| ObjectivesTo explore whether the inflammatory factor TNF-? can affect the differentiation of mesenchymal stem cells(MSCs)into sweat gland cells and reveal its regulatory mechanism from the perspective of epigenetics.Contents1.The effect of TNF-? on the differentiation of MSCs to sweat gland cells(1)MSCs are widely available,easily accessible,immunogenic and multipotent.They are considered as an ideal cell source for stem cell-centered cell therapy.Mesenchymal stem cells can be successfully induced to acquire sweat gland phenotype in vitro by gene editing and co-culture with sweat gland cells in matrix gel,which makes it possible to regenerate sweat gland function in patients with deep burn in the future.In order to solve the problems of low induction efficiency and long induction cycle of MSCs in vitro,and to avoid the miss-target effect of gene editing technology,the effectiveness and safety of delivery system,immune rejection,ethical debate,and the poor mechanical properties,instability and excessive absorption of matrix glue in vivo,we constructed an extracellular matrix-like simulation in vitro by using bioprinting technology.We used bio-printing technology to construct extracellular matrix in vitro to induce the differentiation of MSCs into sweat gland cells by simulating the microenvironment of sweat gland development.(2)Microenvironment can affect the function of MSCs.Studies have shown that inflammatory mediators such as TNF-? and IL-6 can inhibit the differentiation of MSCs into bone,cartilage and adipocytes.In order to effectively induce MSCs to differentiate into sweat glands in burn patients,we simulated the microenvironment in vitro and detected the effect of TNF-? on the differentiation of MSCs into sweat gland cells.2.Epigenetic mechanism of TNF-? regulating the differentiation of MSCs to sweat gland cells(1)Nanog,a pluripotent regulator,is not only related to the differentiation potential of embryonic stem cells and cancer stem cells,but also involved in regulating the differentiation process of MSCs.Studies have shown that knocking down Nanog of MSCs can inhibit their proliferation and differentiation potential.To elucidate the mechanism of TNF-? affecting the differentiation of MSCs into sweat gland cells,we examined the effect of TNF-? on the expression of Nanog.(2)N6-methyladenosine(m6A)is one of the most common internal methylation modifications in mammalian cells.It is regulated by methylase complex METTL3/METTLL14/WATP and demethylase FTO and ALKBH5.Nanog has been proved to contain m6 A modification.Regulation of m6 A level of Nanog mRNA can affect its mRNA abundance and thus determine the fate of embryonic stem cells and cancer stem cells.Based on these findings and in order to elucidate the mechanism of TNF-alpha regulating the expression of Nanog in mesenchymal stem cells,we first investigated the effect of TNF-? on the total RNA m6 A level of MSCs and the methylase or demethylase involved in regulating this change.(3)Meclofenac acid(MA)was found to be a selective inhibitor of FTO,which could compete with FTO to bind m6A-modified nucleic acid to increase the total RNA m6 A level.To verify that the demethylation of FTO is involved in regulating the expression and directional differentiation of TNF-? in MSCs,we used MA2,the form of ethyl ester of MA,to inhibit the demethylation of MSCs FTO and observed its effects on the level of m6 A,FTO expression,Nanog expression and sweat gland differentiation of MSCs.(4)M6A modification accelerates the degradation of RNA and regulates its expression level.To further reveal how the demethylation of FTO under the influence of TNF-? regulates the expression of Nanog,we examined the effect of TNF-? on the m6 A level and half-life of MSCs Nanog.Materials and methods1.The effect of TNF-? on the differentiation of MSCs to sweat gland cells(1)MSCs were induced to differentiate into sweat gland cells in four environments: two-dimensional culture,two-dimensional culture add mouse foot homogenate,extracellular matrix like bionic material,sweat gland extracellular matrix like bionic material and mouse foot dermal homogenate.After 14 days of culture,RT-qPCR and immunofluorescence were used to detect the differences in the expression of sweat gland cell markers in the cells of each group.(2)MSCs were induced to differentiate into sweat gland cells by constructing sweat gland-like extracellular matrix with biomimetic material and mouse foot dermal homogenate.The cells were treated with 20 ng/ml TNF-? and blank respectively.After 14 days of culture,the difference of sweat gland cell markers was detected by RT-qPCR and immunofluorescence.2.Epigenetic mechanism of TNF-? regulating the differentiation of MSCs to sweat gland cells(1)MSCs were treated with 20ng/ml TNF-? and blank treatment respectively in 2D and 3D culture conditions.After 6 hours of continuous culture,the effects of TNF-? on the expression of Nanog in MSCs were detected by RT-qPCR and Western blot.(2)MSCs were treated with 20 ng/ml and 0 ng/ml TNF-?.After 6 hours of culture,the difference of m6 A modification level of total RNA of MSCs between the two groups was detected by immunofluorescence.The difference of methylase and demethylase expression between the two groups was detected by RT-qPCR and Western blot.(3)MA2 inhibits FTO demethylation to verify whether FTO demethylation plays an important role in TNF-alpha regulation of mesenchymal stem cell differentiation.Firstly,the effect of MA2 on total RNA m6 A modification level of mesenchymal stem cells was detected by immunofluorescence assay.Secondly,RT-q PCR and Western blot were used to detect whether MA2 induced changes in FTO expression at the level of mRNA and protein.Then the effects of MA2 on the expression of Nanog in mesenchymal stem cells at the levels of mRNA and protein were examined.Finally,RT-qPCR and immunofluorescence methods were used to verify the difference of sweat cell markers expression between mesenchymal stem cells and sweat gland cells 14 days after induction in sweat-like extracellular matrix.(4)MeRIP-qPCR was used to detect the effect of TNF-? on the m6 A modification level of Nanog in MSCs.Secondly,actinomycin D was used to inhibit cell RNA synthesis,and RT-qPCR was used to verify the effect of Nanog m6 A modification on its half-life.Results1.The effect of TNF-? on the differentiation of MSCs to sweat gland cells(1)Sweat-like extracellular matrix,constructed in vitro with biomimetic materials and mouse foot homogenate as the main components,mimics the microenvironment of sweat gland development and can induce MSCs to differentiate into sweat gland cells and express sweat gland cell markers at the level of mRNA and protein.(2)Compared with the control group,the expression of sweat cell markers in MSCs treated with TNF-? was significantly inhibited after 14 days of induction under the same conditions.The results showed that inflammatory factor TNF-? could inhibit not only the differentiation of MSCs into bone,cartilage and adipocytes,but also sweat gland cells.2.Epigenetic mechanism of TNF-? regulating the differentiation of MSCs to sweat gland cells(1)The results of RT-qPCR and Western blot showed that the expression of Nanog in mesenchymal stem cells treated with TNF-? was inhibited at both the level of gene and protein,suggesting that TNF-? could affect the differentiation of MSCs by regulating the expression of Nanog,which is consistent with the theory that knocking down the expression of Nanog in MSCs could inhibit the proliferation and differentiation potential.(2)Compared with the control group,the m6 A modification level of total RNA was significantly increased after 6 hours of exposure to TNF-? in MSCs,and the expression of demethylase FTO was inhibited at the level of mRNA and protein,but there was no significant difference in the expression of methylase METTL3 and demethylase ALKBH5 genes between the two groups.In conclusion,the inhibition of FTO expression by TNF-? resulted in a significant increase in the m6 A modification level of total RNA of MSCs..(3)Compared with the control group,the m6 A modification level of total RNA in MSCs exposed to MA2 increased significantly only 6 hours later.However,there was no significant difference in the level of FTO between the two groups,which was consistent with the theory that MA2 inhibited FTO demethylation by competing with FTO to bind m6 A modification site.In addition,MA2 inhibited the expression of Nanog at the level of mRNA and protein,resulting in a significant decrease in the expression of sweat cell markers after 14 days of induction of MSCs in the extracellular matrix of sweat-like glands.Therefore,the demethylation of FTO plays an important role in regulating the expression and differentiation of Nanog in MSCs.(4)TNF-? inhibits FTO expression and improves the m6 A modification level of MSCs Nanog gene,resulting in a significant reduction of the half-life of Nanog gene,which is consistent with the theory that m6 A modification accelerates the degradation of mRNA.ConclusionsIn this study,we show that TNF-? inhibits the differentiation of MSCs to sweat glands in a specific sweat gland-inducing environment,accompanied with downexpression of Nanog,a core pluripotency factor.We elucidated that fat mass and obesity-associated protein(FTO)-mediated m6 A demethylation is involved in the regulation of MSCs differentiation potential.Exposure of MSCs to TNF-? reduced expression of FTO,which demethylated Nanog mRNA.Down-expression of FTO increased Nanog mRNA methylation,decreased Nanog mRNA and protein expression,significantly inhibited MSCs capacity for differentiation to sweat gland cells.Our finding is the first to elucidate the functional importance of m6 A modification in MSCs,providing new insight that microenvironment can regulate the multipotency of MSCs at the post-transcriptional level.Moreover,to maintain differentiation capacity of MSCs by regulating m6 A modification suggested a novel potential therapeutic target for stem cell-mediated regenerative medicine. |
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