Study On The Role Of Exosomes In Bone Metastasis Of Prostate Cancer And NDC80 As A Tumor Marker Of Clear Cell Renal Cell Carcinoma

Research background:The vast maj ority of advanced prostate cancer metastases are bone metastases,which are the main cause of death from this disease.Studies have shown that bone metastasis of prostate cancer is related to the activity of osteoblasts and osteoclasts.However,the mechanism of bone metastasis of prostate cancer is still unclear.Exosomes are small vesicles secreted by cells,carrying biological molecules such as nucleic acids,proteins,lipids,etc.,which can reach far away after entering the circulation of body fluids.Recent studies have shown that exosomes secreted by tumor cells can reach the metastatic site to participate in the formation of the pre-metastasis niche and promote the progress of tumor metastasis.Method:In this study,exosomes were obtained in two ways.One is to collect the supernatant of mouse prostate cancer cell RM-1 culture and extract it by ultracentrifugation;the other was the plasma exosomes of prostate cancer patients,extracted using kits.Characterization of exosomes was carried out by means of transmission electron microscope,Western Blotting,and Malvern laser particle size analyzer characterization.Subsequently,MTT assay,alkaline phosphatase detection kits,RT-qPCR test,Von kossa and other experiments were used to detect the effects of RM-1 exosomes and prostate cancer patients’ plasma exosomes on the proliferation and osteogenic differentiation of osteoblastic stem cells MC3T3 influences.RM-1 exosomes and prostate cancer patient plasma exosomes were added to RPMI-1640 medium,and MC3T3 cells were incubated for 20 hours,and then the supernatant was collected to make conditioned medium to culture RM-1 cells.MTT experiment,cell scratch experiment and plate cloning experiment were used to detect the influence of conditioned medium on the proliferation,migration and cloning ability of RM-1 cells.Results:Transmission electron microscopy(TEM),Malvern particle size analyzer,Western Blotting experiment showed that our extract was in the shape of a double concave dish,with a particle size between 30-150nm,carrying exosomal surface markers such as GRP94,CD9,and CD63.Are exosomes;RM-1 exosomes and prostate cancer patients’plasma exosomes affected the proliferation and osteogenic differentiation of MC3T3 cells.In the MTT experiment,it was found that both exosomes can promote the growth of MC3T3 cells to varying degrees.Proliferation activity,alkaline phosphatase detection kit,RT-qPCR test,Von kossa assay showed that it has no obvious effect on the osteogenic differentiation of MC3T3 cells;RPMI-1640 conditioned medium stimulated by MC3T3 could inhibit the proliferation of RM-1 cells And migration ability,but there was no significant difference between the H-exo-CM and M-exo-CM groups.The clone formation experiment showed that there was no significant difference between the C,C-CM,H-exo-CM and M-exo-CM groups.Conclusion:RM-1 exosomes and plasma exosomes of prostate cancer patients can promote the proliferation of MC3T3 cells,but under the experimental conditions,they have no significant effect on the osteogenic differentiation of MC3T3 cells;the secretion of MC3T3 cells can inhibit the proliferation of RM-1 cells And migration has no obvious effect on the cloning ability,but under the experimental conditions of this study,it is impossible to explain whether the exosomes can resist this effect.Background:Renal clear cell carcinoma is a relatively common cancer in the urinary system,and current research on it has not been able to determine its pathogenesis.Renal clear cell carcinoma is a kind of renal tubular epithelial cell adenocarcinoma,and it is also a common cancer of the urinary system.Clinically,this type of tumor is often mixed with granular cell carcinoma and spindle cell carcinoma.Therefore,there are great difficulties in diagnosis and grading,and its pathogenesis has not been proved yet.This project analyzed the factors that affect the grade,staging and prognosis of renal clear cell carcinoma through bioinformatics methods,and determined that NDC80 has the potential to become a biomarker for this type of tumor.Methods:Use Rstudio software to analyze the 4 renal clear cell carcinoma data sets in the GEO database and calculate the differences of highly expressed genes,use Venn Diagram to find common genes;import the selected genes into the TRING database,and use Cytoscape software to screen the core genes;use the CPTAC database to verify the protein expression levels of the above core genes;integrate the gene expression data and patient survival data in the TCGA database to screen The singlefactor and multi-factor survival regression analysis and overall survival analysis of the genes that come out;use GSEA software to analyze the KEGG pathway of the selected genes.Immunohistochemistry was used to detect the expression of NDC80 gene in tumor tissues and adjacent normal tissues;Western Blotting was used to detect the expression level of NDC80 in renal clear cell lines.After knocking down the expression of NDC80 protein in 786-O and Caki-1 cells with siRNA,CCK8,scratch test,Transwell chamber test,flow cytometry and Seahorse test were performed to detect the cell proliferation activity,migration ability,invasion ability,and cell cycle.And mitochondrial respiration.Results:A total of 338 up-regulated genes were screened from 4 GEO data sets,and 10 core genes were selected by protein-protein interaction PPI network analysis,namely ASPM,NUSAP1,CDK1,PBK,NCAPG,NDC80,KIF11,TTK,CCNA2 and DLGAP5.Verified by CPTAC database and TCGA database,ASPM,CDK1,PBK,NCAPG,NDC80 were all highly expressed in clear cell renal cell carcinoma and are related to the poor survival of patients with clear cell renal cell carcinoma.Univariate and multivariate regression survival analysis and overall survival analysis showed that only the expression of NDC80 can predict the survival prognosis of cancer patients.GSEA enrichment pathway analysis showed that high expression of NDC80 is related to cell cycle pathway and DNA replication,and low expression can negatively regulate the process of tricarboxylic acid cycle.NDC80 protein was highly expressed in renal clear cell carcinoma tissues.After knocking down NDC80 in 786-O and Caki-1 cells with siRNA,it inhibited cell proliferation,migration,invasion,cell cy cle and mitochondrial respiratory function.Conclusion:NDC-80 is highly expressed in clear cell renal cell carcinoma tissues,and is closely related to the prognosis of patients with clear cell renal cell carcinoma,and has the potential as a prognostic marker.The results of in vitro experiments show that NDC-80 can promote the function of renal clear cell carcinoma,and may be used as a new anti-cancer target.