The Mechanism Of FTO In Alcohol-induced Osteonecrosis Of Femoral Head Through Regulating Demethylation Of BMP4

ObjectiveThis study aims to investigate the role of 6-methyladenosine(m6A)modification in human alcohol-induced osteonecrosis of femoral head(AIONFH).And to explore the biological function and molecular mechanism of m6 A demethylase fat mass-and obesity-associated protein(FTO).further clarify the role of FTO/BMP4 in alcoholinduced osteonecrosis of femoral head in vivo and in vitro,provides a new experimental theoretical basis for the therapeutic strategy of AIONFH.MethodsThe first part: Collecting clinical femoral head tissue samples of AIONFH and femoral neck fractures that meet the inclusion and exclusion criteria.The clinical femoral head tissue samples with no statistical difference useing age,gender,and body mass index as covariates variables,were screened out through the propensity value matching method.The enrichment biological function of m6 A methylation modification in AIONFH were analysed through m6 A methylation sequencing analysis and bioinformatics analysis.The second part: We cultured bone marrow mesenchymal stem cells as previously reported.FTO knockdown or overexpression were performed in BMSCs.Then,the expression level of FTO were verified through western blot;osteogenic induction,Alizarin red staining and ALP were performed to determine the effect of FTO on osteogenic differentiation;CCK-8 experiment was used to verify the effect of FTO on cells proliferation.BMSCs with alcohol was cocultured to simulate alcohol-induced osteonecrosis of femoral head in vitro.Western blot was performed to verify the expression level of FTO;the effect of alcohol on osteogenic differentiation was verified by osteogenic induction,Alizarin red staining;the effect of alcohol on cells proliferation was verified by CCK-8 experiment.After that,in human clinical samples,the m6 A methylation quantitative experiment was used to verify the level of m6 A methylation modification in human alcohol-induced osteonecrosis of femoral head;the Q-PCR experiment was used to determine the expression level of RNA m6 A methylation-regulated associated genes in human AIONFH.The downstream target genes were screened by Q-PCR experiment in BMSCs transfected with lv-FTO and shFTO.The effect of ethanol and sh-FTO on BMP4 expression level was determined through western blot in vitro.Me RIP-seq and Me RIP-qpcr experiments performed in BMSCs and clinical samples were used to clarify the regulation of FTO on BMP4 m6 A methylation modification.The stability of BMP4 m RNA after FTO-regulated BMP4 demethylation effect was verified by Actinomycin D m RNA tolerance test.The third part: The rats were randomly divided into 4 groups by the random number table method,included Control group(normal rats),Ethanol group(alcohol-induced osteonecrosis of femoral head),sh-FTO group(knockdown FTO group),Ethanol+FTO group(ethanol plus lv-FTO),Ethanol+BMP4 group(ethanol plus lv-BMP4).10 animals in each group.Each group includes 10 rats.The AIONFH model of SD rats was constructed by the Lieber-De Carli liquid diets,which containing 5% alcohol.The changes in the tissue structure of the femoral head between each group were evaluated by HE staining and Masson staining.To reveal the potential therapeutic effect of FTO/BMP4 in AIONFH,the expression level of BMP4 were verified by immunohistochemical experiments.ResultsThe first part: We screened out 40 cases no statistical difference in age(P=0.297),gender(P=1),and body mass index(P=0.811)from 200 clinical tissue samples between alcohol-induced osteonecrosis of femoral head and femoral neck fracture for follow-up experiments.Three of them were selected for Me RIP-seq,and the biological function enrichment analysis of m6 A methylation showed that the regulatory functions of the non-canonical Wnt signaling pathway and the classic Wnt signaling pathway have significantly differences(P<0.05).The second part:(1)Compared with the normal control group,after sh-FTO pretreatment in bone marrow mesenchymal stem cells,the expression of FTO was significantly reduced(P<0.05);After knocking down the expression of FTO,the alizarin red staining showed that the osteogenic differentiation ability of bone marrow mesenchymal stem cells was significantly inhibited.The CCK-8 experiment showed that the proliferation ability of the sh-FTO group was significantly lower than that of the control group(P<0.05).We transferred with lv-FTO in bone marrow mesenchymal stem cells,the expression of FTO was significantly increased compared with the control group(P<0.05).Contrary to knockdown of FTO,overexpression level of FTO can significantly improve the osteogenic differentiation ability of BMSCs.After lvFTO pretreatment,compared with the control group,the proliferation ability of BMSCs was significantly improved.The above results indicate that FTO can promote the osteogenic differentiation and proliferation of BMSCs.(2)After ethanol pretreatment in BMSCs,the expression level of FTO was significantly reduced compared with the control group(P<0.05).Alizarin red staining and CCK-8 analysis results showed that ethanol can inhibit the osteogenic differentiation ability and cell proliferation of BMSCs.That is to say,the inhibition effects of osteogenic differentiation and proliferation in BMSCs performed by ethanol may correlate with FTO.(3)The quantitative analysis of m6 A methylation showed that the overall m6 A methylation level in AIONFH samples was significantly increased compared with femoral neck fractures samples(P<0.05).The results of the Q-PCR experiment showed that in all m6 A methylation modification enzymes,the expression of m6 A demethylation transferase FTO was significantly different,and in alcoholic femoral head necrosis,the expression was significantly reduced(P <0.05),which means,the level of m6 A methylation modification in AIONFH was increased.After overexpression of FTO in vitro,Q-PCR experiments showed that the expressions of Wnt5 b,AKT,BMP2,and BMP4 were significantly increased(P<0.05).In contrast,after knocking down FTO,the expressions of Wnt5 b,AKT,BMP2,and BMP4 were significantly reduced(P<0.05),and the biggest change level is BMP4.(4)The methylation analysis of the BMP4 gene in the Me RIP-seq sequencing data showed that BMP4 is located on chromosome 4,long arm 2,zone 2.Compared with control group,the methylation modification level of the BMP4 gene of AIONFH was significantly increased.The results of the Me RIP-qpcr experiment also showed that the BMP4 methylation modification level in AIONFH samples was significantly higher than control group(P<0.05).Q-PCR results showed that in AIONFH,the expression level of BMP4 was significantly lower than control group(P<0.05).In vitro,BMSCs with alcohol or sh-FTO cocultured.The expression level of FTO was significantly inhibited(P<0.05).After sh-FTO pretreated the BMSCs,the m6 A methylation quantitative test showed that compared with the control group,the overall methylation modification level of the sh-FTO group was significantly increased.In addition,after sh-FTO treatment,the Me RIP-qpcr test showed that the level of BMP4 methylation modification was significantly higher than that of the control group(P<0.05).In contrast,after overexpression of FTO,the m6 A methylation modification level of BMSCs was significantly reduced(P<0.05)and the m6 A methylation modification level of BMP4 was significantly reduced.Which means,BMP4 was a potential target gene of FTO demethylation enzyme.Actinomycin D m RNA stability experiment further analyzed the function of BMP4 m6 A after methylation modification.The results showed that when FTO was overexpressed,the methylation level of BMP4 m6 A decreased,the halflife was prolonged,and the stability of BMP4 m RNA increased.On the contrary,after FTO was knocked down,the half-life was shortened and the stability of BMP4 m RNA decreased.The third part: HE staining showed that the structure of the femoral neck fractured(Control group)was normal,the trabeculae bone were arranged regularly and intact,the cells were evenly distributed,and bone cells could be observed in the bone lacuna.Rats using the alcohol-containing Lieber–De Carli diet(Ethanol group)observed a large amount of vacuole-like necrotic tissue,the trabecular bone structure is destroyed,thinned,and the spacing is sparse.The bone cells in the bone lacuna atrophy and disappear.After transferred sh-FTO in vivo(sh-FTO group),the tissues in the femoral head can also be destroyed.Compared with the Ethanol group,overexpression of FTO can significantly inhibit the destruction of the structure caused by alcohol.We observed the trabecular bones are arranged regularly and there is only a small amount of vacuolelike tissue.Compared with the Ethanol group,Ethanol+BMP4 group also showed clear trabecular structure,only a small amount of vacuole-like tissue.(2)The results of the Masson staining experiment showed that the trabecular bones of the Control group were tightly arranged,the osteogenic tissues accounted for the vast majority,and only a small part was neonatal femoral tissue.In the Ethanol group,the trabecular structure was significantly damaged and disappeared,the vacuole-like tissue increased,and the new bone tissue was less.Compared with the Ethanol group,the trabecular bone structure in Ethanol+FTO group is clear and regular,only a small amount of vacuole-like tissue can be observed,and there are more new bone tissues.The results of the Ethanol+BMP4 group were similar.(3)The results of IHC showed that there were more brown-yellow positive particles in control group,and BMP4 was significantly expressed.The expression of BMP4 in the Ethanol group and sh-FTO group was significantly reduced.After overexpression of FTO,the expression of BMP4 in the Ethanol+FTO group increased significantly.Similarly,after overexpression of BMP4,the expression of BMP4 in the Ethanol+BMP4 group increased significantly(P<0.05).Conclusions:(1)The level of m6 A methylation modification in human alcoholinduced femoral head necrosis femoral head samples was significantly higher than that in the control group.(2)Alcohol inhibit the proliferation and osteogenic ability of BMSCs through regulating the expression of FTO.(3)FTO can regulate the stability of BMP4 m RNA by regulating the level of BMP4 m6 A methylation modification in AIONFH.(4)Overexpression of FTO/BMP4 can inhibit alcohol-induced osteonecrosis of femoral head necrosis in SD rats model,and may play a potential therapeutic effect.