Mechanism Of MiR-33-5p Regulating Mycoplasma Gallisepticum Infection Through JNK Pathway In Chickens

Mycoplasma gallisepticum(MG)is the major pathogenic pathogen of chronic respiratory disease(CRD).CRD is widespread in the poultry industry worldwide,causing huge economic losses.So far,no effective prevention and control measures have been found.Therefore,the investigation of the pathogenic mechanism of MG infection is of great scientific importance for the prevention and control of CRD.Our previous studies have focused on the regulatory mechanisms of miRNA-mRNA interaction networks on MG infection.In recent years,it has been reported that the regulatory network of miRNAs is extremely complex.Lnc RNA,as a competitive endogenous RNA(ce RNA),can act as a sponge for miRNA to mediate miRNA-mRNA regulation.miRNA-mRNAs are also regulated by transcription factors.Previous deep sequencing revealed that miR-33-5p was significantly upregulated after MG infection.In this study,a complex regulatory network of Lnc RNA-miRNA-mRNA and TF-miRNA in the process of MG infection was identified through bioinformatics analysis and experiments.It provides an insight into the role of miR-33-5p in MG infection and its underlying function mechanism,which can help the elucidation of the pathogenesis,diagnosis and prevention of MG infection.The main results of this study are as follows:1.Models of MG-infected chicken embryos and cells were constructed.The qPCR result showed that the expression of miR-33-5p was significantly up-regulated in both MG-infected chicken embryos’ lung tissues and cells(p<0.05).Overexpression of miR-33-5p was able to significantly inhibit the expression of MG adhesion protein,named pMGA1.2,in DF-1 cells compared to the control group,whereas inhibition of miR-33-5p had the opposite effect by western blot,suggesting that miR-33-5p inhibits the adhesion of MG to DF-1 cells by suppressing the expression of pMGA1.2.2.The results of qPCR and Elisa showed that MG-induced expression of TNF-α and IL-1β was significantly down-regulated by overexpressed miR-33-5p,whereas miR-33-5p inhibitor significantly promoted the expression of IL-1β and TNF-α(p<0.05).CCK-8 and apoptosis kit assays showed that miR-33-5p promoted proliferation and inhibited apoptosis in MG-infected DF-1 cells.qPCR and Western blot assays revealed that miR-33-5p significantly inhibited the expression of MG-induced pro-apoptotic genes FAS,CASP3,CASP8 and CASP9(p<0.05)and significantly up-regulated the expression of the apoptosis-suppressing gene BCL-2(p<0.05).These results suggest that miR-33-5p can miR-33-5p JNK significantly inhibit MG-induced inflammatory responses,promote cell proliferation,inhibit apoptosis and thus resist MG infection.3.Bioinformatics software predicted that JNK1 is a target gene for miR-33-5p.The expression of JNK1 was significantly down-regulated in MG-infected chick embryo lung tissues and cells by qPCR and Western blot(p<0.05),while the expression of miR-33-5p was on the contrary.Dual-luciferase reporter assay confirmed that the seed sequence of miR-33-5p binds directly to the JNK1-3’UTR by qPCR and Western blot results showed that overexpression of miR-33-5p significantly reduced the expression levels of JNK1 mRNA and protein(p<0.05),while inhibition of miR-33-5p showed the opposite result.These results demonstrate that JNK1 is a direct target gene of miR-33-5p and its expression is negatively regulated by miR-33-5p.4.The regulation of miR-33-5p on the JNK signaling pathway genes was further explored.The results revealed that overexpression of miR-33-5p significantly inhibited the mRNA as well as protein phosphorylation levels of JUN and FOS(p<0.05)while inhibition of miR-33-5p had the opposite result.Overexpression of JNK1 significantly promoted the mRNA and protein phosphorylation levels of JUN and FOS(p<0.05),but this effect could be attenuated by miR-33-5p.The JNK1 inhibitor SP600125 has a specific inhibitory effect on the JNK pathway.Collectively,the above results suggest that miR-33-5p plays a role in MG infection by targeting JNK1 to inhibit the JNK signaling pathway.5.In addition,Elisa,qPCR,and Western blot results showed that overexpression of JNK1 significantly upregulated the expression levels of pMGA1.2,and pro-inflammatory cytokines IL-1β and TNF-α(p<0.05),whereas miR-33-5p inhibited this effect.CCK-8and apoptosis assays revealed that JNK1 significantly inhibited MG-infected cell proliferation o(p<0.05)and promoted apoptosis cells(p<0.05).In addition,further studies showed that JNK1 significantly increased the expression level of pro-apoptotic genes including FAS,CASP3,CASP8 and CASP9(p<0.05)induced by MG,and obviously inhibited the expression level of the anti-apoptosis gene BCL-2(p<0.01).However,co-transfection of JNK1 and miR-33-5p significantly inhibited the JNK1-induced cell proliferation inhibition and apoptosis(p<0.05).The results were reversed after JNK1 inhibition.Taken together,these results suggest that miR-33-5p inhibits MG proliferation by targeting JNK1 to promote cell proliferation and inhibit inflammatory responses and apoptosis induced by MG.6.The result of qPCR showed that both the relative expression of the primary transcript of miR-33-5p(pri-miR-33-5p)and precursor of miR-33-5p(pre-miR-33-5p)was highly upregulated in MG-infected DF-1 cells(p<0.01),which were consistent with the trend of MG-induced expression of mature miR-33-5p,indicating that MG regulates miR-33-5p expression.The results of the dual-luciferase reporter system showed that the core promoter region of miR-33-5p was identified as 951bp-863 bp upstream of the transcription start site.This promoter region could be bound by STAT5 to enhance its activity.Furthermore,STAT5 was extremely significantly upregulated in MG infection.Up-regulated STAT5 increased the expression of miR-33-5p but significantly repressed the expression of the target gene JNK1.Conclusively,STAT5 is a key transcriptional regulator of miR-33-5p in MG infection and decreases JNK1 expression at the transcriptional level by upregulating miR-33-5p.7.qPCR results showed that the expression of Lnc90386 was significantly down-regulated in MG-infected chick embryo lung tissues and cells(p<0.05).Biological software prediction and qPCR results showed that Lnc90386 had no coding ability and was mainly expressed in the cytoplasm.The dual-luciferase reporter gene results indicated that Lnc90386 competed with JNK1 for binding to miR-33-5p.qPCR and Western blot results showed that Lnc90386 decreased the expression of JNK1 by up-regulating miR-33-5p.These results suggest that Lnc90386 impedes its targeting of JNK1 by competing with JNK1 for binding to miR-33-5p.8.The regulatory role of Lnc90386-miR-33-5p-JNK1 in MG infection has been further analyzed The results of qPCR,Elisa and Western blot showed that overexpression of Lnc90386 led to increases in the levels of pMGA1.2,TNF-α,and IL-1β(p<0.05).Furthermore,co-transfection of Lnc90386 and miR-33-5p inhibited the expression of pMGA1.2,TNF-α,and IL-1β induced by overexpressed JNK1(p<0.05).CCK-8 and apoptosis assays showed that overexpression of Lnc90386 inhibited cell proliferation and promoted apoptosis induced by MG infection(p<0.05).Consistently,overexpression of Lnc90386 significantly promoted the expression of MG-induced pro-apoptotic genes FAS,CASP3,CASP8 and CASP9(p<0.05)and inhibited the expression of the anti-apoptosis gene BCL-2.The effect of Lnc90386 on cell proliferation and apoptosis was inhibited by co-transfection of Lnc90386 and miR-33-5p,while inhibition of Lnc90386 resulted in opposite results.These results suggest that Lnc90386 and JNK1 competitively bind miR-33-5p,thereby affecting the regulation of JNK1 by miR-33-5p.Down-regulated miR-33-5p JNK Lnc90386 inhibits JNK1 expression by targeting miR-33-5p,suppresses MG proliferation and MG-induced inflammatory responses,promotes cell proliferation and inhibits apoptosis to defend against MG infection.Collectively,it was revealed that Lnc90386 mediated the regulation of miR-33-5p-JNK1 through ce RNA via acting as a sponge for miR-33-5p,and STAT5 was a key transcriptional regulator of miR-33-5p.During MG infection,miR-33-5p inhibited the JNK1/JUN/FOS signaling pathway,suppressed inflammatory response and promoted cell proliferation through the co-regulation of Lnc90386-miR-33-5p-JNK1 and STAT5-miR-33-5p-JNK1 to inhibit apoptosis and resist MG infection.