| Transmissible gastroenteritis(TGE)is one of the important diseases in hog industry,and its pathogen is transmissible gastroenteritis virus(TGEV).The small intestine is the main target organ of TGEV,and intestinal epithelial cells are the primary target cells of TGEV.TGEV causes death and shedding of small intestinal mucosal epithelial cells,shortening of intestinal villi,disturbance of water and electrolyte in small intestine,and increase of intestinal osmotic pressure,which leads to severe diarrhea,dehydration,metabolic acidosis and high mortality,causing huge losses to pig industry.Studies reported that TGEV-induced mitochondria damage is the key point of cell death,and mitochondrial permeability transition pore(mPTP)is the switch of mitochondrial damage.The abnormal opening of mPTP can cause mitochondrial damage,release apoptotic factors and activate apoptotic signaling pathways.Virus infection can cause host cells circRNA differential expression,and the differential expression of circRNA plays an important regulatory role in viral infection.Previous studies have shown that TGEV infection can cause mitochondria damage in porcine intestinal epithelial cell jejunum 2(IPEC-J2)and the change of circRNA expression profile.circRNA plays an important role in the opening of mPTP.However,the effect of TGEV on mPTP opening and the mechanism of differentially expressed circRNA induced by TGEV in the opening of mPTP remain unclear.Therefore,this study explored the changes of mPTP opening level and expression of mPTP regulatory molecules after TGEV infection in IPEC-J2 cells.Then,the study filtrated and identified the differentially expressed circRNA,and explored the role and mechanism of circRNA to the mPTP opening.Finally,the mechanism of TGEV regulating circRNA expression was clarified.These results were as following.1.The mPTP opening level and expression of mPTP regulatory molecules after TGEV infection were detected.mPTP opening level was detected after 1 MOI TGEV infection.Results showed that TGEV could significantly increase the concentration of mitochondrial calcium(P ＜ 0.01),reduce the mitochondrial membrane potential(MMP)(P ＜0.01)and induce abnormal opening of mPTP(P ＜ 0.01)in IPEC-J2 cells.q RT-PCR and western blotting results showed that the m RNA and protein levels of VDAC1,Cyp D,ANT1 and ATP5D were significantly increased after TGEV infection in IPEC-J2 cells and in the cells of jejunum of piglet(P ＜ 0.05,P ＜ 0.01).2.Identification of circRNA and its role in abnormal opening of mPTP after TGEV infection in IPEC-J2 cells.q RT-PCR was used to screen differentially expressed circRNA related to mitochondria.Results showed that circ5884 was significantly changed,and it was selected as the research object.Comparing circ5884 sequence with sus scrofa genome sequence in NCBI,results showed that circ5884 was derived from exon 63,exon64,exon 65,and exon 66 of birc6,and it was named as circBIRC6-2 according to its parental gene.PCR,RNase R digestion and FISH results showed that circBIRC6-2 was circular RNA.q RT-PCR and FISH results showed that circBIRC6-2 localized in cytoplasm and nucleus,and circBIRC6-2 was significantly down-regulated after TGEV infection in IPEC-J2 cells and in the cells of piglet jejunum(P ＜ 0.01).mPTP opening level was detected after overexpression and knocking down of circBIRC6-2,and results showed that circBIRC6-2 could significantly inhibit the increase of mitochondrial calcium concentration(P ＜ 0.01),the decrease of MMP(P ＜ 0.01)and the abnormal opening of mPTP(P ＜ 0.01).3.The action mode of circBIRC6-2 regulating the abnormal opening of mPTP.qRT-PCR results showed that circBIRC6-2 did not affect the transcription of parental gene birc6(P > 0.05).RIP results showed that VDAC1,Cyp D,ANT1 and ATP5 D did not directly interact with circBIRC6-2(P > 0.05).Sequence analysis results showed that circBIRC6-2 contained an ORF which can encode 236 amino acid(aa)and two potential IRES sequences.The dual-luciferase reporter assay(DLR)results showed that the two potential IRES sequences could significantly promote protein translation(P ＜ 0.01).In order to demonstrate that circBIRC6-2 could encode proteins,pcircBIRC6-2,pcircBIRC6-2-Flag,pcircBIRC6-2-Flag-Mut and p Flag-BIRC6-236 aa recombinant plasmids were constructed,and they were packaged into lentivirus respectively to infect IPEC-J2 cells.q RT-PCR,western blotting,IP-MS,IF and IEM results showed that circBIRC6-2 could encode a protein of 236 amino acid,and it was named as BIRC6-236 aa,which localized in the cytoplasm,nucleus and mitochondrion.In addition,circBIRC6-2significantly inhibited TGEV-induced increase of mitochondrial calcium concentration(P＜ 0.01),decrease of MMP(P ＜ 0.01)and abnormal opening of mPTP(P ＜ 0.05)by encoding BIRC6-236 aa.4.The mechanism of BIRC6-236 aa inhibiting TGEV-induced abnormal opening of mPTP by targeting VDAC1.IP-MS was used to analyze the proteins interacted with BIRC6-236 aa after overexpression of BIRC6-236 aa in IPEC-J2 cells,and results showed that 91 proteins might interact with BIRC6-236 aa.Bioinformatics analysis results showed that the BIRC6-236 aa interacting proteins enriched in mitochondria-related biological process,and 16 proteins located in mitochondria.Co-IP and PLA results showed that BIRC6-236 aa could interact with VDAC1.In addition,circBIRC6-2 and BIRC6-236 aa could significantly weaken the interaction between VDAC1 and Cyp D(P ＜ 0.05,P ＜0.01).5.The mechanism of circBIRC6-2 expression regulated by TGEV.TGEV structural and nonstructural proteins were overexpressed in IPEC-J2 cells,q RT-PCR results showed that TGEV-M could significantly inhibit the expression of circBIRC6-2(P < 0.01).IP-MS results showed that 117 proteins interacted with TGEV-M.Co-IP and PLA results showed that TGEV-M could interact with hn RNPA1 in the cytoplasm.TGEV could significantly inhibit hn RNPA1 transposition from cytoplasm to nucleus.qRT-PCR results showed that hnRNPA1 could significantly promote the expression of circBIRC6-2(P <0.01).RIP results showed that hn RNPA1 could interact with pre-m RNA of birc6.In addition,TGEV-M could significantly promote the increase of mitochondrial calcium concentration(P < 0.01),decrease of MMP(P < 0.01),abnormal opening of mPTP(P <0.05)and the interaction between VDAC1 and Cyp D(P < 0.05).In this study,we demonstrated that TGEV could induce abnormal opening of mPTP.circBIRC6-2 could inhibit TGEV-induced abnormal opening of mPTP by encoding BIRC6-236 aa which could inhibit the formation of VDAC1 and Cyp D protein complexes.TGEV-M could inhibit the circBIRC6-2 expression by interacting with hn RNPA1.The results elucidated the abnormal opening of mPTP caused by TGEV and the role of circBIRC6-2 in this process.This result could provide a theoretical basis for further revealing the pathogenic mechanism of TGEV. |
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