| Objective:Steroid-induced necrosis of the femoral head(SONFH)is a prevalent orthopedic disease that is associated with the long-term or high-dose application of glucocorticoids.Glucocorticoids can cause vasoconstriction and microvascular embolism in the femoral head,leading to ischemia,hypoxia,and inadequate nutrient supply,ultimately resulting in femoral head necrosis and collapse,and impaired joint function.Currently,the pathogenesis of SONFH is not fully understood,but research has shown that proliferation and osteogenic differentiation disorders of bone marrow mesenchymal stem cell(BMSC)are closely related to the occurrence and development of femoral head necrosis.Methyltransferase 3(METTL3)serves as the central subunit of the methyltransferase complex responsible for regulating N6-methyladenosine(m6A)modification.Current studies have found that m6 A modification plays an essential role in the progression of diseases such as tumors,hematological diseases,and autoimmune diseases.However,the precise role and underlying mechanism of m6 A modification in the pathogenesis of steroid-induced necrosis of the SONFH remain uncertain.The objective of this study is to investigate the involvement and mechanism of METTL3-mediated N6-methyladenosine modification in the proliferation and osteogenic differentiation of BMSC in SONFH.Methods:The study collected bone marrow samples from 23 patients who met the inclusion criteria between September 2020 and September 2021.Of the patients studied,12 were diagnosed with Steroid-induced necrosis of the femoral head and designated as the necrosis group,while the remaining 11 patients had femoral neck fractures and were categorized as the control group.BMSC were isolated and characterized using flow cytometry.The total RNA m6 A modification levels were detected using an RNA methylation quantification kit,and the expression levels of m6 A modification-related genes,including METTL3,METTL14,WTAP,FTO,and ALKBH5,were measured using q RT-PCR in both groups of BMSC.The proliferation activity of BMSC was quantified via the use of a cell counting kit-8(CCK-8)after 24,48,and72 hours of cell culture.The BMSC were stimulated to differentiate into adipocytes or osteoblasts,and the Oil Red O and Alizarin Red staining techniques were employed to visualize the lipid droplets and calcium nodules,respectively.These techniques were employed to explore the impact of METTL3 on the osteogenic activity and proliferation of SONFH,the study upregulated the expression of METTL3 in necrosis group using a METTL3 overexpression plasmid and validated the transfection efficiency using q RT-PCR and Western blot analysis.Moreover,to identify the target genes regulated by METTL3 downstream,RNA sequencing and Me RIP were employed.To compare between two groups,the two-sample independent t-test was employed,while for comparisons among multiple groups,one-way analysis of variance(ANOVA)was utilized,and the linear relationship between METTL3 and glutathione peroxidase 7(GPX7)was tested by Pearson correlation coefficient.Results:The study successfully cultured BMSC in vitro and confirmed their identity using flow cytometry.BMSC were found to be positive for CD29 and CD44 expression,while negative for CD34 and CD45 expression on their surface,which is consistent with the BMSC phenotype.Furthermore,the multipotent differentiation capacity of BMSC was demonstrated through successful induction of adipogenesis and osteogenesis.By employing an RNA methylation quantification kit,it was discovered that the level of m6 A in BMSC in the necrosis group was significantly lower than in the control group((3.56±1.66)% vs.(14.94±2.48)%,t=12.150,P<0.01),indicating a statistically significant difference between the two groups.q RT-PCR analysis revealed that the expression of METTL3 in BMSC from the control group was significantly higher than that in BMSC from the necrosis group(1.13±0.04 vs.0.44±0.06,t=5.579,P<0.01).The relative expression levels of METTL14,WTAP,FTO,and ALKBH5 in the control group BMSC did not differ significantly from those BMSC of the necrosis group(P > 0.05).CCK-8 showed that the absorbance values of the BMSC in the control group after 24,48,and 72 hours of culture were higher than those in the necrosis group(0.41±0.03 vs.0.26±0.02,t=6.759,P<0.01,0.75±0.05 vs.0.44±0.08,t=7.334,P<0.01,1.38±0.07 vs.0.51±0.02,t=9.206,P<0.01),the difference was statistically significant.The staining of Alizarin red demonstrated that the proportion of positively stained cells in the control group was greater compared to that in the necrosis group,where it was lower(0.68±0.06 vs.0.18±0.03,t=7.908,P<0.01),and the difference was statistically significant.After up-regulating the expression level of METTL3 in the necrosis group,CCK-8 experiment showed that the absorbance values after 24,48,and 72 hours of culture were higher than those in the necrotic group before the up-regulation(0.61±0.04 vs.0.23±0.02,t=6.433,P<0.01,1.03±0.08 vs.0.39±0.03,t=8.090,P<0.01,1.62±0.11 vs.0.55±0.06,t=9.387,P<0.01),and the differences were statistically significant.Similarly,the results of Alizarin red staining indicated that the positive staining area/total cell area in the necrosis group was significantly higher after METTL3 overexpression compared to before overexpression(0.83±0.09 vs.0.21±0.04,t=14.630,P<0.01).Further RNA-Seq analysis showed that the expression of GPX7 was significantly increased after the expression level of METTL3 was up-regulated in BMSC in the necrosis group.Me RIP-q PCR confirmed that the level of GPX7 m6 A m RNA modification after METTL3 up-regulation was higher than that in necrosis group cells(2.85±0.85 vs.0.97±0.18,t=10.260,P<0.01),The difference was statistically significant.Conclusion:METTL3-mediated m6 A modification promotes proliferation and osteogenic differentiation of BMSC in SONFH.METTL3 regulates the expression of GPX7 through m6 A modification,and GPX7 may be involved in the pathogenesis of SONFH. |
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