The Role Of Nonsense Mrna Decay In Human Breast Cancer Research

Objective: To study the application of quick and slow transferring methods and different immunoblotting membranes and detecting methods in western blotting.Methods: Proteins were extracted from human breast cell lines (MDA-MB-231 ) for western blotting analysis with different transferring (slow and quick ) methods, different immunoblotting membranes (nitrocellulose, PVDF and nylon membranes) and different (ECL and DAB ) detecting methods.Results:①As for slow and quick transferring methods, the strips of prestained marker in nitrocellulose membrane were stronger than those of nylon and PVDF membranes, and the face and reverso sides of nylon and PVDF membranes were easily confused;②The picture of PVDF through DAB method was slightly clearer than that through nitrocellulose and nylon membrane, and the quick transferring strips were not so regular as the slow transferring strips.③The background of nylon membrane through quick transferring method was obvious, while others had no obvious backgrounds.Conclusions:Different immunoblotting membranes should be chosed according to the needs of experiment; the result of slow transferring method is usually better than that of quick transferring method; ECL is superior to DAB. ECL is a satisfactory method to analyze protein expression with high specificity and sensitivity.PART TWO EXPRESSION AND SIGNIFICANCE OF Y14 AND Upf1 IN HUMAN BREAST CANCER CELLS AND BREAST EPITHELIAL CELLObjective:To study the expression and significance of Y14 and Upf1 in human breast cancer cells and breast epithelial cell.Methods:Western blotting, RT-PCR and Northern blotting were applied to detect the expression of Y14 and Upf1 in human breast cancer cells(MCF-7,ZR-75-30,T47D,MDA-MB-435s,MDA-MB-453,MDA-MB-231)and breast epithelial cell(HBL-100).Results:①Y14 and Upf1 levels of the breast cancer cells were obviously higher than those of breast cell(P<0.05).②Y14 and Upf1 levels of MDA-MB-231 were obviously higher than those of MCF-7.Conclusion: NMD activity increased obviously in the breast cancer cells. NMD activity of MDA-MB-231 is higher than that of MCF-7. PART THREE EXPRESSION AND SIGNIFICANCE OF Y14 AND Upf1 IN HUMAN BREAST CANCER, BENIGN TUMOR AND TUMOR-ADJACENT TISSUESObjective:To study the expression and significance of Y14 and Upf1 in human breast cancer, benign tumor and tumor-adjacent tissues.Methods:Western blotting, RT-PCR and Northern blotting were applied to detect the expression of Y14 and Upf1 in human breast cancer , benign tumor and tumor-adjacent tissues.Results:①Y14 and Upf1 levels of human breast cancer were obviously higher than those of tumor-adjacent tissues (P<0.05).②Y14 and Upf1 levels of the pathological gradeⅢwere obviously higher than those of gradeⅠandⅡ(P<0.05).③Y14 and Upf1 levels of axillary lymph node(+)group were obviously higher than those of axillary lymph node(-)group (P<0.05).④Y14 and Upf1 levels of CerBb-2(+)group were obviously higher than those of CerBb-2(-)group(P<0.05).⑤There were no significant difference of Y14 and Upf1 levels between ER(-)group and ER(+)group (P<0.05).⑥There were no significant differences of Y14 and Upf1 levels between PR(-)group and PR(+)group (P<0.05).⑦There were no significant differences of Y14 and Upf1 levels between≤2cm group and >2cm group (P<0.05).Conclusion:NMD activity increased in the breast cancer tissues, especially in those with high malignancy. Y14 and Upf1 levels increased in pathlogical gradeⅢ, axillary lymph nod(e+) and CerBb-2(+) group. Their expression levels had no relation with ER and PR status as well as tumor size.PART FOUR CONSTRUCTION AND IDENDIFICATION OF RECOMBINED Y14 shRNA PLASMID IN BREAST CARCINOMA CELLSObjective: To construct and idendify the recombined small hairpin RNAs (shRNA) plasmid targeted to Y14 gene, which can knock down the Y14 gene of breast carcinoma cells.Methods: Small hairpin RNAs (shRNA) targeted to Y14 coding gene were designed and synthesized according to the principle mentioned by Elbashir and Reynolds. It was cloned into pGenesil-1 which worked as a transcription vector to construct recombined plasmid named pshRNA1- Y14, pshRNA2- Y14 and pshRNAHK- Y14 (negative control plasmid). The recombined plasmid was extracted in middle quantity and transferred into breast carcinoma cells by LipofectamineTM 2000. RT-PCT and western blot were used to detect Y14 mRNA and protein respectively.Results: Three recombined plasmids: pshRNA1- Y14, pshRNA2- Y14 and pshRNAHK- Y14, were constructed successfully. The transfection rate is 90% by LipofetamineTM 2000. Y14 mRNA was suppressed to 24.16% and 29.72% in contrast to normal cells detected by RT-PCR, and Y14 protein was knocked down to 20.63% and 27.56% measured by western blot in cells transfected by pshRNA1- Y14 and pshRNA2- Y14 plasmid respectively. pshRNA1- Y14 is better than pshRNA2- Y14.Conclusions: Y14 protein in breast carcinoma cells can be knocked down effectively by recombined plasmid pshRNA1- Y14 and pshRNA2- Y14. The effect of pshRNA1- Y14 is better than that of pshRNA2- Y14.PART FIVE PROTEOMICS STUDY ON BREAST CARCINOMA CELLS TREATED WITH Y14 shRNA OR EMITINEObjective: To explore the protein variation point in breast carcinoma cells treated with Y14shRNA or emitine.Methods: To analyze the protein variation point in breast carcinoma cells by two-dimensional gel electrophoresis after being transfected with optimal recombined Y14shRNA plasmid or treated with emitine.Results: Compaired with control group of breast carcinoma cell MDA-MB-231, there were 25 protein variation points in emitine treated group and 20 protein variation points in Y14 shRNA transfected group. Compaired with ER negative breast carcinoma cell MDA-MB-231, there were additional 13 protein variation points in ER positive breast carcinoma cell MCF-7 treated with emitine or Y14 shRNA.Conclusions: Some protein variation points could be detected in breast carcinoma cells by two-dimensional gel electrophoresis after being transfected with optimal recombined Y14shRNA plasmid or treated with emitine.