| Renal cell carcinoma(RCC)is the most common cancer and the deadliest urogenital tumor.However,it is not a single entity,but a collection of multiple tumors,and is considered a phenotype representation of several parts of the renal tubules,each with different histological and genetic characteristics.Renal neoplasms,unlike many other aggressive or invasive neoplasms,are usually globular and glossal,extending into the renal vein,sinus,or perineal tissue,usually without fibrous proliferation or destructive infiltration.Therefore,renal cell carcinoma is less likely to be detected than cancer of other organs.In-depth knowledge of renal anatomy,adequate sampling of tissue,and prediction and careful study of these important findings on gross and microscopic examination.NPM1 belongs to the histone chaperone family,the nucleoprotein/nucleoprotein(NPM)family,which includes multiple functional members(NPM1,NPM2,NPM3 and invertebrate npm-like)and can be found in all multicellular animals.Although the family has good functional characteristics,little is known about the evolution of these genes and proteins.The purpose of this study was to investigate the differences in protein expression profiles between tumor tissues and adjacent tissues of renal clear cell carcinoma(RCC)using the proteomics method,so as to establish the differential expression profiles of RCC,so as to further understand the promoters or inhibitors of the occurrence and development of RCC through the study of some proteins.NPM1 was selected from the differential expression protein spectrum as the target protein,and its expression was verified by Western Blot.Immunohistochemistry was used to analyze the expression of protein NPM1 in renal clear cell carcinoma with different degrees of differentiation.A stable cell line with overexpression and low expression of NPM1 was constructed to observe the effect of RNA interfering expression of NPM1 gene on the proliferation,invasion and migration of 786-o in renal clear cell carcinoma cells,so as to further understand the effect of NPM1 on renal clear cell carcinoma.This study is divided into four parts.Part I: Establishment of differential protein profiles in renal clear cell carcinoma and adjacent tissues Methods:1.10 cases of renal clear cell carcinoma tumor tissue samples and paired adjacent normal tissue samples were selected as the research objects.All the paracancerous tissues were taken from the upper end of the cancer tissue and were more than 10 cm away from the cancer tissue.All patients received no preoperative chemotherapy or radiotherapy,and all tumor tissue samples were pathologically confirmed as renal clear cell carcinoma after surgery.Intraoperative resection group by PBS flush into the liquid nitrogen frozen,again at-80 ℃ refrigerator.2.Extract total proteins from tumor tissues and adjacent tissues,and analyze the protein concentration of tissues.3.Preparation of protein hydrolysis polypeptide mixture.4.Two-dimensional liquid chromatography was used for sample separation.5.The 2-d chromatography-eluted polypeptides were detected by LTQ XL hydrazine ion mass spectrometer.6.Bioinformatics analysis was performed on the mass spectrometry data to find the differentially expressed proteins in cancer tissues and adjacent tissues.Results:1.A total of 846 protein spots were obtained from renal clear cell carcinoma tissue samples by two-dimensional chromatography-mass spectrometry.A total of 535 protein sites were obtained from the adjacent tissues.By comparing these protein sites and searching the database,35 proteins were found to be differentially expressed in cancer tissues and adjacent tissues.2.The up-regulated expression of 20 proteins in tumor tissues were: NPM1,AHNAK2,PDIA3,MCT4,Cyp A,SKP1,CA9,SCGN,KRT18,HAPLN2,TGM2,CPQ,ACY1,POSTN,PAH,FREM2,LAMA4,CSPG4,GBP1,RBP4.3.The down-regulated expression of 10 proteins in tumor tissues were C11orf54,APOA4,SERPINA5,AGXT2,CHDH,NNT,ACE,ISCU,PRDX3,AK2,PNP,NPL,MRPL44,PHYH,GRHPR.4.Through this study,we established the differentially expressed protein spectra of renal clear cell carcinoma tissues and adjacent tissues,providing experimental data for further study on the pathogenesis of renal clear cell carcinoma.5.NPM1 was selected from the differential expression protein spectrum as the target protein for subsequent studies.Part Ⅱ: Validation of differential expression of NPM1 and its correlation with relevant clinicopathological data.Methods:1.The expression of target protein NPM1 in renal clear cell carcinoma and paracancerous tissue was determined by Western Blot in 10 cases of primary renal clear cell carcinoma and 10 cases of paired paracancerous tissue.2.The sections of 100 patients with renal clear cell carcinoma of different stages were collected,and the correlation between the expression of protein NPM1 and the stage of patients was observed by immunohistochemistry.Results:1.In 10 randomly selected groups of renal clear cell carcinoma tissue samples,the expression levels of the target protein NPM1 in the renal clear cell carcinoma tumor tissue and its paired paracancer tissues were compared and analyzed.It was found that the protein NPM1 was up-regulated in the renal clear cell carcinoma tissue.2.Through statistical analysis,the expression of NPM1 protein was considered to be correlated with TNM stage of tumor(p < 0.05).However,there was no correlation between the expression level of NPM1 protein and patients’ gender and age(p > 0.05).Part Ⅲ:Expression of NPM1 in renal clear cell carcinoma cell line and its effect on cell biological function.Methods:1.Preparation of monoclonal cell lines with overexpression and low expression of NPM1.2.The expression of NPM1 protein in 786-O cells transfected with Control plasmid and 786-O cells transfected with NPM1-sh RNA plasmid were observed under a fluorescence microscope.3.Western blot and RT-PCR were used to verify the gene level and protein level of stable cell line NPM1.4.The effects of proliferation,invasion and migration of 786-O cells transfected with plasmid and 786-O cells transfected with NPM1-sh RNA plasmid were observed.Results:1.We successfully constructed stable cell lines with overexpression and low expression of NPM1.2.We observed the effect of NPM1 on the biological behavior of renal clear cell carcinoma cells by observing the growth ability of cells under different expression levels of NPM1 in 786-O.The results of the growth curve showed that compared with the control group,786-O growth ability was significantly enhanced when NPM1 was overexpressed(p < 0.05),while 786-O growth ability was significantly weakened when NPM1 expression was decreased(p < 0.05).3.We observed the effect of NPM1 on the biological behavior of renal clear cell carcinoma cells by observing the migration ability of cells under different expression levels of NPM1 in 786-O.Results of cell migration showed that 786-O migration ability was significantly enhanced when NPM1 expression was overexpressed(p < 0.05),while 786-O migration ability was significantly weakened when NPM1 expression was decreased(p <0.05).4.We observed the effect of NPM1 on the biological behavior of renal clear cell carcinoma cells by observing the invasion ability of cells under different expression levels of NPM1 in 786-O.When NPM1 was overexpressed,786-O’s invasion ability was significantly enhanced(p < 0.05),while when NPM1 was decreased,786-O’s invasion ability was significantly reduced(p <0.05).Part Ⅳ: Tumorigenesis of stable cell lines in nude mice Methods:SPF grade nude mice aged 4 weeks were selected,and the successfully screened stable cell lines were subcutaneous injected into the nude mice.After 30 days,the tumor was killed,the tumor size was measured and weighed,and the expression level of protein NPM1 in the tumor was detected by western-blot,so as to analyze the differences between the tumor bodies with different expression levels of protein NPM1.Results:1.Tumor nodules could be observed subcutaneously in nude mice on the 9th day after inoculation of 786-O cells in each group.With the extension of time,the tumor gradually increased with hard contact and clear boundary with the surrounding area.2.The tumor was weighed,and the results showed that the average tumor weight of NPM1-overexpressed group was significantly higher than that of the control group(p < 0.05).The average tumor weight of NPM1-sh RNA group was significantly lower than that of the control group(p < 0.05).3.Western blot was applied to detect the expression of NPM1 protein in the transplanted tumor tissue of nude mice.The results showed that the expression of NPM1 protein in the NPM1-overexpressed group was higher than that in the control group(p < 0.05).NPM1 protein expression in NPM1-sh RNA group was significantly lower than that in NC group(p < 0.05).Through the above studies,we come to the following conclusions:1.In this study,the differential protein spectra of renal clear cell carcinoma tissues and adjacent tissues were successfully established.2.It was verified that the expression level of NPM1 in renal clear cell carcinoma was significantly higher than that in adjacent tissues;It was found that the expression of protein NPM1 was correlated with distant spread,TNM stage and differentiation level.3.The expression of protein NPM1 can promote the proliferation,invasion and migration of renal clear cell carcinoma cells.4.In vivo tumorigenesis experiments on nude mice verified the effect of protein NPM1 observed in vitro on tumor proliferation. |
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