Transcriptional Regulation Of NPRL2 And Mechanisms To Promote Proliferation In Prostate Cancer Cells

PART 1: BIOINFORMATICS ANALYSIS OF NPRL2 IN PROSTATE CANCER TISSUEObjective: To conduct a bioinformatics analysis on the expression of NPRL2 in prostate cancer tissue and the prognosis of patients.Methods: We analyzed the expression characteristics of NPRL2 in prostate cancer and normal tissues,and its relationship with prognosis based on the TCGA and GEO datasets.Tissue samples from prostate cancer patients in the TCGA database were used for GSEA analysis.Results: In the TCGA database,NPRL2 is highly expressed in prostate cancer tissues than normal tissues.At the same time,we used two GEO data sets(GSE68882 and GSE6956)to find similar patterns.Then,the human protein atlas was used to observe the expression of NPRL2 detected by immunohistochemistry in the prostate cancer tissues and normal prostate tissues.Among them,the expression of NPRL2 was significantly higher than that of normal prostate tissue.Furthermore,we analyzed 485 prostate cancer patients with complete prognostic information in the TCGA database and divided them by the median expression value of NPRL2,which found the disease-free survival(DFS)and overall survival(OS)of prostate cancer patients with high NPRL2 expression was significantly lower than that of patients with low NPRL2 expression.Subsequently,we used the full transcriptome expression data in the TCGA database to divide the patients into two groups with NPRL2 high and low NPRL2 expression based on the median expression value of NPRL2 to conduct GSEA analysis,which indicated that NPRL2 may participate in multiple functions in prostate cancer.Conclusion: NPRL2 gene is highly expressed in prostate cancer tissues,and the prognosis of prostate cancer patients with high NPRL2 expression is worse than the low expression group of PCa,which may be involved in a variety of biological behaviors in the occurrence and development of prostate cancer.PART 2: MOLECULAR MECHANISM OF NPRL2 IN TRANSCRIPTION LEVEL REGULATIONObjective: To explore the transcriptional level regulation mechanism of NPRL2 and reveal the mechanism of high expression of NPRL2 in prostate cancer.Methods: Promoter sequences of different lengths was constructed by PCR.The dual luciferin reporter gene was used to detect the activity of different lengths promoters.Use bioinformatics software to predict the binding of transcription factors in promoter's region and detect the binding of transcription factors to NPRL2 by Western blot,q PCR,dual luciferin reporter gene,and chromatin immunoprecipitation(Ch IP).Results: First,we constructed the-2000 bp promoter sequence upstream of NPRL2 by ordinary PCR and constructed multiple promoter sequences of different lengths based on the-2000 bp sequence,which were loaded by plasmid after transfection into the cells.The activity of the dual luciferin reporter gene was tested,and the promoter activity from-1500 to-1100 bp was found to be the strongest.JASPAR predicted the binding sites of multiple transcription factors and verified by designing si RNA.It was found that when the transcription factor c-Jun was silenced,the m RNA and protein expression levels of NPRL2 were significantly suppressed.Subsequently,the dual luciferin reporter gene and Ch IP proved that c-Juncan bind to the upstream sequence of NPRL2,thereby regulating the expression of NPRL2.We used GEO data set GSE6956 analysis to find that the expression of c-Jun was highly expressed in prostate cancers,similar to NPRL2.The GSE68882 dataset found that the expression of NPRL2 gene was significantly positively correlated with c-Jun.At the same time,according to the median expression of NPRL2 and c-Jun,the relationship between gene expression and the prognosis of prostate cancer patients was explored in the TCGA database.Patients with high expression NPRL2 and c-Jun at the same time had the worst disease-free survival and overall survival.Conclusion: The transcription factor c-Jun could bind to the upstream promoter of NPRL2 at the transcription level to promote the expression of NPRL2.PART 3: THE EFFECT AND MECHANISM OF NPRL2 ON THE PROLIFERATION OF PROSTATE CANCER CELLSObjective: To study the effect of NPRL2 silencing or overexpression on the proliferation of prostate cancer cells in vivo and in vitro.Methods: Lentivirus was used to construct prostate cancer cell lines(LNCa P and PC3)that stably silence or overexpress NPRL2.CCK-8,clone formation,flow cytometry,Western blot were used to verify the effect of NPRL2 on the proliferation activity and apoptotic protein expression of prostate cancer cells.At the same time,silence or overexpress of c-Jun under the premise of silence or overexpression of NPRL2,and again detected the proliferation activity of prostate cancer cells.Results: CCK-8 showed that the proliferation ability of prostate cancer cells PC3 and LNCa P was significantly enhanced after overexpression of NPRL2,while the proliferation ability of silencing NPRL2 prostate cancer cells were significantly reduced.The clone formation also found similar results.When NPRL2 was silenced,the apoptotic rate of PC3 and LNCa P cells increased significantly,a large number of cells were blocked in G1 phase,the expression of apoptosis-related proteins Caspase 3(cleaved)and BAX were significantly reduced,and the expression of anti-apoptotic protein BCL2 was significant up-regulated.Tumor formation experiments in nude mice found that the prostate cancer cells silencing NPRL2 had smaller tumors and lighter weight than the blank cells.It was found that the cell growth was significantly accelerated,the survival ability was enhanced,the apoptosis rate was reduced,and the expression of apoptotic proteins Caspase 3(cleaved)and BAX were reduced in c-Jun and NPRL2 overexpressed group compared with the overexpressed NPRL2 while silencing c-Jun group in PC3 and LNCa P cells.When c-Jun and NPRL2 were silenced at the same time,the cell proliferation activity was significantly weakened,the apoptotic rate was significantly increased,and the expression of apoptotic proteins Caspase 3(cleaved)and BAX were significantly increased.Conclusion: NPRL2 plays a role in promoting proliferation in prostate cancer cells.This role is related to the participation of c-Jun,which could enhance the proliferation effect of NPRL2.PART 4: THE MOLECULAR MECHANISM OF NPRL2 PROMOTING THE PROLIFERATION OF PROSTATE CANCERObjective: To reveal the molecular mechanism by which NPRL2 exerts oncogene function and promotes the proliferation of prostate cancer cells.Methods: We analyzed the differential gene expression of NPRL2 silenced PC3 cells and blank cells by transcriptome sequencing and performed KEGG pathway analysis on the differential expression genes.Then,Western blot was used to detect the difference of pathway molecules after silencing or overexpression of NPRL2,based on pathway inhibitors to verify the expression changes of pathway molecules again.Results: First,we found that the KEGG pathway with the most differential expression genes through transcriptome sequencing in NPRL2-silenced PC3 cells compared with blank cells was the PI3K/AKT pathway,which was involved in the proliferation of a variety of tumors.Western blot results showed that silencing NPRL2 resulted in p-AKT and p-MDM2 significantly reduced,while NPRL2 was overexpressed,the expression of p-AKT and p-MDM2 were significantly increased in prostate cancer cells PC3 and LNCa P.Because PC3 is p53-null cell line,silencing and overexpression of NPRL2 have no significant effect on the expression of p53 in PC3 cell line.In the LNCa P cell line,silencing and overexpression of NPRL2 up-regulate and down-regulate the expression of p53,respectively.Subsequently,we added MK2206,an inhibitor of AKT,to the prostate cancer cells overexpressing NPRL2 group.The results showed that after adding MK2206,the expressions of p-AKT and p-MDM2 were significantly reduced even if NPRL2 was overexpressed.At the same time,the CCK-8 indicated that the proliferation activity of cells was significantly reduced in overexpress NPRL2+MK2206 group than in the blank group cells.Compared with the blank group,the apoptotic proteins Caspase 3(cleaved)and BAX in the overexpression NPRL2 group were significantly reduced,while the expressions of Caspase 3(cleaved)and BAX in the MK2206 group were significantly increased when NPRL2 was overexpressed,and BCL2 expression was opposite to that of Caspase 3(cleaved)and BAX.NPRL2 may have a regulatory effect on CDK2,thereby affecting the AKT pathway,and CDK2 may act as an intermediate effector to mediate the effect of NPRL2 on the AKT/MDM2/p53 pathway.Conclusion: NPRL2 promotes the proliferation of prostate cancer cells by regulating the AKT/MDM2/p53 pathway.PART 5: DEVELOPMENT OF AN AUTOPHAGY-RELATED GENE EXPRESSION SIGNATURE FOR PROGNOSIS PREDICTION IN PROSTATE CANCER PATIENTSObjective: Autophagy-related genes(ARGs)may be involved in a variety of biological functions in prostate cancer.However,there are currently few bioinformatics analyses on ARGs.By analyzing the expression differences and functional characteristics of ARGs in prostate cancer,a prognostic model of autophagy-related genes was constructed to guide the evaluation of patient prognosis.Methods: First,a total of 234 ARGs information were obtained from The Human Autophagy Database.Then,according to the Cancer Genome Atlas(TCGA)database,differentially expressed ARGs were identified in prostate cancer patients.The univariate and multivariate Cox regression analysis was performed to screen the relationship between core prognostic ARGs and overall survival(OS)and disease-free survival(DFS)of prostate cancer patients,and the prognostic model was established.Finally,the correlation between the prognostic model and clinicopathological parameters was further analyzed.Results: Through univariate and multivariate Cox regression analysis,the overall survival-related prognostic model was constructed based on FAM215 A,FDD,MYC,RHEB and ATG16L1,and prostate cancer patients were divided into high-risk and low-risk group according to the model(HR=6.391,95% CI=1.581-25.840,P<0.001).The area under the receiver operating characteristic curve(AUC)of the predictive model was 0.84.The OS-related predictive model scores with stage T3-4 and Gleason score>7was significantly higher than those with stage T1-2(P=0.008),and patients with Gleason score ?7(P=0.015).In addition,based on ARGs(ULK2,NLRC4,MAPK1,ATG4 D,MAPK3,ATG2 A,ATG9B,FOXO1,PTEN,HDAC6,PRKN,HSPB8,P4 HB,MAP2K7,MTOR,RHEB,TSC1,BIRC5,RGS19,RAB24,PTK6 and NRG2),a DFS-related prognostic model was constructed.The AUC was 0.85(HR=7.407,95% CI=4.850-11.320,P<0.001).The DFS-related prognostic model was related to the T stage(P<0.001),N stage(P=0.001),and Gleason score(P<0.001).NPRL2 was mostly related to these prognostic-related autophagy genes,such as MAPK1,FOX01,PRKN,HSPB8,ULK2,TSC1,MTOR,etc.It could be seen that NPRL2 may be involved in multiple links of autophagy-related genes.Conclusions: The prediction model of autophagy-related genes may have good clinical value in predicting the prognosis of prostate cancer patients.In the future,the detection of these genes may be the key to assessing the prognosis of PCa patients.Additionally,it also confirmed the role of NPRL2 in autophagy from the dimension of autophagy-related genes.