| The existence of chronic respiratory diseases in the poultry farming process can seriously affect hatchability,egg production rate and egg quality.The limitations of traditional antibiotics and vaccines have made these diseases bring huge problems to farmers and loss many benefits in chicken industry.Micro RNA is a type of non-coding short-chain single-stranded RNA.Because of its highly conservative evolution and extensive intracellular role,as well as its abnormal expression in airway inflammation,miRNA has become an important factor in controlling the occurrence and development of diseases.Most of the current research is based on the function of abnormal expression of miRNA in diseases,but it ignores the causes of abnormal expression of miRNA.PRMT1,as an epigenetic modification enzyme,has been confirmed to be involved in the occurrence of inflammation of epithelial cells.In our previous research,we confirmed the regulatory effect of PRMT1 on miRNA.However,the specific mechanism of this process is still unknown.Which miRNAs can be regulated by PRMT1 in lung inflammation? Which inflammatory factors can affect the regulation of PRMT1 on miRNA? How does cytokine-regulated PRMT1 affect the occurrence of miRNA? Will PRMT1 cooperate with some transcription factors to affect miRNA expression? Which key miRNA can be regulated during this process? Regarding these issues,we started the research on this subject.Experiment 1 PRMT1 regulates lung inflammation-related snc RNAs.Screen and identify the miRNAs regulated by PRMT1 in airway inflammation.Lung epithelial cells overexpressed PRMT1 for 48 hours for small-RNA seq,and found 73 differentially expressed miRNAs and 436 pi RNAs.comparing these snc RNAs with 5 datasets of microarrays(GEO dataset),23 snc RNAs were related to asthma.Following data validation,12 micro RNAs and 2 PIWI-interacting RNAs.The target genes of these 14 snc RNAs were predicted by KEGG.The signaling pathways analysis of these genes indicated that,Ras/Rap1-mitogen activated protein kinases as a major target for PRMT1 induced snc RNAs.Experiment 2 PRMT1 regulates pri-miRNA.Lung epithelial cells overexpressed PRMT1 at short time points 6h,12 h and 24 h.RT-q PCR detects the expression of pri-miRNA.It was found that PRMT1 can regulate the expression of 10 kinds of pri-miRNAs such as pri-let-7i.RT-q PCR verification after 24 h and 48 h knockdown of PRMT1 using si-PRMT1 revealed that mature miRNA and pri-miRNA(let-7i,let-7d,miR-1296,miR-423,and miR-550a)were down-regulated by PRMT1 knockdown.And the time point of pri-miRNA expression change is before the mature miRNA,suggesting that these five miRNA transcripts(ie pri-miRNA)may be regulated by PRMT1 through a certain molecular mechanism,thereby affecting the expression of mature miRNA.Experiment 3 TGF-β1 regulates PRMT1-miRNA in lung epithelial cells.Lung epithelial cells were added with IL-4(20ng / m L),TNF-α(10g / m L)and TGF-β1(5ng / ml)stimulating factors to detect m RNA and protein expression of PRMT1 after 24 h.The results showed that TGF-β1(5ng / ml)can significantly increase(P <0.1)the expression of PRMT1.Subsequently,TGF-β1 was used to stimulate epithelial cells(0,3h,6h,12 h,24h),and it was found that TGF-β1 significantly increased PRMT1 in a short time(3-12h),and could significantly increase pri-let-7i,pri-let-7d,pri-mir-423 and pri-mir-1296 expression.TGF-β1 miRNA detection at 24 h confirmed that TGF-β1 can regulate the expression of mature miRNAs(let-7d,let-7i,miR-423 and miR-1296).Experiment 4: TGF-β1 stimulates PRMT1 to regulate the expression of pri-miRNA.The online website was used to predict the protein interacting with PRMT1.Four transcription factors(STAT1,RUNX1,p300,and p53)that might interact with PRMT1.BEAS-2B was transfected with pc DNA3.1 and PRMT1 for 48 h to collect cell lysate,and immunoprecipitation(IP)was performed using Ig G antibody and PRMT1 antibody.The results of WB analysis showed that PRMT1 can indeed interact with transcription factors and combined STAT1 and RUNX1 can be significantly increased by overexpressed PRMT1.The addition of TGF-β1 to BEAS-2B cells stimulated by WB,RT-q PCR and cellular immunofluorescence detection proved that it can upregulate the expression of STAT1 and RUNX1.It was further confirmed by Co-IP that TGF-β1 can stimulate and upregulate the interaction of PRMT1 with STAT1 and RUNX1.BEAS-2B cells were transfected with si-PRMT1 and stimulated with TGF-β1 6 hours later.Co-IP results confirmed that inhibition of PRMT1 can effectively reduce the expression of transcription factors that combined with PRMT1,proving that TGF-β1 regulates the expression of RUNX1 and STAT1 through PRMT1.Experiment 5 Study on the mechanism of transcription factors regulating pri-miRNA.Based on the previous experiments,this experiment firstly knocked down STAT1 and RUNX1,then used RT-q PCR technology to detect the expression of pri-miRNA.The results show that STAT1 knockdown can significantly reduce pri-let-7d,pri-let-7i,pri-mir-1296 and pri-mir-423.After knocking down RUNX1,the expression of pri-let-7i and pri-mir-423 is downregulated.The online website predicted the regulatory effects of STAT1 and RUNX1 on pri-miRNA,and found that STAT1 and RUNX1 have binding sites in the pri-let-7i and pri-mir-423 promoters,respectively.Then through the Ch-IP test,it was confirmed that STAT1 can bind to the pri-let-7i promoter region,and RUNX1 can bind to the pri-let-7i promoter regions.In summary,we discovered a new mechanism in the process of airway inflammation.In lung epithelial cells,the abnormal expression of PRMT1 can significantly affect the expression of a variety of inflammation-related mature miRNAs;the cytokine TGF-β1 can activate the regulation of miRNAs by PRMT1;the activated PRMT1 acts as a co-transcription factor to form transcriptional co-transcription factors with the transcription factors STAT1 and RUNX1 The activator further specifically binds and activates pri-let-7i,thereby regulating the expression of mature let-7i. |
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