89.8%±0.012% (t=31.595,P＜0.05),90%,respectively,and The survival of hepatoma carcinoma cell MHCC-97 line were inhibited 21.9% (F = 4.63,P < 0.05).Which have significant difference in statistics.The SER of hepatoma carcinoma cell MHCC-97 line after interference was 1.15.Conclusions RNAi targeting silence N-Ras may increase the radiosensitivity of hepatoma carcinoma cell MHCC-97 line.
ABSTRACT
Objective To explore the effect of BPDE on the expression of N-Ras in the human bronchial epithelial cell line. Methods The levels of mRNA and protein expression in BPDE transformed 16HBE cells(16HBE-T) and untransformed control 16HBE cells(16HBE-N) were examined by using RT-PCR and Western blot. Locations and expression levels of protein in both kinds of cells were analyzed by immunocytochemical method. Results Compared with 16HBE-N, the levels of mRNA and protein of N-Ras significantly increased to 3.616 and 1.600 times in 16HBE-T. Immunocytochemical method showed N-Ras protein in 16HBE-T and 16HBE-N expressed in the cytomembrane and cytoplasm, but the expression level of protein in 16HBE-T was significantly higher than that in 16HBE-N. Conclusion The up-regulated expression of N-Ras oncogene may play an important role in the malignant transformation of 16HBE induced by BPDE
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Objective To study the effects of the RNA secondary structure on the RNA interference efficacy and to optimize the RNAi target sequence selection.Methods N-Ras mRNA in K562 human chronic erythro-leukemia cell line was selected as the target for RNAi experiments.Four siRNAs were designed aiming at secondary structure region and none-secondary structure region separately.The N-Ras expression change and the cell growth was tested by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR),light microscopy,MTT,and flow cytometry.Results Knock-down efficiency was recorded in all of the 4 experiment groups,but the degree varied a lot.The siRNAs targeting none-secondary structure region presented higher silencing efficiency than those targeting secondary structure region.Conclusion The secondary structure of mRNA closely relate to the RNA interference efficiency,which should be considered in the processing of the target sequence selection.
ABSTRACT
Objective: To construct the recombinant vaccinia virus of mutant N-ras/61 gene and enhance the immunogenecity of mutant N-ras／61 protein produced by the recombinant vaccinia virus. Methods: N-ras／61 gene was inserted into P1108 and transfected into CV-1 cell infected with vaccinia virus by Cell FECTIN. PCR and Western blot were used to identify the recombinants. Results: We get recombinant vaccinia virus rV-N-ras/61 by PCR and tk- selecting. The expression of N-ras gene was detected by Western blot. Conclusion: This study is a test for studing effective vaccine of mutant N-ras/61 gene. The efficacy in vivo of the N-ras／61 vaccine in immunotherapy is under investgation.