Difference Analysis Of N~6-Methyladenosine RNA Methylation In Alcoholic Femoral Head Necrosis And Study Of Chinese Herbal Medicine Intervention In Vitro

Objective:To explore the modification pattern of m~6A methylation and the treatment mechanism of Panax Notoginseng Saponins(PNS)for alcohol-induced osteonecrosis of femoral head(AIONFH).Methods:Me RIP-seq was used to identify the differences in m~6A methylation between AIONFH and the control group,and RNA-seq was used to screen the differentially expressed RNA transcripts from the two groups.The AIONFH osteoclast model was established in vitro.After 5 days of intervention with PNS drug serum,the morphology and number of osteoclasts in the intervention group and the control group were observed by TRAP staining.The mRNA and protein expressions of HIST1H3F and METTL3were detected by PCR and WB.Results:In the control group,a total of 17839 m~6A modification sites(peaks)were identified on 6161 mRNAs and 485 peaks on 289 lnc RNAs.A total of 15925 peaks of 5740 mRNAs and 387 peaks of 229 lnc RNAs were identified,and a total of 1007 overlapping mRNA peaks and 30 overlapping lnc RNA peaks were identified between the two groups,suggesting differences in m~6A modification.In the two groups of samples,only one peak accounted for the largest proportion of mRNAs,which accounted for 39.95%of the total in the AIONFH group(about 37.01%in the control group).As the number of peaks increases,the proportion of mRNAs decreases,and most of peaks are located in CDS,followed by 3’UTR and exon.According to the screening conditions of P<0.05 and|Log2FC|≥1,we identified 4130 differentially methylated m~6A sites(DMMSs)in 2602 mRNAs,and 98 DMMSs in 60lnc RNAs.The chromosomes with the highest content of DMMSs are chromosome 1(approximately 10.88%),followed by chromosome 2(approximately 8.47%)and chromosome 5(approximately 7.10%).GO enrichment analysis showed that differential m~6A modification of hypermethylated mRNAs is related to"immune response","T cell receptor signaling pathway","leukocyte migration"and so on.Demethylated mRNAs in differentially m~6A-modified mRNAs are related to"protein folding","protein sumoylation",and"ubiquitin-dependent protein catabolic process".KEGG enrichment shows that hypermethylated mRNAs in differentially m~6A-modified mRNAs are enriched in signal pathways such as"antigen processing and presentation","leukocyte transendothelial",and"T cell receptor signaling pathway".Demethylated mRNAs in differentially m~6A-modified mRNAs are mainly enriched in signaling pathways such as"protein processing in the endoplasmic reticulum","ubiquitin mediated proteolysis".In the correlation analysis between differential m~6A modification and differentially expressed genes(DEGs),we obtained 69 differential m~6A modified DEGs,which are related to biological processes such as“mitosis”and“cell division”.The top 3 genes with m~6A modification multiples are HIST1H3F,DIEXF,IGKV4-1.Cell experiments showed that PNS significantly inhibited the differentiation of osteoclasts,and inhibited the expression of HIST1H3F and METTL3 at the gene and protein levels.Conclusion(s):m~6A methylation may play an important role in the occurrence and development of AIONFH.PNS may up-regulate the expression of HIST1H3F and inhibit osteoclast differentiation by down-regulating METTL3 and negatively regulating the methylation state of HIST1H3F,thus achieving the purpose of treating AIONFH.