| Background and Objectives.Clinical treatment of cartilage defects is a therapeutic challenge in the field of orthopedics.Bone marrow mesenchymal stem cells are considered to be ideal seed cells for cartilage defect repair therapy.How to efficiently promote the targeted differentiation of bone marrow MSCs into cartilage is the key to cartilage tissue repair.The subject group found that wedelolactone,the active ingredient in the famous kidney tonic formula Er Zhi Wan,could promote the chondrogenic differentiation of bone marrow MSCs and significantly upregulate the protein and m RNA levels of Sox9,the core transcription factor of chondrogenic differentiation.However,the mechanism by which wedelolactone upregulates Sox9 expression is unclear and deserves to be explored in depth.In-depth study revealed that wedelolactone could upregulate the expression of RNA methylation enzymes Mettl3 and Nsun4.Based on this,this study proposes the scientific hypothesis that wedelolactone may regulate the chondrogenic differentiation of BMSCs by regulating RNA methylation enzymes Mettl3/Nsun4 and thus causing altered epistasis of RNA methylation modifications.In this study,we explored the role of RNA methylation enzymes Mettl3 and Nsun4 on chondrogenic differentiation and the molecular mechanism of wedelolactone regulation by establishing a targeted differentiation chondrosphere model of BMSCs and an animal model of chondrogenic defects.Methods.1.The promotion effect of wedelolactone on chondrogenic differentiation of BMSCs was investigated by constructing a chondrosphere model by directed differentiation of BMSCs.(1)A chondrosphere model of BMSCs directed into chondrogenic differentiation was constructed by induction of classical induction medium for 7 days,and the expression levels of chondrogenic differentiation marker genes were detected by RT-qPCR and Western blot.(2)Determine the concentration of wedelolactone that produces cytotoxicity to BMSCs by CCK8 and screen the optimal action concentration of wedelolactone by RT-qPCR.(3)After induction of chondrogenic differentiation by wedelolactone at the optimal concentration,the protein expression levels of chondrogenic differentiation marker genes were detected by Western blot.(4)wedelolactone interfered with the chondrocyte model,and the altered levels of RNA methylation m5 C and m6 A were detected by Dot-blot method.(5)Using the chondrocyte goblet model,RNA-seq,RT-qPCR,RNA interference techniques and Dot-blot assay were used to censor the methyltransferases dependent on RNA methylation in chondrogenic differentiation.(6)To add wedelolactone intervention in the chondrocyte model,the protein and m RNA expression of Mettl3 and Nsun4 in each group of cells were detected by Western blot and RT-qPCR.(7)Schr?dinger software was used to predict whether wedelolactone could bind to protein Nsun4 and Mettl3.(8)In vitro detection of targeted binding of wedelolactone to RNA methylation enzymes Mettl3 and Nsun4 using protein purification and SPR techniques.2.To analyze the changes in translation levels of genes related to chondrogenic differentiation of BMSCs by chondrocyte model,combined transcriptome,translatome and proteome.Bioinformatics was used to analyze the principal components and differential genes to compare the variability of each histological assay.The role of translationomics as a bridge was determined by individual histological correlation analysis.Combined transcriptomic and translatomic analysis of altered translational efficiency of genes involved in chondrogenic differentiation.3.The regulatory mechanism of RNA methylation enzyme Nsun4/Mettl3 on Sox9,the core transcription factor of chondrogenic differentiation,was investigated by the chondrosphere model and rat chondrogenic defect model of BMSCs directed into chondrogenic differentiation.(1)A chondrosphere model of targeted induction of BMSCs was constructed,and m5 C and m6 A levels in total m RNA after induction into cartilage were detected by Dot-blot,and m5 C and m6 A levels of Sox9 m RNA were detected by RIP-qPCR.(2)The alteration of protein and m RNA expression of Nsun4 and Mettl3 after chondrogenic differentiation of BMSCs was detected by Western blot and RT-qPCR.(3)Nsun4 and Mettl3 were knocked down by RNA interference fragments to observe the chondrogenic phenotype,and the alterations of marker genes were detected by Western blot and the alterations of m5 C and m6 A levels were detected by Dot-blot.(4)The alteration of Nsun4 and Mettl3 binding Sox9 m RNA amount after chondrogenic differentiation of BMSCs was detected by RIPqPCR.(5)The interactions between Nsun4 and Mettl3 were detected by CO-IP,and the changes in the levels of Nsun4/Mettl3-bound Sox9 3'UTR and the levels of m5 C and m6 A in the Sox9 3'UTR region were detected by RIP-qPCR.(6)The SRAMP website(http://www.cuilab.cn/sramp)predicted the m6 A locus on the Sox9 3'UTR and targeted this range using heavy sulfite sequencing to identify the m5 C locus and RIP-qPCR to identify the m6 A locus.After knocking down Nsu4 and Mettl3 to induce chondrogenic differentiation,the altered levels of m6 A sites and m5 C sites were detected.(7)CO-IP LC/MS was used to detect Nsun4-bound protein,which was further verified by Western blot.And to explore the possible reading proteins of m5 C and m6 A and their regulatory roles on Sox9.(8)Sox93'UTR-driven luciferase reporter gene system was constructed to determine whether the regulation of Sox9 3'UTR by Mettl3/Nsun4/Ythdf2/e EF1a-1 affects its translation process,and to determine by RT-qPCR whether Mettl3/Nsun4 /Ythdf2 on the regulation of Sox9 transcriptional process by RT-qPCR.(9)The Mettl3/Nsun4/Ythdf2/e EF1a-1 protein was purified using a prokaryotic expression system,and the binding mode of the four proteins was explored in vitro using SPR.(10)To construct a rat cartilage defect model,adenoassociated virus was used to overexpress Mettl3 and Nsun4 in BMSCs,and the expression levels of genes related to chondrogenic differentiation were detected by immunofluorescence?Results.1.Small molecules of traditional Chinese medicine promote chondrogenic differentiation of BMSCs part(1)After 7 days of induction culture of BMSCs,the formation of chondrocytes with smooth surface and diameter of about 2 mm could be observed.RT-qPCR and Western blot were performed to detect the expression of Sox9,Aggrecan and Col2,the markers of chondrogenic differentiation,respectively.The results showed that the RNA and protein expression levels of Sox9,Aggrecan and Col2 were significantly higher in the chondrocytes compared with the normal group.(2)The results of CCK8 experiments showed that the working concentration of wedelolactone started to produce toxicity to BMSCs at 160 nm causing cell death,and there was no toxic effect on cells within the range of 80 nm.The results of RT-qPCR showed that the working concentration of wedelolactone at 10 nm significantly upregulated the expression of chondrogenic marker genes Sox9 and Col2,and 10 nm was determined as the optimal working concentration of wedelolactone.(3)In the chondrogenic differentiation of BMSCs,the wedelolactone-induced group could form normal spheres with smooth surface and diameter of about 2 mm.western blot results showed that the wedelolactone-induced group could significantly upregulate the protein expression of chondrogenic marker genes Sox9 and Col2 compared with the induced group.(4)After wedelolactone induction of BMSCs into cartilage differentiation,Dot-blot results showed that m5 C and m6 A levels were upregulated in the wedelolactone-induced group compared to the induced group.The expression of m5 C methyltransferase was censored by RNA-seq and RT-qPCR,which showed a significant decrease in the expression of Nsun4 and a significant increase in the expression of Nsun6.Knockdown of Nsun4 and Nsun6 induced into chondrogenic differentiation,respectively.Dot-blot results showed no differential change in m5 C levels in the knockdown Nsun6-induced group and a significant decrease in m5 C levels in the knockdown Nsun4-induced group compared with the induced group.The m6 A was mainly catalyzed by the methyltransferase Mettl3,and the levels of m6 A were significantly downregulated after knockdown Mettl3 induction relative to the induction group.(5)After wedelolactone intervention into chondrogenic differentiation,Western blot and RT-qPCR results showed that the wedelolactone group significantly upregulated the expression of Nsun4 and Mettl3 protein and RNA relative to the induction group.Further,the molecular docking results showed that wedelolactone could bind Nsun4 with a binding force of-6.235,demonstrating a high binding force,while the binding with Mettl3 was not significant.The Nsun4 protein was purified and the binding of Nsun4 to wedelolactone was verified in vitro by SPR technique,and the results showed that wedelolactone could bind to Nsun4 with a slow binding and slow dissociation binding mode.2.Section on chondrogenic differentiation of bone marrow mesenchymal stem cells and bioinformatics study(1)Principal component analysis of transcriptome and translational group data revealed a clear trend of separation between the normal and induced groups at the transcriptional and translational levels.(2)Analysis of differentially expressed genes revealed 315 differentially expressed genes in the transcriptome and 2384 differentially expressed genes in the translational group.(3)Correlation analysis showed that the Pearson correlation coefficient R2=0.4496 in the normal group and the Pearson correlation coefficient R2=0.5882 in the induced group,and the transcription group was correlated with the translation group.Further,the analysis of the transcriptome and translation group was combined,and the results showed that the ribosome distribution of the overall RNA was significantly higher in the induced group compared with the normal group,and the 3'UTR region was significantly higher compared with other regions.In terms of translation efficiency,the translation efficiency of overall genes increased after induction,and the analysis of genes differing in translation efficiency revealed that there were 2253 genes up-regulated and 142 genes down-regulated after induction,among which the translation efficiency of genes related to chondrogenic differentiation increased significantly.The distribution of ribosomes on the core transcription factor Sox9 RNA was significantly higher after induction,and the difference was statistically significant.3.Study on the mechanism of RNA methylation enzyme Mettl3/Nsun4 epistatic coregulation of Sox9,the core transcription factor of chondrogenic differentiation(1)After chondrogenic differentiation of BMSCs,Dot-blot results showed that m5 C and m6 A levels were significantly upregulated in the induced group relative to the normal group.RIP-qPCR results showed that m5 C and m6 A levels of Sox9 were significantly increased after induced chondrogenic differentiation.(2)By Western blot and RT-qPCR,the protein and m RNA expression levels of Nsun4 were significantly decreased in the induced group compared with the normal group,while the protein and m RNA expression levels of Mettl3 were significantly increased.Further knockdown of Nsun4 and Mettl3 induced chondrogenic differentiation separately,and the results showed that both knockdown Nsun4 and Mettl3 chondrocytes showed cavities and failed to form spheres.Moreover,the Western blot results confirmed that the expression of Sox9,Aggrecan and Col2,the markers of chondrogenic differentiation,was significantly decreased in the knockdown Nsun4 and Mettl3-induced group compared with the induced group.Dot-blot experiments confirmed that m5 C levels were significantly decreased in the knockdown Nsun4-induced group compared to the induced group,and m6 A levels were significantly decreased in the knockdown Mettl3-induced group compared to the induced group.(3)RIP-qPCR results showed that the levels of Nsun4 and Mettl3 binding Sox9 RNA were significantly increased in the induced group in comparison to the normal group.(4)The results of CO-IP Western blot and Dot-blot showed that Nsun4 and Mettl3 could form a complex after induction into chondrogenic differentiation.And the RIP-qPCR results confirmed that the levels of Nsun4 and Mettl3 bound to Sox9 3'UTR were significantly increased after induction,and the levels of m5 C and m6 A in the Sox9 3'UTR region were significantly increased.(5)Ribo-seq results showed that the distribution of ribosomes was significantly elevated in the 1950nt-2250 nt range of the Sox9 3'UTR region after induction.The SRAMP website predicted m6 A sites on Sox9 RNA,and the results indicated the presence of potential m6 A sites in the Sox9 3'UTR region(2020nt-2250 nt range).Meanwhile,the heavy sulfite sequencing results revealed that the position of 2062 nt on Sox9 RNA is the m5 C site.The methylation level of the m5 C site was significantly elevated after induction,and the methylation level of the m5 C site was significantly reduced after knockdown of Nsun4 with Mettl3 induction.Further,RIP-qPCR experiments confirmed that 2030 nt on the Sox9 RNA is the m6 A site,which is similar to the position of the m5 C site,and the methylation level of m6 A at this site was significantly reduced after knockdown of Nsun4 and Mettl3.It indicates that Nsun4 and Mettl3 can bind to the 3'UTR region of Sox9 and epitopically modify the m5 C and m6 A sites.(6)The binding proteins of Nsun4 were explored by CO-IP LC/MS,and the results showed that during chondrogenic differentiation,Nsun4 binds Mettl3 with translation-related proteins,including translation elongation factor e EF1a-1.CO-IP Western blot further confirmed that after induction,Nsun4 binds Mettl3 with e EF1a-1.(7)m5C and m6 A can affect the translation process of RNA by recruiting reading proteins,and the Ythdf2 family is a conserved m6 A reading protein.CO-IP Western blot results confirmed that Mettl3 significantly binds Ythdf1-3 after induction,while Nsun4 binds Ythdf2,indicating that Ythdf2 specifically binds to the complex Nsun4/Mettl3.Knockdown of Ythdf2 induced into chondrogenic differentiation,and Western blot results showed that the Sox9 protein expression level was significantly decreased in the knockdown Ythdf2-induced group relative to the induced group,and the RIP-qPCR results indicated that the level of Ythdf2-bound Sox9 m RNA was significantly increased after induction.(8)The results of dual luciferase reporter genes showed that the translation level of Sox9 was significantly decreased after knocking down Mettl3,Nsun4,Ythdf2 and e EF1a-1,respectively.Meanwhile,there was no significant difference in the change of m RNA level of Sox9 relative to the induced group after knockdown of Mettl3,Nsun4 and Ythdf2 induction,respectively.(9)A prokaryotic expression system was used to express Mettl3,Nsun4,Ythdf2 and e EF1a-1 and purify the corresponding proteins for Biacore experiments to explore the binding modes of the four proteins,and the results showed that Mettl4,Nsun4 and Ythdf2 bind to each other two by two to form a complex,followed by e EF1a-1 binding to Ythdf2.(10)AAV viruses with no-load and overexpression of Nsun4 and Mettl3 were transferred into the defect of rat cartilage defect model by infecting BMSCs and reared for 6weeks for sampling.The expression of chondrogenic marker genes was detected by immunofluorescence.The results demonstrated that the protein expression of Sox9,Aggrecan and Col2 was significantly increased in the overexpression Nsun4 with Mettl3 group compared with the null group.Conclusions.1.Wedelolactone,a small molecule of traditional Chinese medicine,can target binding to RNA methylation enzyme Nsun4,regulate the expression of Nsun4,and then regulate the m5 C methylation level of RNA to play a role in promoting chondrogenic differentiation of BMSCs.2.Transcriptomic and translatomic analyses revealed that the translation of overall RNA was enhanced and the ribosome distribution in the 3'UTR region of RNA was increased after chondrogenic differentiation of BMSCs.Meanwhile,the translation efficiency of genes increased at the overall level,and the translation efficiency of genes associated with chondrogenic differentiation was significantly enhanced.Translation of Sox9,a core transcription factor of chondrogenic differentiation,was enhanced.3.During chondrogenic differentiation of BMSCs,the RNA methylesterase Nsun4 forms a complex with Mettl3 to target binding and methylation modifying the 3'UTR region of the core transcription factor Sox9.Further recruitment of reading protein Ythdf2 and translation elongation factor e EF1a-1 regulated the translational reprogramming of Sox9,thereby promoting chondrogenic differentiation of BMSCs.The assembly pattern of the complex NMYE(Nsun4/Mettl3/Ythdf2/e EF1a-1)was also validated in vitro. |
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