METTL14-mediated N6-methyladenosine Modification Of MAP2 In Human RPE Cells

Retinal pigment epithelium(RPE)is a neatly arranged monolayer of hexagonal cells located in the outermost layer of the human retina.It is arranged in polarity and is located between the photoreceptor cells of the retina and Bruch’s membrane.The main function of RPE is to swallow and digest the outer disc membrane of photoreceptor cells,provide them with nutrients,and maintain metabolism.Abnormalities in the structure and function of RPE can lead to apoptosis and necrosis of retinal photoreceptor cells,leading to serious diseases such as retinitis pigmentosa(RP)and age-related macular degeneration(AMD),which is called RPE-associated degenerative ocular diseases.RP represents a group of hereditary retinal diseases with different incidence,progression and severity,resulting in vision loss.The disease is characterized by progressive damage of RPE and photoreceptor cells with a high degree of genetic heterogeneity.Studies have found that many patients with RP have RPE phagocytosis and tight junction dysfunction;AMD is a serious eye disease,involving the outer layer of the retina,pigment epithelium and choroid,resulting in progressive damage of central vision.The cause of AMD is complex,and it is currently believed to be the result of a combination of environmental and genetic factors.At present,a large number of studies have been carried out on the pathogenesis of RPE-associated degenerative ocular diseases.NEUROD1is one of the pathogenic genes of RP,but its specific function and molecular mechanism in the pathogenesis of RP are still unclear.The MAP2 gene has been reported to be highly expressed in the retina of AMD patients.In addition,epigenetic factors such as DNA methylation,histone modification,chromatin remodeling and non-coding RNA regulation have been widely reported in the pathogenesis of RPE-associated degenerative ocular diseases.RNA methylation is a new type of epigenetic modification,which has been widely studied in many diseases.As a common RNA methylation modification,N6-methyladenine(m~6A)accounts for more than 80%of RNA methylation and is widely present in various eukaryotic organisms(such as mammals),mainly in regulation of gene expression at the post-transcriptional level.The common motif of m~6A is RRACH.The modification level of transcript m~6A can be dynamically regulated by methyltransferases(writers),binding proteins(readers)and demethylases(erasers).The writers“write”methylation modification into RNA,and mediates the modification process of RNA methylation.METTL3 and METTL14 are the most common methyltransferases,which can catalyze the m~6A methylation of m RNA(and other nuclear RNA)in vivo and in vitro.The erasers"erase"the methylation of RNA,and ALKBH5 and FTO can remove m~6A methylation from m RNA(and other nuclear RNA).The readers"read"the information of RNA methylation modification and participates in the translation and degradation of downstream RNA.For example,the recognition and binding of YTHDF domain proteins to m~6A m RNA can shorten the half-life of m RNA and promote its degradation.m~6A binding proteins in the cytoplasm include YTHDF1(YTH domain family member 1),YTHDF2(YTH domain family member 2),YTHDF3(YTH domain family member 3)and YTHDC2(YTH domain 2).A large number of studies have shown that m~6A methylation is related to the pathogenesis of many diseases,such as type 2 diabetes,neurological diseases,mitochondrial-related diseases and various tumors.The latest research shows that the level of m~6A methylation is down-regulated in the brain striatum of the neurodegenerative disease Parkinson’s disease(PD)model mice.Down-regulation of m~6A can lead to the death of dopaminergic neurons,which in turn induces PD.RP and AMD are both neurodegenerative diseases,however,the relationship between these two diseases and m~6A methylation has not been reported yet.Therefore,I speculate that m~6A RNA methylation is also involved in the pathogenesis of RPE-associated degenerative ocular diseases,and plays a certain role.In order to verify this hypothesis and detect the role of m~6A RNA methylation in the pathogenesis of RPE-associated degenerative ocular diseases,I studied the GEO database of two RPE-associated degenerative ocular diseases samples,RP and AMD,and found that the expression of METTL14 in RP patient samples was significantly reduced.The relationship between m~6A RNA methylation and RPE-associated degenerative ocular diseases is worthy of further study.Purpose:Using RNA interference technology,construct a model of RPE cells down-regulated by METTL14,and observe whether m~6A methylation regulated by METTL14 affects the phenotypes of RPE cells such as phagocytosis,tight junctions,apoptosis and cell cycle.And further study the specific mechanism of METTL14 molecular regulation,so as to explore the role of N6-methyladenosine(m~6A)RNA methylation in human RPE cells and RPE-associated degenerative ocular diseases.Methods:Phagocytosis,apoptosis and cell cycle of ARPE-19 cell line were detected by lentivirus-mediated METTL14 short hairpin RNA(sh RNA).The differentially expressed genes and differential m~6A peak were screened by RNA-seq and Me RIP-seq.The overlapping genes of RNA-seq and Me RIP-seq were found by setting the threshold of differentially expressed genes and Wayne diagram analysis.Furthermore,protein-protein interaction analysis(PPI analysis)was carried out between these overlapping genes and more than 110 pathogenic genes of RPE-associated degenerative ocular diseases,and the target genes regulated by METTL14 and downstream genes with protein interaction with the target genes were found.The target gene was overexpressed in human primary RPE cell(h RPE).Apoptosis,phagocytosis,tight junction and cell cycle were observed to detect the function of the target gene in h RPE.The interaction mechanism of the above-mentioned upstream and downstream genes was verified by RNA pull-down assay and CO-IP analysis.Finally,RIP-q PCR was used to find the reading proteins involved in the regulation of METTL14 target genes.Results:Down-regulation of METTL14 expression significantly decreased the m~6A rate of ARPE-19 cell line.After sh-METTL14treatment,phagocytosis of ARPE-19 cell line decreased,apoptosis increased and cell cycle arrest,indicating that down-regulation of METTL14 could lead to phenotypic pathological changes of ARPE-19cell line.RNA-seq results showed that 3250 genes were significantly up-regulated and 4262 genes were significantly down-regulated after METTL14 down-regulation.The results of Me RIP-seq showed that the m~6A peak abundance of 346 genes decreased and the m~6A peak abundance of 685 genes increased.A total of 6 genes(AJAP1,MAP2,LRRN1,FMN2,CDH4 and KANK2)were found to overlap in the results of RNA-seq and Me RIP-seq.PPI analysis of these overlapping genes with more than 110 pathogenic genes of RPE-associated degenerative ocular diseases found that only MAP2 interacts with the RP pathogenic gene NEUROD1.The results also showed that the m~6A peak of MAP2 in h RPE cells decreased significantly after sh-METTL14 treatment.The q RT-PCR results showed that the expressions of MAP2 and NEUROD1were up-regulated in h RPE cells treated with sh-METTL14.Therefore,MAP2 was selected as a candidate target for sh-METTL14-mediated m~6A modification.Further studies showed that interfering with METTL14 or up-regulating MAP2 could decrease the phagocytosis and tight junction function of h RPE cells,increase apoptosis and block the cell cycle.Down-regulation of METTL14 can lead to a significant increase in MAP2 and NEUROD1,and overexpression of MAP2 can lead to a significant up-regulation of NEUROD1.It was found that in h RPE cells that have been down-regulated by METTL14,down-regulation of MAP2can increase phagocytic function,decrease apoptosis,improve cell cycle,enhance tight junction function,and save cell function;RNA pull-down assay showed that METTL14 could directly bind to the m RNA of MAP2.CO-IP analysis confirmed the direct combination of MAP2 and NEUROD1.After down-regulation of YTHDF2,the expression of MAP2m RNA and protein increased significantly.RIP-q PCR results showed that METTL14 inhibited the expression and translation of MAP2 through YTHDF2.Conclusion:This study found that the expression of METTL14 in RP patients was significantly reduced,and the down-regulation of METTL14expression led to the pathological changes of the RPE cell phenotype;METTL14 regulated the expression of MAP2 through the m~6A-YTHDF2pathway,and down-regulation of METTL14 could lead to an increase in the expression of MAP2.MAP2 combined with the pathogenic gene NEUROD1 and then induced pathological changes in h RPE cells.This study lays the foundation for further research on the role and mechanism of m6A gene modification in RPE-associated degenerative ocular diseases.