Study On The Role Of CCAAT Enhancer-Binding Protein Homologous Protein In Molecular Mechanism Of Huntington’s Disease

Objective: Huntington’sdisease(HD)is an autosomal dominant inherited neurodegenerative disease.The main cause was repeated amplification of CAG trinucleotide sequence in IT15 gene on chromosome 4 of patients,which produced mutant Huntingtin(Htt)containing abnormal enlarged polyglutamine fragment(Poly Qs)after translation.It gradually accumulates in cells to form protein inclusion bodies,and accumulates specifically in neurons to affect the function of nerve cells,which can lead to premature death of striatum projective GABA neurons and cerebral cortex pyramidal cells.At present,there is a lack of effective and feasible treatment for Huntington’s disease.The study found that transcriptional dysregulation,endoplasmic reticulum stress(Endoplasmic Reticulum Stress ERstress)and autophagy is strongly associated with the onset of HD,including autophagy in gradually be taken seriously in recent years.In recent years,it has been found that CCAAT enhancer binding protein homologous protein(CHOP)plays an important role in neurodegenerative diseases.Some previous studies have confirmed that reducing CHOP expression level has a protective effect on nerve cells,and may be a potential treatment method for neurodegenerative diseases.However,its role in the pathogenesis of HD has rarely been reported.In this study,HDPC12 cells were used to explore the molecular mechanism related to CHOP in HD pathogenesis.Methods: The amino terminal fragment(N-Htt)of EGFP-labeled Huntington protein induced by Tet-on regulation contained 23 poly Qs(n-HTt-Q23,normal type)and74 poly Qs(n-HTt-Q74),respectively.Variant)in a rat adrenal neuropheochromocytoma PC12 cell model(David C.Courtesy of Professor Rubinsztein).The above cells were routinely cultured in vitro,and doxycycline(Dox)was added to induce them for different times.HDPC12 cell lines transfected with mi R-224-5p overexpression or silenced plasmid were constructed,and HDPC12 cell lines transfected with CHOP overexpression plasmid or silenced small interfering RNA were constructed.TRB3 silences HDPC12 cell lines transfected with small interfering RNA.The expression changes of mi R-224-5p,CHOP and TRB3 in HDPC12 cells were detected by q RT-PCR.CHOP,TRB3,Beclin-1,p62 and other proteins were detected in HDPC12 cells by Westernblot.Inclusion body formation and lysosome expression were observed by immunofluorescence.Cell apoptosis was detected by flow cytometry.We use transmission electron microscopy to observe autophagosomes.Dual luciferase reporter analysis verified the specific binding site between mi R-224-5p and CHOP.Chromatin immunoprecipitation confirmed direct binding of CHOP to the TRB3 promoter region.Results: 1.After Dox induction,the mutant protein inclusion bodies were formed in N-htt-Q74 cell line,but the protein was uniformly expressed in N-htt-Q23 cell line all the time,no inclusion body formed.2.Mutant N-htt was expressed in N-HTt-Q74 cell line,but not in N-HTt-Q23 cell line;Inclusion bodies were formed in N-htt-Q74 cells,but not in N-htt-Q23 cell line.3.In N-htt-Q74 cell line,Beclin-1 protein level was significantly decreased,p62 protein level was significantly increased,and autophagosome formation was significantly decreased.4.When CHOP protein expression level was up-regulated,Beclin-1 protein level was significantly reduced,p62 protein level was significantly increased,autophagosome formation was significantly reduced,and apoptosis was increased.When CHOP protein expression level was decreased,Beclin-1 protein level was significantly increased,p62 protein level was significantly decreased,autophagosome formation was significantly increased,and apoptosis was decreased.5.The expression levels of TRB3 protein and m RNA in N-htt-Q74 cell line were significantly increased compared with those in N-htt-Q23 cell line.6.CHOP expression was up-regulated,and TRB3 protein expression was increased.When CHOP expression level was down-regulated,the expression level of TRB3 protein was decreased.CHOP has direct binding effect with TRB3 promoter.7.TRB3 is involved in CHOP-mediated autophagy regulation.8.CHOP affects the formation of intracellular inclusion bodies by regulating TRB3.9.The expression level of mi RNA-224-5p in N-htt-Q74 cell line was significantly decreased compared with that in N-htt-Q23 cell line.10.When the expression level of mi RNA-224-5p was increased,the protein level of Beclin-1 was significantly increased,while the protein level of p62 was significantly decreased.When the expression level of mi RNA-224-5p was decreased,the protein level of Beclin-1 was significantly decreased,while the protein level of p62 was significantly increased.11.The expression level of mi RNA-224-5p was increased,and the expression level of CHOP was decreased.CHOP protein expression was increased when mi RNA-224-5p expression was decreased.CHOP is modulated by mi RNA-224-5p targeting.12.Mi RNA-224-5p regulates autophagy of N-htt-Q74 cells by CHOP.13.Mi RNA-224-5p affects the formation of intracellular inclusion bodies by regulating CHOP.Conclusion: 1.CHOP protein and m RNA expression levels were significantly increased while autophagy activation level was inhibited by N-htt variation.Down-regulated CHOP expression can activate autophagy activation level of N-htt-Q74 cells,significantly inhibit the formation of intracellular inclusion bodies,reduce cell apoptosis,and protect N-htt-Q74 cells.2.TRB3 is the target gene of CHOP.CHOP regulates autophagy and influences the formation of intracellular inclusion bodies by targeting TRB3.CHOP/TRB3 signaling activation plays an important role in autophagy and inclusion body formation.3.CHOP is the target gene of mi R-224-5p,and mi R-224-5p has a negative regulatory effect on CHOP.Increasing the expression level of mi R-224-5p can reduce the expression level of CHOP,activate the autophagy level of N-htt-Q74 cells,reduce the formation of intracellular inclusion bodies,promote cell survival,and have neuroprotective effects.4.Mi RNA-224-5p regulates autophagy and affects inclusion body formation by regulating CHOP/TRB3.