Mechanism Of FOXA1/INMT Signal Axis Inhibiting Proliferation And Promoting Apoptosis Of Prostate Cancer

Objective: Prostate cancer is the second most common malignant tumor in men and the fifth most common cause of cancer death in the world.About 10%(307000)of male cancer-related deaths are caused by prostate cancer.Prostate specific antigen(PSA)can be used to screen prostate cancer,but its specificity and sensitivity are limited.Therefore,there is an urgent need for new biomarkers to be more accurately used in the diagnosis and prediction of prostate cancer.Multiomics can not only more accurately predict the response of cancer cells to chemotherapy or immunotherapy,but also find biomarkers with more diagnostic / prognostic value.INMT is down regulated in prostate cancer,but its specific mechanism is not clear.The study of its molecular mechanism may help to deepen the understanding of tumorigenesis and development,and find new targets in various cancers.FOXA1,as a driving factor in the pathogenesis and progression of breast cancer and prostate cancer,is involved in hormone receptor dependent transcription processes to regulate prostate or mammary gland specific gene expression and disease progression.This study will take the joint analysis of metabolomics and transcriptomics of prostate cancer as the starting point,jointly verify the biological role of INMT in inhibiting the proliferation and promoting apoptosis of prostate cancer cells from in vitro and in vivo experiments,prove that FOXA1 as a transcription factor inhibits the transcription of INMT,screen and identify the effects of INMT on MAPK,TGFβ and Wnt signaling pathways,deeply reveal the molecular mechanism of INMT in prostate cancer,and strongly prove the important role of INMT in the occurrence and development of prostate cancer.Methods: 1.Extensive targeted metabolome technology,extensive targeted metabolome and transcriptome combined analysis technology Collect fresh prostate specimens: 30 pairs of prostate cancer tissue samples and adjacent tissue samples,30 cases of benign prostatic hyperplasia tissue samples,and 30 cases of prostate cancer tissue samples after endocrine therapy.Through the identification of metabolites and the quality control analysis of sample data,and screen out some different metabolites,so as to predict and analyze the relevant functions of metabolites in the sample.Further,through correlation analysis,metabolic pathway enrichment and other bioinformatics techniques,the molecular biological functions and pathway regulation mechanisms of metabolome and transcriptome of prostate cancer were comprehensively and systematically analyzed,and the biological model of molecular mechanism was constructed.From the above results,significant metabolic pathways,differential metabolic enzymes and differential metabolites were selected and analyzed in-depth experiments to reveal their biological mechanism or clinical application.2.Bioinformatics analysis Based on TCGA data resources and GSE46602 data set,the correlation between INMT m RNA expression and clinical data was analyzed.Use GSEA to elucidate the functional cascade associated with INMT RNA expression in prostate cancer;The most significant biological and molecular functions of INMT were revealed by the analysis of INMT co-expression m RNA;The correlation between tumor microenvironment and immune infiltration was analyzed by CIBERPORT,CIBERPORT-ABS,QUANTISEQ and other algorithms.3.Lentivirus transfection technique was used to verify the effect of INMT on the proliferation and apoptosis of prostate cancer cells The prostate cancer cell line was transfected with INMT overexpression lentivirus.The overexpression efficiency of INMT was verified by Western Blot and RT-q PCR.The ability of cell proliferation and apoptosis was detected by Western Blot,RTCA,CCK-8 and flow cytometry.4.In vivo experiments verify the biological function of INMT in prostate cancer The prostate cancer cell lines with stable overexpression of INMT were constructed by lentivirus transfection technology,and the subcutaneous tumorigenesis experiments were carried out in nude mice.Firstly,Balb/c nude mice for 4-6 weeks were injected with prostate cancer cells under the right armpit to observe the growth of the transplanted tumor.After 30 days of tumor formation,they were killed,and different groups of tumors were weighed.5.Transcriptional regulation of FOXA1 and INMT The upstream transcription factors and binding sites of INMT were predicted by UCSC and JASPAR database;After transfection of FOXA1 or INMT si RNA,the changes of INMT and FOXA1 protein levels were detected by Western Blot to determine the upstream and downstream relationship of FOXA1 and INMT;Ch IP-q PCR experiment was used to verify the combination of FOXA1 and INMT promoter region and its transcriptional regulation function.6.Transcriptional changes of prostate cancer cells transfected with INMT virus After constructing a prostate cancer cell line with stable overexpression of INMT by lentivirus transfection technology,transcriptome analysis was carried out to identify the differential genes and pathways compared with the negative control group.The changes of MAPK,TGFβ and Wnt signaling pathways were verified by Western Blot.7.INMT affects glycosylation modification of prostate cancer After knockdown and overexpression of inmt in prostate cancer cell lines 22RV1 and PC-3,O-Glc NAc Multi Mab antibody was used to detect the effect of INMT on glycosylation modification.Result: 1.Based on the widely targeted metabolome technology and the joint analysis technology of widely targeted metabolome and transcriptome,we found that 68 metabolites were up-regulated and one metabolite was down-regulated in prostate cancer.There were differences in multiple metabolic pathways including tryptophan metabolism,including Melatonin,N-Acetyl-5-Hydroxytryptamine and 2-(Formylamino)Benzoic Acid;The differential gene is INMT.2.Through bioinformatics analysis,we found that the expression of INMT in prostate cancer was lower than that in normal prostate tissue.Prostate cancer patients with low expression of INMT are more likely to have lymph node metastasis,increased Gleason score,increased PSA level and lower survival benefit.INMT expression is lower in most cancer tissues,such as such as bladder urothelial carcinoma,breast invasive carcinoma,ceramic square cell carcinoma and endocervical adenocarcinoma,colon adenocarcinoma,and head and neck squamous cell carcinoma,and kidney chromophobe.With the increase of INMT expression,tumor stage decreased,tumor purity decreased,estimate score increased,immune score increased,matrix score increased,immune response intensity increased,and a variety of immune cells and tumor microenvironment components increased.3.Western Blot showed that INMT was lower in androgen sensitive prostate cancer cell lines(LNCa P、22RV1 and VCa P)and higher in androgen resistant prostate cancer cell lines(PC-3,PC-3M and DU145).After lentivirus transfection of PC-3 and 22RV1 cell lines stably overexpressed INMT,Western Blot showed that the level of PCNA decreased,the level of Bcl-2 decreased and the level of cleaved-caspase 3 increased.Through RTCA and CCK-8,it was found that the proliferation ability of prostate cancer cells decreased after INMT overexpression.Through the detection of apoptosis by flow cytometry,we found that the apoptosis of prostate cancer cells increased significantly after INMT overexpression.4.The subcutaneous tumorigenesis experiment of nude mice showed that the tumor formed by injecting the overexpressed 22RV1 and PC-3 cells into the subcutaneous of nude mice was smaller and lighter than that of the control group.5.FOXA1 was predicted as the upstream transcription factor of INMT by UCSC and JASPAR database.After transfection of FOXA1 or INMT si RNA,the changes of INMT and FOXA1 protein levels were detected by Western Blot,which confirmed that FOXA1 was the upstream factor of INMT;Ch IP-q PCR experiment was used to verify the combination of FOXA1 and INMT promoter region and its transcriptional regulation function.6.22RV1 lentivirus transfection stably overexpressed INMT.Transcriptomic analysis showed that 561 genes were down-regulated and 731 genes were up-regulated compared with the negative control.The enrichment pathway is significantly related to biological processes such as DNA replication,mitosis,cell cycle and apoptosis.Western Blot confirmed that phosphorylated p38 MAPK in MAPK pathway was significantly up-regulated after INMT overexpression,but the total amount of p38 MAPK did not change significantly.The expression of Smad4,a key factor in TGFβ signaling pathway,was significantly reduced.In PC-3 cell line,the expression of TGFβ decreased after overexpression of INMT.Correlation factors of Wnt signaling pathway(β-catenin and cyclin D1)were down-regulated.7.After knockdown and overexpression of INMT in prostate cancer cell lines 22RV1 and PC-3,O-Glc NAc Multi Mab antibody was used to detect.It was found that O-Glc NAc modification was up-regulated in different molecular weight proteins after knockdown of INMT,while O-Glc NAc modification was down-regulated in different molecular weight proteins after overexpression of INMT.Conclusion: There are abnormalities in various metabolic pathways including tryptophan metabolism in prostate cancer.Low expression of INMT is associated with lymph node metastasis,high Gleason score,high PSA and poor survival benefit.INMT inhibits the proliferation of prostate cancer cells and promotes apoptosis.INMT is related to tumor microenvironment,immune infiltration and drug sensitivity.FOXA1,as a transcription factor of INMT,inhibits its transcription.INMT affects MAPK and TGFβ And Wnt signaling pathway,of which the effect on MAPK pathway is the most significant.INMT may be involved in glycosylation modification of prostate cancer.