Our previous study has shown that the transcription factor Krüppel-like factor 7(KLF7) promotes peripheral nerve regeneration and motor function recovery after spinal cord injury.KLF7 also participates in traumat...Our previous study has shown that the transcription factor Krüppel-like factor 7(KLF7) promotes peripheral nerve regeneration and motor function recovery after spinal cord injury.KLF7 also participates in traumatic brain injury,but its regulatory mechanisms remain poorly understood.In the present study,an HT22 cell model of traumatic brain injury was established by stretch injury and oxygenglucose deprivation.These cells were then transfected with an adeno-associated virus carrying KLF7(AAV-KLF7).The results revealed that,after stretch injury and oxygen-glucose deprivation,KLF7 greatly reduced apoptosis,activated caspase-3 and lactate dehydrogenase,downregulated the expression of the apoptotic markers B-cell lymphoma 2(Bcl-2)-associated X protein(Bax) and cleaved caspase-3,and increased the expression of βIII-tubulin and the antiapoptotic marker Bcl-2.Furthermore,KLF7 overexpression upregulated Janus kinase 2(JAK2) and signal transducer and activator of transcription 3(STAT3) phosphorylation in HT22 cells treated by stretch injury and oxygenglucose deprivation.Immunoprecipitation assays revealed that KLF7 directly participated in the phosphorylation of STAT3.In addition,treatment with AG490,a selective inhibitor of JAK2/STAT3,weakened the protective effects of KLF7.A mouse controlled cortical impact model of traumatic brain injury was then established.At 30 minutes before modeling,AAV-KLF7 was injected into the ipsilateral lateral ventricle.The protein and m RNA levels of KLF7 in the hippocampus were increased at 1 day after injury and recovered to normal levels at 3 days after injury.KLF7 reduced ipsilateral hippocampal atrophy,decreased the injured cortex volume,downregulated Bax and cleaved caspase-3 expression,and increased the number of 5-bromo-2'-deoxyuridine-positive neurons and Bcl-2 protein expression.Moreover,KLF7 transfection greatly enhanced the phosphorylation of JAK2 and STAT3 in the ipsilateral hippocampus.These results suggest that KLF7 may protect hippocampal neurons after traumatic brain injury through activation of the JAK2/STAT3 signaling pathway.The study was approved by the Institutional Review Board of Mudanjiang Medical University,China(approval No.mdjyxy-2018-0012) on March 6,2018.展开更多
Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds.A regulatory sequence ...Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds.A regulatory sequence of EPSPS gene was isolated previously,and an alteration in its 5´-untranslated region(UTR)pyrimidine(Py)-rich stretch element is involved in the regulation of EPSPS expression in glyphosate-resistant(GR)Eleusine indica.However,the transcription factors involved in this regulatory sequence remain to be elucidated.In this study,we investigated the regulatory network of EPSPS overexpression associated genes in a GR E.indica population by RNA-seq.The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2752 unigenes and a decrease in the expression of 4025 unigenes in the GR E.indica,compared to the glyphosatesusceptible(GS)E.indica.Among them,1373 unigenes were identified to be co-expressed with the EPSPS gene in GR E.indica.GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway,chlorophy II and peroxisome metabolism processes.Notably,the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate(S3P,a substrate of EPSPS),was also up-regulated.Eight transcription factors were identified as likely to be involved in the regulation of the EPSPS expression,and three of them(ARF2,ARF8 and BPC6)showed more binding sites because of a(CT)n insertion of the 5´-UTR Py-rich stretch element in GR.However,the yeast one-hybrid assay illustrated that ARF8 and BPC6 could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS,but could not bind to the mutated form.Our data suggests that the transcriptional regulation of EPSPS expression is complex and was significantly altered in GR E.indica.These discoveries provide new references for further study of the EPSPS overexpression mechanism that endows glyphosate resistance.展开更多
基金supported by the National Natural Science Foundation of China,No.81870977 (to YW)the Natural Science Foundation of Heilongjiang of China,No.H2018068 (to WYL)+3 种基金the Basic Research Operating Expenses Program of Heilongjiang Provincial Universities of China,No.2019-KYYWFMY-0018 (to WYL)the Graduate Innovative Research Projects of Mudanjiang Medical College of China,No.YJSCX-MY22 (to DM)Key projects of Education Department of Hebei Province of China,No.ZD2020178 (to XMF)Hebei Key Laboratory of Nerve Injury and Repair of China (to XMF)。
文摘Our previous study has shown that the transcription factor Krüppel-like factor 7(KLF7) promotes peripheral nerve regeneration and motor function recovery after spinal cord injury.KLF7 also participates in traumatic brain injury,but its regulatory mechanisms remain poorly understood.In the present study,an HT22 cell model of traumatic brain injury was established by stretch injury and oxygenglucose deprivation.These cells were then transfected with an adeno-associated virus carrying KLF7(AAV-KLF7).The results revealed that,after stretch injury and oxygen-glucose deprivation,KLF7 greatly reduced apoptosis,activated caspase-3 and lactate dehydrogenase,downregulated the expression of the apoptotic markers B-cell lymphoma 2(Bcl-2)-associated X protein(Bax) and cleaved caspase-3,and increased the expression of βIII-tubulin and the antiapoptotic marker Bcl-2.Furthermore,KLF7 overexpression upregulated Janus kinase 2(JAK2) and signal transducer and activator of transcription 3(STAT3) phosphorylation in HT22 cells treated by stretch injury and oxygenglucose deprivation.Immunoprecipitation assays revealed that KLF7 directly participated in the phosphorylation of STAT3.In addition,treatment with AG490,a selective inhibitor of JAK2/STAT3,weakened the protective effects of KLF7.A mouse controlled cortical impact model of traumatic brain injury was then established.At 30 minutes before modeling,AAV-KLF7 was injected into the ipsilateral lateral ventricle.The protein and m RNA levels of KLF7 in the hippocampus were increased at 1 day after injury and recovered to normal levels at 3 days after injury.KLF7 reduced ipsilateral hippocampal atrophy,decreased the injured cortex volume,downregulated Bax and cleaved caspase-3 expression,and increased the number of 5-bromo-2'-deoxyuridine-positive neurons and Bcl-2 protein expression.Moreover,KLF7 transfection greatly enhanced the phosphorylation of JAK2 and STAT3 in the ipsilateral hippocampus.These results suggest that KLF7 may protect hippocampal neurons after traumatic brain injury through activation of the JAK2/STAT3 signaling pathway.The study was approved by the Institutional Review Board of Mudanjiang Medical University,China(approval No.mdjyxy-2018-0012) on March 6,2018.
基金This study was supported by the National Natural Science Foundation of China(31871984)the Guangdong Provincial National Natural Science Foundation,China(2017B030311006)+1 种基金the Department of Science and Technology of Guangdong Province,China(2019B121201003)the special fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science,China(202105TD,R2020PYJX005).
文摘Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds.A regulatory sequence of EPSPS gene was isolated previously,and an alteration in its 5´-untranslated region(UTR)pyrimidine(Py)-rich stretch element is involved in the regulation of EPSPS expression in glyphosate-resistant(GR)Eleusine indica.However,the transcription factors involved in this regulatory sequence remain to be elucidated.In this study,we investigated the regulatory network of EPSPS overexpression associated genes in a GR E.indica population by RNA-seq.The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2752 unigenes and a decrease in the expression of 4025 unigenes in the GR E.indica,compared to the glyphosatesusceptible(GS)E.indica.Among them,1373 unigenes were identified to be co-expressed with the EPSPS gene in GR E.indica.GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway,chlorophy II and peroxisome metabolism processes.Notably,the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate(S3P,a substrate of EPSPS),was also up-regulated.Eight transcription factors were identified as likely to be involved in the regulation of the EPSPS expression,and three of them(ARF2,ARF8 and BPC6)showed more binding sites because of a(CT)n insertion of the 5´-UTR Py-rich stretch element in GR.However,the yeast one-hybrid assay illustrated that ARF8 and BPC6 could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS,but could not bind to the mutated form.Our data suggests that the transcriptional regulation of EPSPS expression is complex and was significantly altered in GR E.indica.These discoveries provide new references for further study of the EPSPS overexpression mechanism that endows glyphosate resistance.
