| Objective:To study the effect of MHL on the methylation level of N6-methyl adenine（m6A）in the process of osteogenic differentiation of bone marrow mesenchymal stem cells（BMSCs）and the regulation of methyltransferase Mettl3（Methyltransferase-like3）,Mettl14（Methyltransferase-like 14）、and WTAP（Wilms tumour 1-associated protein）,we detected the mRNA and protein expression levels of m6A methylation and methyltransferase during the osteogenic differentiation of BMSCs in the control group,OS group and MHL group,analyzed the difference of m6A methylation level and the expression levels of Mettl3,Mettl14 and WTAP during the osteogenic differentiation of BMSCs cultured by MHL,and explored the function of mettl3 in the regulation of osteogenic differentiation by MHL.Methods:MTT assay,acid phosphatase assay,Alizarin red staining,alkaline phosphatase staining and alkaline phosphatase detection were used to detect the proliferation rate and alkaline phosphatase（ALP）activity of BMSCs in control group,osteogenic inducer（OS）group and different concentrations of Eclipta herba extract（MHL）group.The mRNA expression levels of Osteocalcin（OCN）and Osterix in BMSCs treated with complete medium,OS,1μg/ml MHL,2μg/ml MHL and 5μg/ml MHL for 9 days were detected by QRT PCR.And then we analyzed the differences of proliferation ability,bone differentiation and bone mineralization ability of BMSCs treated with different treatments.The methylation level of m6A in total RNA of BMSCs cultured in complete medium,OS and 5μg/ml MHL for 9days was quantitatively detected by colorimetric method.RNA methylated RNA immunoprecipitation sequencing（Me RIP-seq）method was used to analyzed the m6A modified omics of BMSCs in control group,OS group and 5μg/ml MHL group.Then we analyzed the modification characteristics of m6A regulated by Eclipta herba extract.The mRNA and protein expression levels of Mettl3,Mettl14 and WTAP in BMSCs treated with complete medium,OS,1μg/ml MHL,2μg/ml MHL and 5μg/ml MHL for 9 days were detected by q RT-PCR and Western blot.BMSCs were infected by lentivirus containing mettl3-shctrl and mettl3-sh to obtain BMSCs Mettl3-shctrland BMSCs Mettl3-sh.Fluorescence microscopy,PCR and Western blot were used to identify the infection effect.We used alkaline phosphatase activity assay to detecte ALP activity in BMSCs Mettl3-shctrland BMSCs Mettl3-shtreated with OS and 5μg/ml MHL for 9 days,and analyzed the difference of ALP activity in BMSCs treated with OS and 5μg/ml MHL.Results:1.MHL had no significant effect on proliferation of BMSCs（p>0.05）.MHL promoted the osteogenic differentiation and mineralization of BMSCs in a dose-dependent manner（p<0.05）,and promoted the mRNA expression of osterix and OCN（p<0.05）.2.Compared with the control group,the methylation level of m6A in BMSCs of OS group was higher（p<0.05）;compared with OS group,the methylation level of m6A in BMSCs of MHL group was increased（p<0.01）.In control group,OS group and MHL group,more than 90%of the m6A peak was located in GGAC sequence,and the m6A site of BMSCs was mainly located near the translation terminator codon.MHL mainly changed the m6A modification of HIF-1α,hippo and other genes related to osteogenic differentiation.3.Compared with OS,MHL significantly increased the mRNA and protein expression of Mettl3 and Mettl14（p<0.05）,but had no effect on the expression of WTAP（p>0.05）.4.Mettl3 knockdown cell line BMSCs mettl3 sh was successfully established（p<0.01）.The ALP activity in MHL group was higher than that in OS group after 9 days of culture with OS and 5μg/ml MHL（p<0.01）.Compared with BMSCsMettl3-sh Ctrl,the ALP activity of BMSCs Mettl3-shwas significantly decreased after 5μg/ml MHL treatment（p<0.01）.Conclusions:MHL increases the methylation level of total RNA and promotes the expression levels of methyltransferase Mettl3 and Mettl14 in osteogenic differentiation of BMSCs,suggesting that mettl3/14 mediated m6A modification may play an important role in MHL promoting osteogenic differentiation of BMSCs. |
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