Study On The Role And Mechanism Of M6A RNA Methyltransferase METTL3 In Promoting The Development Of Esophageal Squamous Cell Carcinoma

Objective: Esophageal cancer(ESCA)is a common malignant tumor of the digestive tract,ranks in the seventh morbility and in the sixth mortality worldwide.The main pathologic types of ESCA are esophageal adenocarcinoma and ESCC.ESCC is consisted of 80-90% of ESCA in China.Although great progress had been made in the treatment of surgery,chemotherapy,radiotherapy and tumor immunotherapy in recent years,the overall survival of ESCA patients was still poor.At present,the relevant molecular mechanism of ESCC including occurrence,metastasis and recurrence remained unclear.It had been shown that m6 A RNA methylation was an important epigenetic modification that played an vital role in many biological processes,such as lipogenesis,spermatogenesis,tumor and other diseases.Up to now,the expression level,biological function and mechanism of m6 A RNA methylation related enzymes in ESCA had not yet been reported.The expression level of m6 A RNA methylase METTL3 in ESCA is unknown.Since ESCC is a common pathological type in China,the expression of METTL3 in ESCC tissues and cells is still unknown.The correlations between METTL3 and the clinicopathological characteristics and survival prognosis of ESCC patients are unknown.The exact biological function of METTL3 in the development of ESCC in vivo and in vitro is unknown.The molecular mechanism of METTL3 affect the progression of ESCC is unclear.In conclusion,this study aims to explore the role and the potential molecular mechanism of METTL3 in the development of ESCC.It might be provided a new theoretical basis for the prevention and treatment of ESCC.Methods: In the part ?,the expression of m6 A RNA methylation related enzymes in ESCA was analyzed by bioinformatics,using two online databases of Oncomine and UALCAN.In the part ?,Western blot and RT-qPCR were used to verify the expression of METTL3 in ESCC tissues and cells.In the part ?,immunohistochemistry was used to analyze the correlation between METTL3 and the clinicopathological characteristics and survival prognosis of ESCC patients.In the part ?,the knock-downed METTL3 ESCC cell lines KYSE140 and Eca109 and the over-expressed METTL3 ESCC cell lines Eca9706 and KYSE510 were constructed.Next,the effect of METTL3 expression on the proliferation was investigated in the constructed ESCC cells by CCK8 assay.Then,the effect of METTL3 expression on the migration was again investigated in the constructed ESCC cell line using the scratch test method.Finally,the subcutaneous tumorigenesis model of nude mice was constructed to verify the effect of METTL3 in vivo.In the part ?,the potential molecular mechanism of METTL3 that promoted the development of ESCC was analyzed.Results: In the part ?,METTL3 was consistently and highly expressed in esophageal cancer in both Oncomine and UALCAN databases,which might be the key m6 A methylase in ESCA.In the part ?,both the protein and RNA expression of METTL3 in ESCC tissues were higher than those in adjacent normal tissues,as well as in ESCC cells and normal esophageal epithelial cells.In the part ?,the expression of METTL3 in ESCC was significantly correlated with the tumor T stage(P= 0.003),and the overall survival of ESCC patients with high expression of METTL3 was significantly shortened(P=0.0148).In the part ?,knock-downed METTL3 inhibited the proliferation and migration of ESCC cells,while over-expressed METTL3 promoted the proliferation and migration of ESCC cells.Stable knock-down expression of METTL3 inhibited subcutaneous tumor formation of ESCC cells in nude mice.In the part ?,METTL3 modified c-Myc through m6 A methylation and then promoted the expression of c-Myc,while c-Myc regulated the expression of PD-L1 by binding to the promoter of PD-L1 in ESCC.Besides,PD-L1 promoted the the correlation between METTL3 and the clinicopathological characteristics and survival prognosis of ESCC patients of ESCC which might be closely associated to the extensive lymph node metastasis.Conclusions: 1.METTL3 was highly expressed in ESCC and was closely related to the T stage of ESCC.It was associated with a poor prognosis in patients with ESCC.2.METTL3 promoted the proliferation and migration of ESCC in vivo and in vitro.3.The METTL3/c-Myc/PD-L1 signal axis might be a potential molecular mechanism in the progression of ESCC.