Effects Of M/Z 6455.5da Protein On Renal Cell Carcinoma And Its Possible Mechanism

BackgroundRenal cell carcinoma(RCC)is one of the most common urological tumors.It originates in the Renal cortex.It accounts for 80%to 85%of malignant Renal diseases and 2%to 3%of all cancers,and its incidence is increasing year by year.Patients with advanced RCC have a extremely poor prognosis,with a median survival of only 18 months,poor sensitivity to radiotherapy and chemotherapy,and easy drug resistance.Therefore,it is of great significance to find a safe,effective and less side effect biotherapy for the treatment of patients with advanced RCC."Proteomics",first proposed by Wilkins et al.In 1996,refers to the protein composition of a specific tissue or organ at a specified time and under specific physiological(or pathological)conditions.With the continuous progress of mass spectrometry,proteomics research has been rapidly developed,and the research on the diagnosis and treatment of cancer has been continuously deepened.Proteomics is a comprehensive understanding of disease occurrence and cellular metabolism at the protein level.The study of proteomics can screen the differential protein spectrum of different tissues or body fluids through mass spectrometry for qualitative and quantitative analysis,which has important clinical application value in the search for tumor biomarkers,the study of tumor pathogenesis and the search for new and effective anti-tumor drugs.Our team compared Wilms' tumor children serum with healthy children serum by Surface enhanced laser desorption/ionization time of flight mass spectrometry(SELDI TOF MS)technology screening in the two groups have significant difference,the protein-M/Z 6455.5 Da and proved its in children with Wilms' tumor staging diagnosis and prognosis monitoring role,discovered the M/Z 6455.5 Da protein in Wilms' tumor are inhibition in vitro,the newly discovered in the future in the diagnosis and treatment of pediatric Wilms' tumor is of great significance.Therefore,the study of these safe,effective and less side effects of small molecular peptides may bring hope for the treatment of patients with advanced RCC.In this study,M/Z 6455.5Da protein was mainly used to intervene renal cell carcinoma cells,so as to explore the effect and mechanism of M/Z 6455.5Da protein on RCC.Purpose1.To study the effects of M/Z 6455.5Da protein on the proliferation and apoptosis of human renal cell carcinoma cells 786-O and ACHN.2.To investigate the molecular mechanism of M/Z 6455.5Da protein inducing the apoptosis of 786-O and ACHN in human renal cell carcinoma.Materials and Methods1.materialHuman renal cell carcinoma cells:786-O,ACHN,purchased from Chinese academy of sciences cell bank.M/Z 6455.5Da protein:this research group design,production by LifeTein LLC artificial synthesis(-20?,dark storage).2.methods?.Different concentrations of M/Z 6455.5Da protein were used to act on human RCC cells 786-O and ACHN,and Cell Counting Kit-8 test was used to detect the growth inhibition of M/Z 6455.5Da protein on human RCC cells 786-O and ACHN,and the Cell growth curve was plotted.FITC-labeled M/Z 6455.5Da protein at different concentrations were used to act on human RCC cells at 786-O and ACHN,and the localization of the peptides on the cells was detected by cell indirect immunofluorescence technique.M/Z 6455.5Da protein with concentrations of 0 mg/mL,0.5 mg/ml and 1.0 mg/ml were selected to intervene in renal carcinoma cells,and cell apoptosis was detected by flow cytometry.?.M/Z 6455.5Da protein at concentrations of 0 mg/ml,0.25 mg/ml,0.75 mg/ml and 1.25 mg/ml was selected to act on 786-O and ACHN of RCC cells,and the expression patterns of Bax,bcl-2 and PCNA proteins were obtained by Western blot assay,to preliminarily investigate the molecular mechanism of M/Z 6455.5Da protein inducing apoptosis of RCC cells.Results1.CCK-8 test showed that the growth inhibition rate of human renal cell carcinoma cells 786-O and ACHN(24 h,48 h,72 h)increased rapidly with the increase of polypeptide concentration under the same culture time with the treatment of human kidney cancer cells with M/Z 6455.5Da protein of 0,0.25,0.5,0.75,1.0,1.25,1.5 mg/ml,and the diference was statistically significant(P<0.05).Under the same polypeptide concentration,the growth inhibition rate of human renal cell carcinoma cells increased rapidly with the increase of time,and the difference was statistically significant(P<0.05).The results showed that the M/Z 6455.5Da protein could significantly inhibit the proliferation of renal carcinoma cells,showing a concentration-time dependent effect.Cell immunofluorescence assay showed that FITC-labeled M/Z 6455.5Da protein acted on the cell membrane or cytoplasm of renal cell carcinoma 786-O and ACHN cells in a concentration-dependent manner.In Annexin V-FITC/PI cell apoptosis assay,M/Z 6455.5Da protein acted on renal cell carcinoma cells 786-O and ACHN.Compared with the control group of 0 mg/ml,the apoptosis rate of 0.5 mg/ml and 1.0 mg/ml peptide concentration group gradually increased with the increase of peptide concentration,and the diference was statistically significant(P<0.05).The results showed that M/Z 6455.5Da protein induced apoptosis of human renal cell carcinoma in a concentration-dependent manner.2.The expression of Bax,bcl-2 and PCNA proteins in human renal cancer cells was detected by Western blot after the cells was treated with M/Z 6455.5Da proteins at concentrations of 0 mg/ml,0.25 mg/ml,0.75 mg/ml and 1.25 mg/ml for 48 h.It was found that Bcl-2 and PCNA protein expression in cells of the experimental group with concentrations of 0.25 mg/ml,0.75 mg/ml and 1.25 mg/ml were significantly lower than those of the control group with concentrations of Omg/ml M/Z 6455.5Da protein polypeptide(p<0.05).At the same time,with the increase of the concentration of M/Z 6455.5Da protein,the expression of Bcl-2 and PCNA proteins gradually decreased,and the difference between the low-concentration group and the high-concentration group was statistically significant(p<0.05).The results showed that M/Z 6455.5Da protein could inhibit the expression of Bcl-2 and PCNA protein in human RCC cells in a concentration-dependent manner.Bax protein expression in the cells of each experimental group was also found to be higher than that of the control group,with statistically significant differences(p<0.05).Moreover,the higher the concentration of M/Z 6455.5Da protein was,the higher the Bax protein expression was.The difference between the low-concentration group and the high-concentration group was statistically significant(p<0.05).The results showed that M/Z 6455.5Da protein could up-regulate Bax protein expression in a concentration-dependent manner.In summary,the apoptosis induced by serum marker M/Z 6455.5Da protein may be regulated by up-regulation of Bax expression and down-regulation of bcl-2and PCNA expression.Conclusion1.M/Z 6455.5Da peptide can inhibit the proliferation of 786-O and ACHN cells and induce the apoptosis of human kidney cancer cells.2.M/Z 6455.5Da peptide may play a role in inducing the apoptosis of 786-O and ACHN in human renal cancer cells by up-regulating the expression of pro-apoptotic protein Bax and down-regulating the expression of bcl-2 and PCNA protein.