The Role And Mechanism Of TREM2 In The Tumor Microenvironment And The Migration Of Prostate Cancer

Prostate cancer(PCa),also known as prostate adenocarcinoma(PRAD),is one of the most common malignancies of the male reproductive system and is the leading cause of cancer-related deaths in men worldwide.According to the latest data from the American Cancer Society and the National Cancer Institute,in 2020 there were approximately 191,930 new cases of prostate cancer and33,330 related deaths in the United States,accounting for 21%of all tumor incidence and 10%of mortality.In China,although the incidence of prostate cancer is slightly lower than that in Europe and the United States,with the aging of the population and improved diagnostic techniques,the incidence of prostate cancer has increased significantly and has a tendency to become progressively younger.According to the latest statistics in China,the annual incidence of prostate cancer exceeds 60,000 cases and 26,600 patients die from prostate cancer.This shows that prostate cancer has become a global public problem that threatens men’s health and lives.Although radical prostatectomy(RP)has proven to be the primary treatment for patients with clinically limited prostate cancer,approximately 20%of prostate cancer patients experience biochemical recurrence(BCR),become resistant to androgen deprivation therapy(ADT),and eventually develop metastatic debulking resistant prostate cancer(m CRPC).m CRPC patients have a median overall survival(OS)of 13-32months,with a 5-year survival rate of 15%.Therefore,it is imperative to explore the molecular mechanisms of prostate cancer to slow down the progression of prostate cancer and improve patient survival.In recent years,immunotherapeutic strategies based on targeting the tumor microenvironment(TME)have emerged as a promising approach for tumor treatment,which is considered to be one of the key factors in tumor initiation and spread and plays an important dynamic role in tumor develo-pment.In addition,the immune escape mechanism in the tumor microenviron-ment plays a key role in tumor development,metastasis and treatment resistance.Clinical studies have demonstrated that various immune checkpoint inhibitors,such as programmed cell death protein 1(PD-1)and cytotoxic T-lymphocyte-associated antigen 4(CTLA4),are widely used in the treatment of various malignancies,such as melanoma,kidney cancer and non-small cell lung cancer.However,compared to immune responsive cancers,prostate cancer is considered to be an immune cold cancer.Due to its low tumor mutation burden(TMB),complex TME and immunogenicity,prostate cancer patients have shown low responsiveness in clinical trials with anti-CTLA4 or anti-PD-1.Therefore,there is an urgent need to explore the molecular mechanisms related TME and to improve immunotherapy responsiveness in prostate cancer.Recently,the role of triggering receptors expressed on myeloid cells 2(TREM2)in tumors has been of great interest to scholars.Studies have shown that TREM2 is aberrantly expressed in a variety of malignancies,including kidney cancer,gastric cancer,and glioma,and serves as a potential prognostic marker as a pro-oncogene.More importantly,TREM2 acts as an immunosup-pressive receptor that regulates TEM and can stimulate immunosuppressive cells such as bone marrow-derived suppressor cells and tumor-associated macrophages(TAMs),while suppressing anti-tumor immune responses.Currently,the expression level,prognostic impact and molecular regulatory mechanisms of TREM2 in prostate cancer are not fully understood.In this study,we aimed to investigate the role of TREM2 in regulating the immune microenvironment and migration invasion of prostate cancer and its molecular mechanism.Firstly,based on TCGA,Oncomine,HPA and GEO database data and in vitro experiments,we used bioinformatics to detect the expression and clinical significance of TREM2 in prostate cancer.Meanwhile,the correlation between TREM2 and many factors of the immune microenvironment in prostate cancer was analyzed,and then the effect of TREM2 on the immune microenvironment of prostate cancer was elucidated.Finally,we explored the effect of TREM2 on migration and invasion in prostate cancer through in vitro experiments with molecular regulatory mechanisms centered on post-trans-criptional modifications of m~5C.This study was divided into four parts:Part One TREM2 expression in prostate cancer and its clinical signifi-canceObjective:To explore the expression of TREM2 in prostate cancer tissues and cell lines and its effect on the prognosis of patients with prostate cancer.Methods:1.Based on TCGA,Oncomine,HPA and GEO database data,bioinfor-matics methods were used to analyze the expression level of TREM2 in patients with prostate cancer.2.The expression level of TREM2 in prostate cancer and adjacent tissues was detected by immunohistochemistry,real-time quantitative PCR(q RT-PCR)and Western blot.3.Based on TCGA database data,Kaplan-Meier curve,Cox regression analysis,ROC curve and nomogram methods were used to evaluate the effect of TREM2 on clinicopathological characteristics and its prognostic value.Results:1.Based on TCGA,Oncomine,HPA and GEO database data analysis,TREM2 m RNA expression was up-regulated in patients with prostate cancer and pan-cancer.2.The expression level of TREM2 protein in prostate cancer tissue samples and cell lines was detected by immunohistochemistry,Western blot-ting and RT-PCR.The results showed that TREM2 protein expression was significantly up-regulated in prostate cancer tissues compared with adjacent normal tissues,and high TREM2 expression was correlated with Gleason score,but the difference was not statistically significant.3.Based on TCGA database data analysis,high expression of TREM2 was significantly correlated with clinical T stage,N stage,M stage,Gleason score,treatment outcome,residual tumor,age and progression free interval(PFI).4.Based on TCGA database data,Kaplan-Meier curve,Cox regression analysis,ROC curve and nomogram analysis showed that TREM2 high expression was associated with progression-free interval.Conclusions:TREM2 is up-regulated in prostate cancer tissue samples and cell lines,and is closely related to the poor clinicopathological characte-ristics of prostate cancer.TREM2 can be used as an independent prognostic factor for PFI in patients with prostate cancer.Part Two The effect of TREM2 on the tumor microenvironment of prostate cancerObjective: To explore the effect of TREM2 on the tumor microenvironment of prostate cancerMethods:1.Gene enrichment analysis(GSEA)was used to analyze TREM2 related signaling pathways.2.Functional annotation of TREM2 related genes was analyzed based on protein interaction network.3.Based on the TIMER database data,the ss GSEA algorithm was used to analyze the correlation between TREM2 and tumor immune cell infiltration.4.Based on TISIDB database data,the correlation between TREM2 and chemokines and/or chemokine receptors was analyzed.5.ESTIMATE algorithm was used to calculate the immune score and stromal score of TREM2 in tumor microenvironment.6.Based on TCGA database data,the correlation of TREM2 immune checkpoint was analyzed.7.Based on TISIDB database data,the correlation between TREM2 and immunomodulators was analyzed.Results:1.The TREM2 high expression group was significantly enriched in PI3K/Akt axis and cancer pathway.More importantly,multiple immune-related signaling pathways were significantly associated with TREM2 overexpression,including cytokine-cytokine receptor interaction,cytokines,inflammatory response,B-cell receptor signaling pathway,and chemokine signaling pathway.2.The expression level of TREM2 was significantly correlated with B cells(r=-0.273),CD8+T cells(r=0.069),CD4+T cells(r=0.344),macrophages(r=0.323),neutrophils(r=0.281)and dendritic cells(r=0.281)0.433).3.TREM2 expression is significantly correlated with a variety of tumor cell-related chemokines and their receptors.These genes included CCL3(r= 0.437),CCL17(r=0.405),CCL18(r=0.445),CCR2(r=0.359),CCR5(r= 0.429)and CXCR4(r=0.416).4.TREM2 expression was significantly correlated with estimated score(r=0.590),immune score(r=0.540)and matrix score(r=0.530).5.TREM2 was positively correlated with the expression of PD-1(r= 0.280),PD-L1(r=0.260)and CTLA4(r=0.320).6.TREM2 had the highest correlation with the six immunomodulators.HAVCR2(r=0.732),LGALS9(r=0.653),TGF-B1(r=327 0.6553),CSF1R(r= 0.51),ADORA2R(r=0.374)and LAG3(r=0.312),respectively.Conclusions: TREM2 expression is significantly correlated with many factors in the TME of prostate cancer,such as tumor infiltrating immune cells,tumor chemokines and their receptors,immune-matrix score,immune checkpoints and immunosuppressants.Based on this,it is speculated that TREM2 plays a crucial role in the TME of prostate cancer.Part Three The effect of TREM2 on the migration and invasion of prostate cancer cellsObjective: To explore the role and molecular mechanism of TREM2 in regulating the migration and invasion of prostate cancer cells.Methods:1.To detect the functional phenotype of TREM2 in prostate cancer by cell transfection with si RNA specific knockdown of TREM2.2.The effect of TREM2 on the migration and invasion of prostate cancer cells was detected by cell scratch test and Transwell test.3.Western blot was used to detect the expression of EMT-related proteins(such as vimentin,β-catenin and MMP-9),PD-1 and PI3K/Akt path-way.Results:1.To evaluate the effect of TREM2 on the migration and invasion of prostate cancer cell lines,TREM2-sirna was transfected into C4-2 and PC-3 cells.Knockdown of TREM2 expression significantly decreased the scratch healing ability of C4-2 and PC-3 cells compared with control cells.Similarly,the migration and invasion abilities of C4-2 and PC-3 cells were significantly decreased after silencing TREM2.2.The expression of EMT-related proteins vimentin,β-catenin and MMP-9 decreased after knocking down TREM2.3.the expression of PD-1 decreased after knocking down TREM2.4.TREM2 knockdown significantly reduced the expression of p-AKT and p-PI3 K,but there was no significant change in total AKT and PI3 K.Conclusions: TREM2 knockdown inhibits the migration and invasion of prostate cancer cell lines.TREM2 plays a role in promoting cancer through Akt/PI3 K signaling pathway.In addition,TREM2 is involved in the immune response of prostate cancer by regulating PD-1 expression.Part Four TREM2 promotes the migration and invasion of prostate cancer cells through RNA methyltransferase NSUN2Objective: To explore the mechanism by which TREM2 promotes the migration and invasion of prostate cancer cells by up-regulating the expression of NSUN2.Methods:1.The overall level of m5 C modification was examined using m5 C Me RIP-q PCR.2.Western blot was used to detect the expression of EMT-related pro-teins(such as vimentin,β-catenin and MMP-9),NSUN2 and Snail proteins.3.The level of NSUN2-enriched Snail m RNA was detected by RNA immunoprecipitation q PCR(RIP-q PCR).4.Methylated RNA immunoprecipitation q PCR(Me RIP-q PCR)was used to detect the role of NSUN2 in Snail post-transcriptional modification.5.The expression levels of Snail precursor and mature m RNA were determined by RT-q PCR.6.Immunohistochemical staining was used to detect the expression of NSUN2 in prostate cancer tissues.Results:1.NSUN2 mediated TREM2 migration and invasion in a m5 C modified manner.To determine whether TREM2 carcinogenic effects are dependent on m5 C modification,we examined the overall level of m5 C modification using m5 C Me RIP-q PCR.The results showed that knockdown of TREM2 significantly reduced the level of m5 C modification in C4-2 cells compared with normal control cells.2.NSUN2 enhances Snail m RNA stability by m5 C modification.