Mesenchymal Stem Cells Affect The Progression Of Prostate Cancer Through PKMYT1-CCNB1/CCNE1 Pathway

Objective Prostate cancer is the most common malignant tumor in men worldwide,and its occurrence and development involve a variety of mechanisms.Recent studies have shown that mesenchymal stem cells(MSCs)can be recruited to the tumor site during the process of tumorigenesis and development,and become an important part of the tumor microenvironment.mesenchymal stem cells participate in the process of tumorigenesis and development.However,the role of mesenchymal stem cells in the progression of prostate cancer and the mechanism by which mesenchymal stem cells affect prostate cancer are not well understood.Therefore,this study aims to clarify the mechanism by which mesenchymal stem cells affect prostate cancer cells in the progression of prostate cancer.Methods 1.First,we screened genes associated with malignancy in prostate cancer.Through R-package GDCRNATools,we downloaded RNA sequencing data of 52 normal prostate tissue samples and 495 prostate cancer tissue samples in the TCGA database.We screened differentially expressed genes between prostate cancer tissues and normal prostate tissues.We performed a Weighted Correlation Network Analysis(WGCNA)on the RNA sequencing data of all prostate cancer tissues to find the module most relevant to the Gleason score.We performed a survival analysis on genes used for differential expression analysis to identify genes that significantly affect overall survival.Finally,we crossed the differentially expressed genes,the genes in the module most relevant to the Gleason score,and the genes that significantly affected the overall survival.We found a gene related to cell cycle that has not been reported yet,PKMYT1.2.We examined the expression of PKMYT1 in 27 prostate cancer tissues and 27 benign prostatic hyperplasia tissues by immunohistochemistry.We analyzed the expression levels of PKMYT1 in four prostate cancer cells LNCa P,C4-2,PC-3 and DU 145 by real-time quantitative PCR and Western Blot.Furthermore,we knocked down PKMYT1 by RNA interference in PC-3 cells of which the PKMYT1 expression was highest and evaluated the proliferation,cycle,colony forming ability,and migration of PC-3 cells after knockdown.We predicted possible downstream genes of PKMYT1 by protein-protein interaction analysis and gene expression correlation analysis,and verified them in PC-3 cells after knocking down PKMYT1.We also performed an online search and validation of PKMYT1 inhibitors.3.We knocked down PKMYT1 in LNCa P cells and evaluated the proliferation,cell cycle,PKMYT1 and downstream gene expression of LNCa P cells after knockdown.The mesenchymal stem cells and LNCa P cells were co-cultured by Transwell.The proliferation,cycle,and expression of PKMYT1 and its downstream genes were evaluated in the co-cultured LNCa P cells.At the same time,we investigated the sensitivity of LNCa P cells to enzalutamide before and after co-culture with mesenchymal stem cells by Cell Counting Kit-8.Result1.Through differentially expressing genes for TCGA prostate cancer data,we obtained 1794 differentially expressed genes.After WGCNA analysis,we obtained a purple module with 367 genes most relevant to the Gleason score.After survival analysis,we obtained 687 genes that have a significant effect on the overall survival of prostate cancer patients.After crossing the above three groups of genes,we obtained 7 genes,of which PKMYT1 has not been reported in prostate cancer.And it is a gene associated with the cell cycle.2.Immunohistochemistry results showed that the expression of PKMYT1 in prostate cancer tissues was significantly higher than that in benign prostatic hyperplasia tissues.Among the four prostate cancer cells LNCa P,C4-2,PC-3 and DU 145,PC-3 cells had the highest PKMYT1 expression.After knocking down PKMYT1 in PC-3 cells,cell proliferation was significantly inhibited,the proportion of cells in G0/G1 phase was significantly increased,the proportion of cells in G2/M phase was significantly decreased,colony forming ability was weakened,and migration ability was weakened.We got two downstream genes of PKMYT1,CCNB1 and CCNE1,by protein-protein interaction analysis and gene expression correlation analysis.It was verified that the expression of CCNB1 and CCNE1 was decreased after knocking down PKMYT1.We found a PKMYT1 inhibitor,Fostamatinib,and found that it significantly inhibited the proliferation of PC-3 cells.3.After knocking down PKMYT1 in LNCa P cells,cell proliferation was significantly inhibited,cell cycle changes of LNCa P cells were similar to that of PC-3 cells,and the expressions of CCNB1 and CCNE1 were also decreased.After co-culture with mesenchymal stem cells,the proliferation of LNCa P cells was significantly inhibited,the proportion of G0/G1 phase cells was significantly increased,and the proportion of G2/M phase cells was significantly decreased.These results were similar to the results of knocking down PKMYT1.Moreover,the expressions of PKMYT1,CCNB1,and CCNE1 in LNCa P was also significantly decreased after co-culture with mesenchymal stem cells.Treating LNCa P cells with enzalutamide revealed that LNCa P cells were less sensitive to enzalutamide after co-cultured with mesenchymal stem cells.Conclusions1.PKMYT1 is a gene that promotes the progression of prostate cancer,and it promotes cell proliferation by promoting the expression of CCNB1 and CCNE1.2.The inhibitor of PKMYT1,Fostamatinib,significantly inhibited the proliferation of prostate cancer cell line PC-3.3.When co-cultured with LNCa P cells,mesenchymal stem cells exert an inhibitory effect on LNCa P cells by inhibiting the expression of PKMYT1.4.Mesenchymal stem cells can induce LNCa P cells to enter a dormant state.Thereby LNCa P cells are less sensitive to enzalutamide.