Study On The Mechanism Of RNA N6-methyladenosine Methylation In A Mouse Model Of Scleroderma

Background:Scleroderma is a connective tissue disease with the highest mortality rate,but its pathogenesis is not clearly understood.Current studies indicate that multi-organ fibrosis caused by immune abnormalities and vascular damage is a recognized pathophysiological feature under the dual role of genetic and environmental factors.In recent decades,traditional treatments have not significantly changed scleroderma survival rates.Therefore,it is the focus of great attention to develop new therapeutic methods through in-depth understanding of its pathogenesis.Epigenetics refers to changes in the level of gene expression without changing the nucleotide sequence.Among them,DNA methylation,RNA methylation,chromatin remodeling,histone modification and nc RNA are important components of epigenetics,but RNA methylation lags behind other epigenetics.In recent years,with the breakthrough of Me RIP-seq and other related technologies,RNA methylation has rapidly become a research hotspot.Of the more than 170 RNA modifications identified so far,N6-methyladenosine is the most common methylation modification in eukaryotes,i.e.,methylation of the sixth N atom on the adenine of RNA.m6 A modification can affect various aspects of RNA regulation,including RNA stability,localization,transport,splicing and translation.A large number of studies have reported that m6 A modification plays an important role in the diagnosis,treatment and prognosis of tumors,neurological diseases and cardiovascular diseases,but the mechanism of m6 A modification in scleroderma remains unclear.PartⅠ Changes in m6 A methylation landscape of a mouse model of scleroderma and its underlying mechanism Objective: To investigate the changes of m RNA&lnc RNA&small RNA m6 A modification profile and its potential mechanism in bleomycin-induced scleroderma mouse model.Methods:(1)Bleomycin-induced scleroderma mouse model was established,which were divided into control group and BLM group.The morphological changes of skin were observed by HE staining and Masson staining,and the m RNA expression levels of Col1α1 and Col3α1 were detected by RT-PCR.(2)The modification and expression profiles of m RNA,lnc RNA and small RNA transcripts were screened by epigenome microarray.(3)GO&KEGG analysis of modified difference m RNA transcript enriched entries.(4)The modification and expression profiles of m RNA transcripts were analyzed jointly,and the protein interaction was analyzed.(5)Me RIP-q PCR verified differentially modified m RNA and lnc RNA,namely Hras,Lama3,Stat3,Tnc,Ccl3,Ccl9,Saa1 and Il1 b,ENSMUST00000136927,R＿040273,ENSMUST00000143429 and ENSMUST00000162565.(6)The differentially expressed m RNAs,namely Hras,Tnc,Ccl3,Ccl9,Saa1 and Il1 b,were verified by RT-PCR.(7)lnc RNA-mi RNA-m RNA ce RNA network was constructed using mi RDB and mi Rwalk database.(8)Clustering heat maps of differentially modified lnc RNA and m RNA of adjacent genes were constructed.(9)pri-5-mmu-mir-6923 、 pri-5-mmu-mir-7071 、 pri-3-mmu-mir-3071 、pri-5-mmu-mir-149 、 mmu-mir-7064 、 mmu-mir-7665 、 mmu-mir-6239 、mmu-mir-376b、mmu-mir-7087 were analyzed using mi Rwalk database and mi RDB database.Results:(1)HE staining showed that in BLM group,dermal thickness increased,subcutaneous fat layer thickness decreased,and a large number of inflammatory cells infiltrated.Masson staining showed that collagen deposition increased in BLM group and extended to subcutaneous adipose layer,or even partially replaced adipose tissue.RT-PCR showed that the m RNA expression levels of Col1α1 and Col3α1 in BLM group were significantly increased.The changes of body weight in mice showed that the body weight of BLM group decreased significantly.(2)The modification profile data of the epigenome microarray showed that the modification levels of 733 m RNA transcripts were up-regulated and 110 m RNA transcripts were down-regulated;The modification level of 85 lnc RNA transcripts was up-regulated,and the modification level of 16 lnc RNA transcripts was down-regulated.80 small RNA transcripts were up-regulated and 19 small RNA transcripts were down-regulated.(3)The expression profile data of the epigenome microarray showed that 1991 m RNA transcripts were significantly up-regulated and 1913 m RNA transcripts were significantly down-regulated,and the modification level and expression level of the m RNA transcripts were negatively correlated.Protein interaction analysis of these m RNA transcripts showed that Il1 b interacted with Hras,Ccl3 and Saa1,which may play an important regulatory role in scleroderma.(4)Me RIP-q PCR showed that the modification levels of Hras,Lama3,Tnc and Stat3 were significantly up-regulated,while the modification levels of Ccl3,Ccl9,Saa1 and Il1b were significantly down-regulated.RT-PCR showed that the expression and modification levels of Hras,Ccl3,Saa1 and Il1 b showed a reverse trend.Hras,Tnc,Ccl3 and Ccl9 have corresponding RNA-binding proteins,and the expression levels of Mbnl1,Mbnl3 and Taf15 transcripts are significantly different in the expression profile.(5)RT-PCR showed that there was no significant difference in the expression levels of METTL3,METTL14,WTAP and ALKBH5,but the expression level of FTO was down-regulated.The role of FTO and Tnc in scleroderma is the content of the second part.(6)The expression level of lnc RNA transcripts was negatively correlated with the modification level.Me RIP-q PCR showed that the modification levels of ENSMUST00000136927 and NR＿040273 were up-regulated,and the modification levels of ENSMUST00000162565 and ENSMUST00000143429 were down-regulated.(7)The ce RNA network of ENSMUST00000066545,ENSMUST00000197091 and ENSMUST00000147045 indicated that these three lnc RNAs might affect the expression of related target genes by competitively binding mi RNAs.(8)Adjacent gene analysis of differential-modified lnc RNA provided possible target genes of Galns,Lilr4 b,Msn,Saa3,Cflar,Dmpk,Frmd4 b,Otub2,Sdccag3 and Il1f6.(9)Modification levels of some pri-mi RNA,pre-mir NA,sno RNA and sn RNA transcripts changed,and more m RNAs were screened through prediction of target genes of 9 precursor mi RNAs.Conclusions: In this study,the m RNA&lnc RNA& small RNA modification profiles of a mouse model of scleroderma were constructed by transcripome microarray for the first time,and the mechanism of m6 A modification on scleroderma was explored through bioinformatics analysis and molecular biology experiments.Part Ⅱ Regulation of FTO-mediated Tnc m6 A modification of skin fibrosis in a mouse model of scleroderma Objective: To explore the mechanism of FTO-mediated Tnc m6 A modification of skin fibrosis in a mouse model of scleroderma.Methods:(1)Me RIP-q PCR and RT-PCR were used to verify the modification level and expression level of Tnc in scleroderma patients.(2)The Tnc knockdown scleroderma mouse model was constructed by recombinant adenovirus vector.The morphological changes of skin were observed by HE&Masson staining,the m RNA expression levels of Col1α1 and Col3α1 were detected by RT-PCR,and the content of hydroxyproline in skin was detected by alkaline hydrolysation.(3)A mouse model of scleroderma was constructed with the intervention of FTO recombinant protein,and the effects of FTO overexpression on Tnc gene and protein levels were detected by Western blot and RT-PCR.(4)A model of scleroderma mice in which FTO and Tnc recombinant proteins intervened together were constructed,and FTO and Tnc were overexpressed simultaneously.The morphological changes of skin were observed by HE&Masson staining,the m RNA expression levels of Col1α1 and Col3α1 were detected by RT-PCR,and the hydroxyproline content of skin was detected by alkaline hydrolysation to confirm whether FTO-mediated m6 A modification can affect the function of Tnc in scleroderma.Results:(1)Me RIP-q PCR and RT-PCR showed that the modification level and expression level of Tnc in scleroderma patients were up-regulated.(2)HE staining showed that knocking down Tnc could reduce dermal thickness,increase subcutaneous fat thickness and relieve inflammatory cell infiltration in scleroderma mice.Masson staining showed that knocking down Tnc could reduce the area of collagen deposition in scleroderma mice.RT-PCR showed that knocking down Tnc could down-regulate the m RNA expression levels of Col1α1and Col3α1.In addition,knocking down Tnc can reduce the content of hydroxyproline in skin lesions.(3)Me RIP-q PCR showed that FTO overexpression could down-regulate the modification level of Tnc,as well as the expression level of Tnc gene and protein.(4)HE staining showed that FTO overexpression could decrease dermal thickness and increase subcutaneous fat thickness in scleroderma mice.Masson staining showed that FTO overexpression could reduce the skin collagen deposition area in scleroderma mice.RT-PCR showed that FTO overexpression down-regulated Col1α1 and Col3α1 m RNA expression levels and hydroxyproline content.(5)The results of HE&Masson staining,Col1α1&Col3α1 m RNA and hydroxyproline content showed that Tnc overexpression could antagonize the alleviating effects of FTO overexpression on skin fibrosis in scleroderma mice.Conclusion: FTO-mediated m6 A modification could inhibit Tnc modification and expression level,thus alleviating skin fibrosis in a mouse model of scleroderma.