SPDEF Transcription Activates CCL28 Expression And Inhibits Macrophage M2 Polarization To Prevent Immune Escape Of Colorectal Cancer Cells

Background:Colorectal Cancer(CRC)is one of the common malignant tumors in our country.In recent years,the incidence and mortality of colorectal cancer in our country have maintained an upward trend.According to the 2020 Cancer Statistics Report of China,the incidence and mortality of colorectal cancer ranked second and fifth respectively in all malignant tumors in our country.The current treatment of colorectal cancer patients with surgery and radiotherapy seriously affects the quality of patients’ survival.In recent years,through continuous exploration of the pathogenesis of colorectal cancer,several studies have confirmed that the immune system has a close relationship with the occurrence and development of colorectal cancer,and tumor cells can resist the recognition of the immune system through genetic and epigenetic changes or mutations(antigen loss or emergence of new antigens)constituting immune activities.Genetic and epigenetic alterations can also form neoantigens and immune microenvironment of tumor cells,and tumor immune editing makes immune cells "incompetent" or difficult to play anti-tumor role,which develops into immune escape of tumor and therefore greatly affects the therapeutic effect of tumor.Therefore,it is important to investigate the molecular mechanism of immune escape in colorectal cancer cells for the treatment of tumors.During tumor progression,tumor-associated Macrophages(TAMs)mainly exhibit the characteristics of M2 type macrophages,supporting tumor growth through pro-angiogenic,anti-inflammatory,and stromal remodeling functions.They function by secreting different growth factors such as VEGF,PDGF,EGF,chemokines,and cytokines such as IL-10,TGF-Β,CCL2,and CXCL12.In addition,macrophages are able to secrete matrix remodeling molecules to help tumor invasion,such as metalloproteinases and histone proteases.These functions make macrophages a new target for adjuvant antitumor therapy.The CCL28(mucosal associated epithelial chemokine)gene encodes a chemokine,which is expressed in many tissues in the human body.Studies have shown that CCL28 is closely associated in tumorigenesis and development.Several studies have shown that the expression level of CCL28 is also associated with the prognosis of many tumors such as breast cancer,colon cancer,and gastric cancer.In addition,CCL28 can be involved in the immune response of tumors by affecting the chemotaxis and activation of immune cells.Studies have shown that CCL28 can attract a variety of immune cells,such as T cells,B cells,and natural killer cells,to participate in the immune response of tumors.ccl28 can also promote the maturation and activation of dendritic cells and enhance their ability to present antigens,thus enhancing the immune response of tumors.In conclusion,the CCL28 gene may play a role in tumorigenesis and development,but its specific mechanism needs further study.SPDEF(SAM pointed domain containing Ets transcription factor)is a transcription factor that plays important biological functions in normal cells,including regulating processes such as cell differentiation,proliferation and apoptosis.Recent studies have shown that SPDEF also plays an important role in suppressing cancer development and progression.the expression level of SPDEF is down-regulated in cancers including prostate,lung,breast and colorectal cancers.In addition,SPDEF deficiency has been associated with tumor aggressiveness and poor prognosis.It was found that SPDEF inhibits the proliferation and invasion of cancer cells through multiple pathways,including inhibition of cell cycle progression,regulation of apoptosis,and inhibition of cell migration and invasion.In addition,SPDEF can inhibit the self-renewal and proliferation of tumor stem cells,thereby reducing tumor recurrence and metastasis.Therefore,SPDEF is considered as a potential oncogene,but its molecular mechanism is still unclear.Methods:1.COX regression analysis to screen 18 key Immune-related genes(IRGs)that predict prognosis of CRC patients in the TCGA database,integration of clinical prognostic data and gene expression data of CRC patients in the TCGA dataset to construct a multifactorial prognostic risk model and calculate the risk value for each sample,assess the accuracy of the model using ROC curves.2.12 key IRGs may be closely related to the clinicopathological characteristics of CRC patients,WGCNA analysis of 3 GEO microarrays to screen out 2 CRC gene co-expression modules,intersect the IRGs of TCGA database and GEO database to obtain 5 key IRGs.3.Select CCL28 for further analysis,screen of transcription factors of CCL28 gene,4 transcription factors were found to be co expressed with CCL28,it is speculated that the transcription factor of CCL28 gene is SPDEF based on scientific research results and references.4.Analyze the relationship between SPDEF and CCL28 and the expression of M2 macrophages in CRC clinical samples.5.Verify the activation of CCL28 expression by SPDEF transcription in vitro experiments.6.Verify that SPDEF upregulates CCL28 expression to prevent M2 macrophage polarization and inhibit CRC cell growth in vitro experiments.7.Verify that SPDEF can upregulate CCL28 expression,prevent M2 macrophage polarization,and inhibit CRC cell tumorigenesis in NOD-SCID mice in vivo experiments.Results:1.COX regression analysis screened 18 key IRGs that could predict prognosis of CRC patients(1)A total of 477 IRGs were found to be differentially expressed in CRC samples through the TCGA database,of which 297 IRGs were down-regulated and180 IRGs were up-regulated.(2)Univariate and multifactorial COX regression analysis screened 18 key IRGs,12 of which were closely associated with clinicopathological characteristics of CRC patients.2.5 key IRGs obtained by combining the results of TCGA and GEO database CRC correlation analysis(1)WGCNA analysis of 3 GEO microarrays screened for 2 CRC gene co-expression modules;(2)Using the GEO database dataset and TCGA database,a total of 5 key IRGs were finally found to be validated in GEO data(CCL28,ESM1,FABP4,STC2 and VIP).3.CCL28 may regulate macrophage infiltration through SPDEF transcription factors involved in CRC cell immune escape(1)Tumor associated macrophages(TAMs)are deeply involved in the occurrence and development of tumors,the results of immune infiltration analysis showed that CCL28 in CRC cells may be involved in CRC cell immune escape by affecting macrophage infiltration,we choose CCL28 for subsequent analysis.(2)Tumor-associated transcription factors were obtained using the Cistrome website,and 21 transcription factors differentially expressed in CRC were screened.Correlation analysis showed that CCL28 was positively regulated with transcription factors NR5A2,KLF4 and SPDEF,while negatively regulated with transcription factor MYH11.We speculate that SPDEF may transcriptionally activate the expression of CCL28 through the validation of TCGA and GEO databases and relevant literature.4.The expression of SPDEF and CCL28 was verified by CRC clinical sample analysis(1)RT-qPCR,WB and immunohistochemistry were used to analyze the clinical samples of CRC.It was found that the expression of SPDEF and CCL28 was decreased and the polarization of M2 type macrophages was increased in the samples of CRC compared with the normal tissues.5.It was confirmed that SPDEF could activate the expression of CCL28 in vitro.(1)CHIP-qPCR results showed that SPDEF was more enriched in the P1 region of the CCL28 promoter.(2)Double luciferase assay showed that CCL28-WT luciferase activity increased significantly after overexpression of SPDEF,while CCL28-MUT did not change significantly.(3)RT-qPCR showed that SPDEF gene was over-expressed in CT26 and HCT-116 cells,and the expression of SPDEF and CCL28 was increased,but the effect of over-expression of SPDEF on CCL28 could be reversed by interfering CCL28.6.In vitro,overexpression of SPDEF could up-regulate the expression of CCL28 and inhibit M2 polarization of macrophages,thus inhibiting the growth of CRC cells.Interference of CCL28 could reverse the effect of overexpression of SPDEF.(1)The results of RT-qPCR,WB,Elisa and Flow cytometry showed that SPDEF could up-regulate the expression of CCL28 and inhibit M2 polarization in macrophages.(2)The results of CCK8 test,Transwell test and Flow cytometry test suggested that SPDEF could up-regulate the expression of CCL28 and inhibit M2 polarization of macrophages,thus inhibiting the growth of CRC cells.7.SPDEF could up-regulate CCL28 expression to prevent M2 macrophage polarization and inhibit CRC cell tumorigenesis in NOD-SCID mice.(1)a subcutaneous xenograft model of NOD-SCID mice was constructed and found that tumor growth was slower and tumor weight was reduced in the oe-SPDEF group compared with the oe-NC group,but compared with the oe-SPDEF group,tumor growth and tumor weight increased in the oe-SPDEF+sh-CCL28 group.(2)the m RNA and protein expressions of Il-10,Arg-1,SPDEF,CCL28 and CD163 were detected by RT-qPCR,WB,immunohistochemistry and Elisa,SPDEF could up-regulate CCL28 expression,prevent M2 macrophage polarization and inhibit CRC cell tumorigenesis in NOD-SCID mice.Conclusions:1.In the bioinformatics analysis,18 key IRGs were identified,12 of which were closely related to the clinicopathologic features of CRC patients.Five IRGs,including CCL28,were screened,which may be involved in immune escape of CRC cells.The prognostic risk model was constructed and the accuracy of the model was evaluated by ROC curve.2.SPDEF transcriptionally activated CCL28 expression was confirmed by analysis of CRC clinical samples and in vitro experiments.3.SPDEF can transcriptionally activate CCL28 expression,inhibit M2 polarization of macrophages and prevent immune escape of CRC cells.It provides new ideas and potential therapeutic targets for the immunotherapy of CRC.