The Mechanism Of METTL14-mediated M6A Modification Of RUNX2 MRNA In Regulating Choroidal Melanoma Cell Metastasis

Objective: Choroidal melanoma(CM)is an intraocular malignancy with a high mortality and metastasis rate that is common in adults.However,the mechanism of CM migration remains unclear.Therefore,it is of great significance to explore the molecular mechanisms involved in CM transfer.m6A stands for N6-methylated adenosine,which is the most common form of modification in higher living m RNAs.It is involved in the progress of various cancers and plays an important role in the occurrence and development of tumors.Methyltransferase-like 14(METTL14),as one of m6 A methyltransferases,participates in the dynamic reversible process of m6 A modification.METTL14 plays a significant role in the metastasis of various cancers.However,the expression and function of METTL14 in choroidal melanoma have not been reported.Transcription factor 2(Runt-related transcription factor 2),the downstream target gene of METTL14,is a key regulator of osteogenesis and is highly expressed in melanoma,but its expression and function in choroidal melanoma have also not been reported.The results of transcriptomic sequencing showed that Stearoyl-Co A desaturase 1(SCD1)was A significant downstream factor of stearoyl-Co A desaturase 1(SCD1),a key enzyme in lipid synthesis that converts saturated fatty acids(SFA)to monounsaturated fatty acids(MUFA).SCD1 is highly expressed and associated with poor prognosis in many tumor tissues.Therefore,SCD1 may be a key target molecule for CM therapy.Therefore,it is significant to explore the role of these three factors in the metastasis of choroidal melanoma and the specific regulatory relationship between them,which can provide a new direction for the diagnosis and treatment of CM.Methods:1.Clinical specimens were collected and western blotting was used to detect the expression level of METTL14 protein in choroidal melanoma tissues and normal choroidal tissues.After knockdown and overexpression of METTL14,the changes of migration and invasion ability of choroidal melanoma cells were investigated in vitro.In vivo experiments were conducted to investigate the change of metastasis ability of choroidal melanoma cells after METTL14 knockdown in nude mice.Western blotting assay was used to detect the changes of key proteins of Wnt/β-catenin signaling pathway after METTL14 overexpression.2.After METTL14 was silenced and overexpressed in choroidal melanoma cells,the protein content of RUNX2 in choroidal melanoma cells was detected by western blotting assay,and the m RNA expression of RUNX2 was detected by q RT-PCR assay.Potential modification sites of m6 A in RUNX2 3’UTR were identified through literature review and nucleic acid sequence analysis.After modifying METTL14 expression in choroid melanoma cells,changes in m6 A modification content in RUNX2 3’UTR were detected by methylated RNA immunoprecipitation(Me RIP)assay,and luciferase activity in wild-type and mutant RUNX2 3’UTR was detected by dual luciferase reporter assay.Bioinformatics analysis detected the expression correlation between RUNX2 and the m6 A writer METTL14 in uveal melanoma tissue from a public database.The association between RUNX2 and prognosis in UM was analyzed using the TCGA database.Clinical specimens were collected,and the expression level of RUNX2 protein in choroidal melanoma tissues and normal choroidal tissues was detected by western blotting.After knockdown of RUNX2,the changes of migration and invasion ability of choroidal melanoma cells were investigated in vitro.In vivo experiments were conducted to investigate the change of metastasis ability of choroidal melanoma cells after RUNX2 knockout in nude mice.In vitro experiments were conducted to determine the response effect of interfering RUNX2 expression on the migration and invasion ability of choroidal melanoma cells mediated by silencing METTL14.3.Transcriptional sequencing was used to search for RUNX2-regulated downstream genes.After RUNX2 was silenced and overexpressed in choroidal melanoma cell lines,western blotting was used to detect changes in the protein content of the downstream genes.Based on the predicted transcription-binding sites,the specific binding capacity of RUNX2 for DNA fragments of the corresponding promoters of downstream genes was confirmed by Chromatin Immunoprecipitation(Ch IP)precipitation.Western blotting assay was used to detect the expression levels of downstream genes in choroidal melanoma tissues and normal choroidal tissues.Using the GEPIA data of uveal melanoma,the relationship between the expression of downstream genes and the prognosis of the disease was analyzed.In vitro experiments were conducted to investigate the changes of migration and invasion ability of choroidal melanoma cells after knockdown of downstream genes.Results:1.METTL14 protein was significantly up-regulated in the choroidal melanoma tissues we collected.The decrease and increase of METTL14 expression can inhibit and promote the migration and invasion of choroidal melanoma cells in vitro.Choroidal melanoma cells with stable METTL14 knockdowns showed decreased metastasis efficiency in nude mice compared with negative controls.Overexpression of METTL14 may activate the Wnt / β-catenin signaling pathway in choroidal melanoma by phosphorylation of GSK3β.2.Silencing and overexpression of METTL14 can down-regulate and up-regulate RUNX2 protein and m RNA expression.There is a potential m6 A modification site in RUNX2 3’UTR.RUNX2 3’UTR can be modified by m6 A,and overexpression of METTL14 can up-regulate the modification level.Overexpression of METTL14 increased the luciferase activity of wild-type RUNX23’UTR,but had no effect on the luciferase activity of mutant RUNX2 3’UTR at m6 A.In uveal melanoma,RUNX2 was positively correlated with METTL14 expression.In the TCGA database,patients with high RUNX2 expression in uveal melanoma had a poor prognosis.RUNX2 protein was significantly upregulated in all the choroidal melanoma tissues we collected.The decrease of RUNX2 expression can inhibit the migration and invasion of choroidal melanoma cells in vitro.The metastasis efficiency of choroidal melanoma cells with stable RUNX2-knockout was reduced in nude mice compared with negative controls.Silencing METTL14 inhibited the migration and invasion of choroidal melanoma cells,which was significantly recovered after overexpression of RUNX2.3.According to the transcriptional sequencing results of choroidal melanoma cell lines,SCD1 was identified as the downstream gene of RUNX2.Silencing and overexpression of RUNX2 down-regulated and up-regulated the expression of SCD1 protein.Ch IP assay showed that RUNX2 could transcriptionally regulate the expression of SCD1.The GEPIA analysis showed that patients with high SCD1 expression had shorter survival times.In vitro experiments confirmed that the migration and invasion ability of CM cells decreased significantly after SCD1 knockdown.Conclusions: 1.METTL14 can promote the migration,invasion and metastasis of choroidal melanoma cells,and activate the Wnt / β-catenin signaling pathway in choroidal melanoma.2.METTL14 can promote the migration,invasion and metastasis of choroidal melanoma cells by modifying RUNX2 3’UTR with m6 A.3.RUNX2 regulates the expression of SCD1 through transcriptional regulation.SCD1 is highly expressed in CM tissues and is associated with poor prognosis in patients.