| Background:Bladder cancer(BC)is one of the common malignant tumors in urology.In the world,the incidence of bladder cancer ranks ninth among malignant tumors.In Europe and the United States,the incidence of bladder cancer ranks fourth in male malignant tumors.According to the statistics in 2018,the death toll of bladder cancer in the world is as high as 199,922.In our country,BC incidence and mortality have increased annually in recent years,which is extremely dangerous to human health in China.However,at present,there is still a lack of effective treatment for advanced bladder cancer,resulting in a poor prognosis and high mortality,and the 5-year overall survival rate is only 25%-35%.Therefore,important research directions include exploring the molecular mechanism of the occurrence,progression,and prognosis of bladder cancer,finding new targets for the treatment of BC,and searching for personalized and accurate treatment methods for bladder cancer.The occurrence and development of bladder cancer is a complex process,which is affected not only by internal genetic factors,but also by external environmental factors.Among them,the growth and apoptosis genes of tumor cells are out of control,which leads to the continuous growth of tumor cells.In order to adapt to the rapid growth,tumor cells promote the rapid proliferation of tumor by changing the metabolic mode of the cell itself.One-carbon unit metabolism connects amino acid metabolism with the biosynthesis of nucleotides,which is of great significance to the rapid proliferation of tumors.Methylenetetrahydrofolate dehydrogenase 2(MTHFD2),as one of the key enzymes of carbon metabolism,also plays an important role in many kinds of tumors.For example,MTHFD2 is highly expressed in head and neck squamous cell carcinoma,glioma,hepatocellular carcer,esophageal squamous cell carcinoma and renal cell carcinoma,and is closely related to the prognosis of patients.It is considered to be an independent biomarker to predict the prognosis.Some studies have found that MTHFD2 is involved in the process of tumor immune cell infiltration.Tumor microenvironment(TME)plays an important role both in understanding the occurrence,progressive and metastasis of tumor and in the diagnosis,prevention and prognosis of tumor.Among them,tumor immune cell infiltration plays an important role in the occurrence,progressive and prognosis of tumor.Immune escape has become an important factor in the poor effect of tumor therapy.The combination of programmed death ligand 1(PD-L1)on the surface of tumor cells and programmed death receptor-1(PD-1)receptor on the surface of immune cells has become an important mechanism of tumor cell immune escape.At present,it has become a hot topic in a variety of tumor immunotherapy.However,there are few reports about the effects of MTHFD2 expression and immune cell infiltration and PD-L1 expression on the progression and prognosis of bladder cancer.Objective:The purpose of this study was to observe the expression of MTHFD2 in bladder cancer and its relationship with clinical stage and prognosis of bladder cancer.To explore the effect of MTHFD2 on the proliferation of BC cells,and to clarify the relationship and mechanism between the expression of MTHFD2 and immune cell infiltration and PD-L1 expression in BC.To find an effective indicator of drug sensitivity of bladder cancer and to provide new ideas and methods for the diagnosis and treatment of BC.Methods:1.In order to determine the correlation between the expression of MTHFD2 and prognosis in BC.We analyzed the expression of MTHFD2 in BC and adjacent tissues by TIMER and TCGA online database;detected the expression of MTHFD2 in BC and adjacent tissues by immunohistochemistry,reverse transcription-quantitative PCR(RT-PCR)and Western blotting(WB);and detected the expression of MTHFD2 in BC cell line and SV-HUC-1 by RT-PCR.The relationship between the expression of MTHFD2 and clinical stage and prognosis was analyzed by TCGA database and bladder cancer tissue microarray(TMA),and the possible factors affecting the overall survival rate of bladder cancer were explored by univariate and multivariate Cox regression analysis.2.To determine the correlation between the expression of MTHFD2 and immune cell infiltration in BC.We used TIMER database to explore the relationship between the expression of MTHFD2 and immune cell infiltration and immune-related molecules in BC.The score of tumor microenvironment was calculated by ESTIMATE.Immunohistochemical staining was used to detect the expression of CD8 and PD-L1 in TMA of bladder cancer.3.To further confirm the role of MTHFD2 in promoting tumor proliferation in BC.We used lentivirus transfection method to knock down the MTHFD2 gene in bladder cancer cell lines(T24 and UMUC3)and verified by RT-PCR and WB.The ability of cell proliferation was detected by CCK-8 and colony formation assay.The changes of apoptosis and cell cycle were detected by flow cytometry.The tumor growth of bladder cancer cells with low expression of MTHFD2 was observed by an in vivo experiment in nude mice.Finally,the effect of low expression of MTHFD2 on the expression of PD-L1 was confirmed by WB and immunohistochemistry.4.To further explore the mechanism of the effect of MTHFD2 on the proliferation of bladder cancer.We used TCGA database to analyze differentially expressed genes and bioinformatics methods to analyze functional protein enrichment related the signal pathways.At the same time,the differentially expressed genes and enriched signal pathways in bladder cancer cells with silencing MTHFD2 were confirmed by the second generation of high-throughput mRNA sequencing(RNAseq)analysis.WB assay confirmed the expression of P13K/AKT(phosphatidylinositol kinase/protein kinase B)signal pathway related proteins(PI3K,p-PI3K,AKT,p-AKT)and PD-L1 in bladder cancer cells with low expression of MTHFD2.Then a series of functional recovery experiments confirmed that PI3K agonist(740Y-P)can restore the proliferation ability and PD-L1 expression of bladder cancer cells with low expression of MTHFD2.5.In order to understand the relationship between the expression of MTHFD2 and clinical drug sensitivity.We used the Genomics of Drug Sensitivity in Cancer(GDSC)database to analyze the correlation between clinical commonly used chemotherapeutic drugs and related targeted drugs and MTHFD2 expression.To provide reference value for guiding clinical drug use.Results:1.MTHFD2 was highly expressed in bladder cancer and associated with poor prognosis.First,we found that MTHFD2 was highly expressed in various tumors by analyzed TIMER online database,including BC.In the analysis of TCGA data,the unpaired tissue comparison revealed that MTHFD2 expression was increased in BC samples when 414 BLCA samples were compared to 19 normal bladder tissue samples.Similar results were obtained when 19 BLCA samples were compared to their matching normal bladder tissue samples.Secondly,bladder cancer TMA also confirmed that the expression of MTHFD2 in BC tissues was higher than normal tissues.At the same time,RT-PCR showed that the expression of MTHFD2 in BC cell lines(UMUC3,T24,J82,EJ,BIU,5637)was higher than that of SV-HUC-1.In addition,24 pairs of clinical BC samples and adjacent tissues were detected by RTPCR,and 6 pairs of high-grade bladder cancer and adj acent tissues were detected by WB.The expression of MTHFD2 in BC was higher than that in adjacent tissues.Finally,in TCGA database and TMA,we observed that the expression of MTHFD2 was strongly correlated with TNM stage and AJCC stage and poor prognosis.In the univariate Cox regression analysis,AJCC Stage,TNM-T stage,TNM-N stage,Age and MTHFD2 expression were significantly associated with the OS of patients with BC.In the multivariate Cox regression analysis,Age remains significant and independent,the expression of MTHFD2 showed a trend to correlate with patient OS.2.The relationship between MTHFD2 expression and immune cell infiltration in bladder cancer.Firstly,we investigated the relationship between MTHFD2 expression and immune cell infiltration in BLCA using the TIMER database.We found that MTHFD2 was negatively correlated with tumor purity and positively correlated with the infiltration of neutrophils,CD8+T cells,dendritic cells and macrophages.Meanwhile,we analyzed differences in immune infiltration between the low-and high-MTHFD2 expression groups for 22 immune cells using CIBERSORT.The distribution of B cell memory,B cell plasma,T cell follicular helper,Tregs,Monocyte,Macrophage M0,Macrophage M1,Macrophage M2,T cell CD4+naive,T cell CD4+memory activated,Myeloid dendritic cell activated,Mast cell activated,Mast cell resting,and Neutrophil was significantly different between the low-and high-MTHFD2 expression groups.In addition,we found the significant correlation between MTHFD2 expression and various immune-related molecules using the TIMER database,including immune-stimulators,immune inhibitors,MHC molecules,chemokines,and receptors.Especially,the association between MTHFD2 and immune checkpoint molecules was shown,MTHFD2 expression was strongly related with the expression of CD274,CTLA4,HAVCR2,LAG3,PDCD1,PDCD1LG2,TIGIT,and SIGLEC15.We found a significant positive correlation between TMB and MTHFD2 expression in the TCGA-BC cohort.Compared the tumor microenvironment(TME)related scores between the low-and high-MTHFD2 expression groups,it was apparent that samples with the high-MTHFD2 expression had higher Estimate,Immune,and Stromal scores.Finally,we verified the correlation between MTHFD2 and CD8/PD-L1 expression in the TMA cohort.The results again showed that MTHFD2 expression was significantly and positively correlated with CD8 and PD-L1 expression.3.MTHFD2 exerted oncogenic effects in BLCA.We constructed T24 and UMUC3 cell lines with stable knockdown of MTHFD2 expression.RT-PCR and WB assay showed that the expression of MTHFD2 was significantly decreased in T24 and UMUC3 cells after viral transfection.Then,CCK8 and colony formation assay showed that the proliferation ability of T24-shRNA2 and UMUC3-shRNA2 cells was significantly decreased.Flow cytometry showed that the number of cells in G1 phase increased in the T24-shRNA2 and UMUC3-shRNA2 groups,which made the cells stagnant in G0/G1 phase,and the later apoptosis of cells with knockdown of MTHFD2 was higher than that of the control group.We verified these findings that the xenografts in T24-shRNA2 group were significantly smaller and lighter in an in vivo experiment.We also detected PD-L1 expression in xenografts using IHC,and consistent with the cell lines,PD-L1 expression was significantly downregulated in the T24-shRNA2 group.4.Knockdown of MTHFD2 inhibited the proliferation of BLCA cells through inactive PI3K/AKT signaling pathways.First,we found that 2319 upregulated and 639 down-regulated DEGs by analyzing the transcriptome data of the TCGA cohort.Through functional enrichment analysis,we can find that the function of MTHFD2 was related to multiple metabolic pathways and associated with immune regulation,such as T-cell activation,cytokine production and interactions,as well as DNA replication and cell cycle regulation.And in terms of signaling pathways,PI3K/AKT/mTOR regulated pathways were significantly enriched.Similarly,a total of 367 up-regulated and 120 down-regulated DEGs were found in RNA-seq.And in the enrichment analysis,we found many familiar terms in the enriched functions and pathways,such as cytokine activity,chemokine receptor binding and metabolic process.Using GSVA(ssGSEA)analysis,we verified a significant positive correlation between MTHFD2 expression and PI3K/AKT pathway activation.Therefore,to further confirm the mechanism of MTHFD2 affecting the proliferation of BC cells by inhibiting PI3K/AKT signal pathway.WB assays showed that p-PI3K and p-AKT decreased significantly after silencing MTHFD2 in T24 cells,but no significant differences in total PI3K and AKT,which resulted in the decreasing ratio of p-PI3K/PI3K and p-AKT/AKT.When T24-shRNA2 cells were treated with 740Y-P,the protein levels of p-PI3K/PI3K and p-AKT/AKT were increased in a dose dependent manner.Finally,we conducted a series of functional recovery experiments using the 740Y-P.The results showed that the proliferation and colony formation ability inhibited by silencing of MTHFD2 in T24-shRNA2 cells restored after 740Y-P stimulation.Meanwhile,the later apoptosis induced by silencing of MTHFD2 was partially inhibited after 740Y-P stimulation.Moreover,PD-L1 expression decreased by silencing of MTHFD2 in T24-shRNA2 cells partially restored after 740Y-P stimulation.These results suggest that MTHFD2 functions in bladder cancer in part through the PI3K-AKT signaling pathway.5.MTHFD2 could be an effective indicator for chemotherapy and targeted therapy sensitivity of BC.We predicted the association between MTHFD2 expression and multiple drug sensitivity(IC50)in BC using the GDSC database.These drugs fall into two main categories,those recommended in clinical guidelines for conventional chemotherapy and those targeting the PI3K/AKT pathway.There was a significantly negative correlation between MTHFD2 expression and IC50 of conventional chemotherapy drugs,including doxorubicin,cisplatin,methotrexate,mitomycin C,5-fluorouracil,camptothecin,vinblastine,and gemcitabine.There was also a significantly negative correlation between MTHFD2 expression and IC50 of PI3K/AKT pathway targeting drugs,including A.443554,PF.4708671,AZD6482,TGX221,PIK-93,and YM201636,while a positive correlation with AKT.inhibitor.Ⅷ.These results suggest that MTHFD2 can be used as a potential indicator of drug sensitivity in the treatment of bladder cancer.Conclusion:We found that MTHFD2 was highly expressed in bladder cancer,and it was closely related to tumor stage,poor prognosis,tumor immune cell infiltration and PDL1 expression.The proliferation ability of bladder cancer cells with knockdown of MTHFD2 were significantly decreased,and the later apoptosis of cells increased significantly.We also found that Knockdown of MTHFD2 inhibited the proliferation of BC cells and expression of PD-L1 through inactive PI3K/AKT signaling pathways.And the expression of MTHFD2 is related to traditional chemotherapeutic drugs and targeting the PI3K/AKT signal pathway drugs sensitivity.To sum up,MTHFD2 regulating bladder cancer cell proliferation and PD-L1 expression via the PI3K/AKT Signaling Pathway.MTHFD2 may be a potential marker and therapeutic target for chemotherapy and immunotherapy of bladder cancer. |
