The Study Of Gp96 In Acute Alcoholic Liver Injury In Mice By CRISPR/Cas9 Technology

Objective With the support of CRISPR/Cas9 technology,this study inhibits gene expression by gene editing Gp96 sequence,and studies the role and regulation of Gp96 in acute alcoholic liver injury.Methods(1)Design plasmid: Search for Gp96-related gene sequences in NCBI,and find the same Gp96 gene sequence in human and mouse in exons,the target sequence(CRISPR)targeting the mutation was designed in exons 4-9 according to the protein conserved functional domain positions of the two,sequence alignment of exon4 to exon 9 revealed a total of 6 identical CRISPR sequence positions.However,after analyzing the off-target rate,three CRISPR sequences were finally designed and three plasmids were constructed.(2)Effective sgRNA screening and validation: The synthesized three plasmids were mixed 1:1:1 with mouse myoblast C2C12,and the successfully transfected cells were screened by puromycin.The DNA was extracted for PCR amplification,electrophoresis,gel recovery,sequencing and effective sgRNA protein verification.(3)In vitro experiments: Mouse myoblast C2C12 was cultured and divided into three groups: Normal group: no treatment;Normal+alcohol group:Directly give alcohol culture to induce damage.Gp96-sgRNA+alcohol group: After effective transfection of effective sgRNA,alcohol culture is carried out to induce damage.CCK-8 cell activity proliferation assay.Hoechst 33258 apoptosis staining.Immunoblotting to detect expression of related factors: Gp96,PCNA,Caspase-3,HSP70,P4502E1(CYP2E1),p-STAT3.(4)In vivo experiment: Healthy and clean Kunming male mice were randomly divided into 3 groups: Normal group(n=12):normal feeding mice;saline+alcohol group(n=12): physiological fluid in tail vein(same volume as effective sgRNA);Gp96-sgRNA+alcohol group(n=12): effective sgRNA was injected into the tail vein.The mice were fed normally for 3 days after the tail vein injection.At the end of the third day,56 degree red star Erguotou(24mL/kg)was intragastrically administered orally,acute alcoholic liver injury in mice was induced by fasting for 24 h and then sacrificed together.Blood was taken from the mouse eyeball,and serum was separated to detect changes in AST and ALT;Hematoxylin-eosin staining(HE staining)was used to detect liver injury in mice;The changes of liver glycogen in mice were detected by periodic acid Schiff staining(PSA staining);Apoptosis of hepatocytes was detected by apoptosis staining of Hoechst33258;Immunoblotting to detect expression of related factors: Gp96,PCNA,VEGF,Bcl-2,Bax,Caspase-3,HSP70,HSP27,P4502E1(CYP2E1),p-STAT3,TNF-?.Results DNA sequencing results showed that the gene sequences of sgRNA1 and sgRNA2 transfected C2C12 cells were not different from the normal sequences,but the gene sequences of sgRNA3-transfected cells showed a large difference compared with the normal sequence.The results of Western blot showed that the expression of Gp96 in sgRNA3-transfected cells were significantly lower than that in normal cells(P<0.01),therefore sgRNA3 was identified as a valid guide RNA.In vitro experiments:1.CCK-8 results showed that the survival rate of the Normal plus alcohol Cells and the Gp96-sgRNA3 plus alcohol Cells were lower compared with the normal cells,(P<0.05 or P<0.01),while the survival rate of Gp96-sgRNA3-transfected cells were significantly lower than that in normal cells after alcohol stimulation(P<0.01).2.Hoechst 33528 staining results: Compared with the normal cells,both the Normal plus alcohol cells and the Gp96-sgRNA3 plus alcohol cells showed significant apoptosis(P<0.01),and the apoptosis was more significant in the Gp96-sgRNA3 plus alcohol cells than that in the Normal plus alcohol cells(P<0.01).3.Western blot results:Compared with the Normal plus alcohol group,the protein expression levels of CYP2E1,Caspase-3,HSP70 in the Gp96-sgRNA3 plus alcohol cells were significantly increased(P<0.05 or P<0.01),however,the expression levels of Gp96,p-STAT3,PCNA were significantly decreased(P<0.05 or P<0.01).In vivo experiments: 1.Changes in serum transaminase: AST,ALT levels in serum of saline plus alcohol mice and Gp96-sgRNA3 plus alcohol mice were significantly higher than those in the normal mice(P<0.05 or P<0.01),while the serum levels in Gp96-sgRNA3 plus alcohol mice were significantly higher than those in saline plus alcohol mice(P<0.01).2.Hematoxylin-eosin staining showed that saline plus alcohol mice and Gp96-sgRNA3 plus alcohol mice had significant liver damage than normal mice(P<0.01),while Gp96-sgRNA3 plus alcohol mice had higher hepatocyte necrosis scores than saline plus alcohol mice(P<0.01).3.The results of periodic acid Schiff reagent staining showed that saline plus alcohol mice and Gp96-sgRNA3 plus alcohol mice presented significant hepatic glycogen consumption compared with Normal group after 24 h of alcohol treatment(P<0.01),while Gp96-sgRNA3 plus alcohol mice had more significant hepatic glycogen reduction than saline plus alcohol mice(P<0.01).4.The Hoechst33528 staining result showed that hepatocyte apoptosis was observed in both saline plus alcohol mice and Gp96-sgRNA3 plus alcohol mice afteralcohol treatment(P<0.01)and in the Gp96-sgRNA3 plus alcohol mice,hepatocyte apoptosis was more significant than that in the saline plus alcohol mice(P<0.01).5.Western blot results: Compared with the saline plus alcohol mice,the protein expression levels of CYP2E1,Bax,Caspase-3 and TNF-? in liver of Gp96-sgRNA3 plus alcohol mice were significantly increased(P<0.05 or P<0.01),however the protein expression levels of Gp96,HSP27,HSP70,p-STAT3,PCNA,VEGF and Bcl-2were significantly decreased(P<0.05 or P<0.01).Conclusions Gp96 alleviates alcohol-induced acute liver injury by regulating the expression of related genes such as cell proliferation,apoptosis and stress metabolism.Targeting inhibition of mice Gp96 gene expression by CRISPR/Cas9 technology can promote alcohol-induced acute liver injury in mice.