METTL3 Promotes Proliferation And Migration Of ESCC Cells By Regulating AMIGO2

Research background and purpose:Esophageal cancer is one of the most common malignant tumors,and esophageal squamous cell carcinoma(ESCC)is the most common subtype in esophageal cancer.Dysregulation of numerous genes has been considered as the most fundamental events in the occurrence and development of ESCC.Therefore,it is of great clinical significance to explore the molecular mechanisms of the key genes involved in driving ESCC progression,which will facilitate the identification of novel diagnostic markers and therapeutic targets to improve ESCC prognosis.Recent evidences indicated that dysregulation of RNA m6A closely contributes to the progression of ESCC development.Methyltransferase-like 3(METTL3),a core m6A methyltransferase,has been proved to be up-regulated in ESCC and is closely related to its poor prognosis.However,the downstream target genes and the biological role of METTL3 in ESCC progression has not yet been well illustrated.Adhesion molecule with Ig like domain 2(AMIGO2),as a member of the AMIGO protein family,has been proved to be involved in the occurrence and development of numerous tumors through various signaling pathways.However,the role of AMIGO2 in ESCC has not yet been illustrated.In this study,we aim to explore whether METTL3 regulates the proliferation and migration of ESCC cells through modulating AMIGO2 expression and its underlying mechanism involved.Methods:1.Lentiviral-mediated stably METTL3 knocking down Eca-109 and TE1 cell models were constructed.CCK-8 assay,EdU cell proliferation assay,Wound healing assay and Transwell assay were performed to study the effect of METTL3 knockdown on the viability,proliferation and migration of ESCC cells.2.Tumor xenograft models were constructed by subcutaneously injecting TE1 cells with stable knockdown of METTL3 into nude mice to evaluate the effect of METTL3 knockdown on xenograft tumor growth in vivo.3.Methylated RNA immunoprecipitation sequencing(MeRIP-seq)and RNA-seq were performed to analyze the genes with differentially methylated m6A peak and differential expression in METTL3 knocking down Eca-109 cells.Then we established the gene set overlaps of the data between MeRIP-seq and RNA-seq to explore the differential expression genes with differential m6A peaks.The result was verified by MeRIP-qPCR,RT-qPCR or Western Blot.4.Lentiviral-mediated stably AMIGO2 knocking down and overexpressing Eca-109 and TE1 cell models were constructed.EdU cell proliferation assay,Wound healing assay and Transwell assay were performed to study the effect of knockdown and overexpression of AMIGO2 on the proliferation and migration of ESCC cells.5.Rescue assays were applied to evaluate the effects of AMIGO2 overexpression in METTL3 knocking down ESCC cells or METTL3 overexpression in AMIGO2 knocking down ESCC cells on cell proliferation and migration by EdU cell proliferation assay,Wound healing assay and Trans well assay.6.Lentiviral-mediated stably YTHDC1 knocking down ESCC cell model was constructed.RNA-seq was performed to explore the differential expression genes in YTHDC1 knocking down cells.Conjoint analysis of the data from RNA-seq analysis between METTL3 knocking down cells and YTHDC1 knocking down cells was performed to explore the overlap differential expression genes.The result was verified by RT-qPCR and Western Blot.7.RIP-qPCR was applied to detect whether YTHDC1 interact with the mRNA of AMIGO2 in ESCC cells.RT-qPCR was performed to detect the level of pre-mRNA and mature mRNA of AMIGO2 in YTHDC1 knocking down and the control cells.Results:1.Stably METTL3 knocking down ESCC cell models were successfully established.We showed that METTL3 knockdown decreased ESCC cell viability,inhibited ESCC cell proliferation and migration.Results from tumor xenograft models indicated that METTL3 depletion repressed xenograft tumor growth in vivo.2.Conjoint analysis of MeRIP-seq and RNA-seq data in METTL3 knocking down cells showed a total of 60 differential expression genes with differential m6A peaks,including AMIGO2,a member of the AMIGO protein family.Results from MeRIP-qPCR,RT-qPCR and Western Blot analysis further revealed that METTL3 knockdown down-regulated the levels of mRNA m6A modification,mRNA and protein of AMIGO2 in METTL3 knocking down cells.3.Stably AMIGO2 knocking down and overexpressing ESCC cell models were successfully established.We showed that AMIGO2 knockdown inhibited ESCC cell proliferation and migration,whereas AMIGO2 overexpression enhanced ESCC cell proliferation and migration.4.Data from rescue assays demonstrated that AMIGO2 overexpression partially reversed the inhibition of METTL3 knockdown-mediated ESCC cell proliferation and migration,whereas METTL3 overexpression partially abrogated the inhibition of AMIGO2 knockdown-mediated ESCC cell proliferation and migration.5.Stably YTHDC1 knocking down ESCC cell model was successfully established.Conjoint analysis of the data from RNA-seq analysis between METTL3 knocking down cells and YTHDC1 knocking down cells demonstrated a total of 91 overlap differential expression genes,which contained AMIGO2.We further validated that YTHDC1 knockdown decreased the expression of AMIGO2 at mRNA and protein level in Eca-109 cells.RIP-qPCR analysis showed that the mRNA levels of AMIGO2 enriched by antiYTHDC1 antibodies significantly decreased in METTL3 knocking down Eca-109 cells.In addition,the ratio of AMIGO2 pre-mRNA to its mature mRNA by RT-qPCR analysis in YTHDC1 knocking down cells obviously increased when compared to that in the control cells.Conclusions:1.METTL3 knockdown inhibited ESCC cell proliferation and migration,and repressed xenograft tumor growth in vivo.2.METTL3 knockdown downregulated AMIGO2 expression in an m6A-YTHDC1mediated manner in ESCC cells,thereby inhibited ESCC cell proliferation and migration.