Role And Mechanism Of METTL3 In The Cancer-promoting Phenotype Of Pancreatic Cancer-associated Fibroblasts

Pancreatic cancer is a gastrointestinal neoplasm with a very high degree of malignancy and a poor prognosis.The chemoresistance of pancreatic cancer is prominent.There are many interstitial components in pancreatic cancer,and the drug is extremely difficult to penetrate.It is of great significance to deeply study the role and specific molecular targets of the interstitial components of pancreatic cancer in facilitating the incidence and development of cancer.The role of the tumor microenvironment in the progress of cancer has been widely demonstrated.The most prominent feature of the tumor microenvironment of pancreatic cancer is the proliferation of interstitial tissue,and the most common cells are cancerassociated fibroblasts(CAFs).CAFs are highly heterogeneous in pancreatic cancer,and can be divided into tumor-promoting and tumor-suppressing types according to their functions.Non-selective targeting of CAFs will accidentally damage tumor-suppressing CAFs,thereby affecting the efficacy of pancreatic cancer treatment.Cancer-promoting CAFs and tumor-suppressing CAFs can transform into each other under certain conditions.Therefore,it is important to target the key molecules that mediate the tumorpromoting/tumor-suppressing phenotype transformation of CAFs.Long non-coding RNA(Lnc RNA)is an RNA with a length of more than 200 nucleotides without coding function,which plays a key role in the pathogenesis of pancreatic cancer.Lnc RNAs also play a key regulatory role in mediating the cellular communication between CAFs and tumor cells.Studying the expression and related biological functions of lnc RNAs in pancreatic cancer CAFs provides new ideas for therapeutic methods targeting pancreatic cancer interstitial tissue.m6A methylation modification is one of the most common epigenetic modifications in eukaryotes.Recently,it was found that the key regulators of m6 A methylation in pancreatic cancer and the level of m6 A methylation modification has significant changes in pancreatic cancer tissues and cells,but few studies have been conducted in pancreatic cancer stromal components and tumor-associated fibroblasts.METTL3,as the core regulatory gene of m6 A methylation modification,has been found to have a cancerpromoting effect in related studies,but the expression level and biological significance of METTL3 in CAFs have not yet been elucidated.There are also m6 A modification on lnc RNA,and they have regulatory effect on the production,transcription,degradation and other processes of lnc RNA.Studying how METTL3 regulates the expression of downstream lnc RNA through m6 A methylation modification provides new ideas for the mechanism and target molecules of methylation modification in CAFs.In this subject,we first isolated,extracted and identified CAFs in pancreatic cancer,and identified the functional typing of CAFs by co-culture with pancreatic cancer cells.The cancer-promoting CAFs were selected to be co-cultured with pancreatic cancer cells,and METTL3 was screened out through cell proliferation experiments to maintain the cancer-promoting phenotype of CAFs,which was verified by in vivo and in vitro experiments.Immunofluorescence staining of pancreatic cancer tissue in tissue microarray showed that METTL3 was significantly expressed in pancreatic cancer stroma,and the expression level of METTL3 increased with tumor grade.The m6 A sequencing and transcriptome sequencing of CAFs revealed that METTL3 regulates the expression of the downstream lnc RNA molecule MALAT1 through m6 A methylation modification in CAFs.The results of gene set enrichment analysis showed that METTL3 played a tumorpromoting role in CAFs through the IL-6/STAT3 pathway.MALAT1 can regulate the expression and secretion of IL-6 in CAFs.Through the above research results,we have discovered and revealed the role of METTL3 in pancreatic CAFs in maintaining the cancer-promoting phenotype mediated by CAFs and the specific regulatory mechanism,which provides new ideas for the future treatment of precisely targeting CAFs in pancreatic cancer.Part I : Role of METTL3 in the maintenance of cancerpromoting phenotypes in pancreatic cancer-related fibroblasts and its clinical significanceObjective: To study the expression level of METTL3 in pancreatic cancer interstitial tissue and cancer-associated fibroblasts,the clinical significance of METTL3 expression in interstitial tissue by tissue microarray was analyzed,and in vitro and in vivo experiments were used to explore the role of METTL3 in CAFs maintaining the cancer-promoting phenotype.Methods: The primary CAFs of pancreatic cancer were isolated and identified,and they were co-cultured with pancreatic cancer cells to determine whether they had a cancerpromoting phenotype,and then the CAFs with cancer-promoting effects were selected for research.Multicolor in situ hybridization staining of pancreatic cancer tissue microarray(90 cases)and immunohistochemical staining of clinical specimens were used to compare the expression differences of METTL3 in pancreatic cancer interstitial tissue and adjacent tissue.The correlation between the expression of METTL3 in tumor interstitial tissue of patients with pancreatic cancer and the clinicopathological parameters and prognosis of patients were studied in 90 cases.The role of METTL3 in CAFs in maintaining a proliferative phenotype of cancer cells was studied by co-culture of pancreatic cancer cells and CAFs cells in vitro and subcutaneous tumor inoculation of nude mice in vivo.Results: The results of multicolor in situ hybridization staining of tissue microarray and immunohistochemical staining of clinical specimens showed that the expression level of METTL3 in pancreatic cancer interstitial tissue was higher than that in adjacent tissue.The statistical analysis of METTL3 expression in tumor interstitial tissue of pancreatic cancer patients and the clinicopathological features of patients showed that the expression of METTL3 in tumor interstitial tissue was closely related to the pathological grade of pancreatic cancer.Both in vitro and in vivo experiments demonstrated that METTL3 maintains the pro-proliferative phenotype of CAFs.Conclusion: METTL3 is highly expressed in pancreatic cancer interstitial tissue and cancer-associated fibroblasts.METTL3 plays a critical role in maintaining a cancerpromoting phenotype in pancreatic cancer-associated fibroblasts.Part II:METTL3 maintains a cancer-promoting phenotype in CAFs via the IL-6/STAT3 pathwayObjective: To explore the signaling pathway through which METTL3 maintains the cancer-promoting phenotype in CAFs.Methods: GSEA analysis was performed in the transcriptome sequencing results of down-regulated METTL3 expression in CAFs,and the most obvious signaling pathway affected by METTL3 in CAFs was found.The supernatant of CAFs after knockdown of METTL3 expression were detected by inflammatory cytokine protein microarray and compared with the supernatant of NFs cells,and the cytokines with the most significant changes in CAFs after knockdown of METTL3 expression were screened and verified by q PCR and ELISA.The screened cytokines were added to stimulate pancreatic cancer cells to observe whether they had an accelerating effect on the proliferation phenotype of pancreatic cancer.Results: The results of GSEA analysis showed that the most obviously downregulated pathway after knockdown of METTL3 expression in CAFs was the inflammation-related pathway,among which the IL-6/JAK/STAT3 pathway was the most significantly down-regulated.Some pancreatic cancer-related pathways were also significantly downregulated.Real-time quantitative PCR showed that the m RNA expression levels of pivotal inflammatory factors IL-6 and LIF in the IL-6/JAK/STAT3 pathway were significantly decreased.Inflammatory cytokine protein microarray analysis showed that the cytokine whose expression level was greater than 300 and decreased most significantly in CM from CAFs after down-regulation of METTL3 was IL-6,while the expression level of IL-6 in CM from NFs increased after down-regulation of METTL3 expression.ELISA experiments verified that the alterations of IL-6 in the supernatants of CAFs was consistent with the results of microarray.Co-culture experiments showed that CAFs CM and exogenous IL-6 could stimulate the expression of phosphorylated STAT3 in pancreatic cancer cells and the proliferation ability of pancreatic cancer cells,and this promotion could be inhibited by IL-6 neutralizing antibody.Conclusion: METTL3 in pancreatic cancer CAFs plays a role in promoting pancreatic cancer through the IL-6/STAT3 signaling pathway.IL-6 in CAFs CM can activate the STAT3 pathway and proliferation ability of pancreatic cancer cells.Part III: METTL3 in pancreatic CAFs plays a role in promoting cancer by regulating MALAT1 expressionObjective: To explore the downstream molecular mechanism of METTL3 in tumorpromoting effect in CAFs cells.Methods: After knockdown of METTL3 expression in cancer-promoting CAFs cell lines,m6 A sequencing and transcriptome sequencing(including m RNA and lnc RNA)were performed.By combining the two sequencing results,the levels of m RNA and m6 A methylation modification in lnc RNA molecules both decrease after knockdown of METTL3 expression were screened.Me RIP-PCR and RNA stability experiments were used to verify whether METTL3 regulates the expression and stability of the screened lnc RNA molecules through m6 A modification.The correlation between METTL3 and the screened lnc RNA was analyzed by TCGA and GEO public databases.In vitro and in vivo experiments were used to explore that METTL3 mediates its cancer-promoting phenotype in CAFs by maintaining the expression of screened lnc RNA molecules.To observe whether knockdown of METTL3 expression in CAFs affects their overall methylation level by m6 A dot blot.Results: The m6 A dot blot experiments verified that the overall methylation level decreased significantly after down-regulation of METTL3 expression in CAFs.The results of m6 A sequencing showed that the distribution of methylation modification sites on m RNA was mainly in the 3’UTR region,followed by the CDS region,and the least distribution was in the 5’UTR region.After METTL3 knockdown in CAFs,m6 A sequencing and transcriptome sequencing were performed.The combined analysis showed that m RNA level and m6 A methylation level of MALAT1 significantly decreased after down-regulation of METTL3 expression.Me RIP-PCR and RNA stability experiments confirmed that METTL3 enhanced the stability of MALAT1 and promoted the expression of MALAT1 by enhancing m6 A methylation in CAFs.Analysis of datasets in public databases TCGA and GEO revealed that the association of METTL3 with MALAT1 was positively correlated in multiple datasets.MALAT1 was expressed in pancreatic cancer interstitial tissue and cancer-associated fibroblasts.CCK8 and colony formation assays showed that overexpression of MALAT1 in CAFs enhanced the ability of promoting pancreatic cancer cell proliferation,while down-regulation of MALAT1 expression in CAFs inhibited the ability to promote pancreatic cancer cell proliferation.Overexpression of MALAT1 in CAFs promotes the expression and secretion of autologous IL-6,while decreasing MALAT1 level inhibits the expression and secretion of autologous IL-6.Both in vitro cell experiments and in vivo nude mouse subcutaneous tumor experiments demonstrated that MALAT1 was involved in mediating the effect of METTL3 on the proproliferation of pancreatic cancer cells in CAFs cells.Conclusion: METTL3 regulates the expression of MALAT1 through m6 A modification level in CAFs.MALAT1 is expressed in pancreatic cancer interstitial tissue.MALAT1 plays a tumor-promoting role in pancreatic CAFs.MALAT1 in CAFs can regulate the expression of its own and secreted inflammatory factor IL-6.METTL3 mediates the maintenance of its own cancer-promoting phenotype through MALAT1 expression in cancer-promoting CAFs.