Screening,Identification Of Cytoglobin Interaction Proteins And Analysis Human Hepatocyte Transcription Before And After Cytoglobin Overexpression And Knockout

Cytoglobin was first named as stellate activator protein(STAP),later renamed Cytoglobin(CYGB),a globulin discovered by Japanese scientists during proteomic analysis of fibrotic liver;CYGB is the fourth members of globulin family,as carriers of the body’s storage and transport of oxygen,have terminal oxidase,scavenging reactive oxygen species(ROS)and nitric oxide dioxygenase activities.In recent years,there have been more and more reports on cytoglobin,which is related to the development of various diseases such as fibrosis in liver,kidney,pancreas and other tissues or organs,glaucoma,gastric tube reflux and some inflammation and nerves degenerative diseases,etc.Studies have shown that cytoglobin has the function of proto-oncogenes and tumor suppressor genes,which may be related to the methylation of CYGB.Previous work of the research group showed that recombinant human Cytoglobin can reverse liver fibrosis,hyperlipidemia and atherosclerosis in rats,in order to further study the potential function and molecular mechanism of cytoglobin,we performed yeast two-hybrid system screening to identify 17 proteins interacting with CYGB using Mate&PlateTM Library-Universal Human(Normalized).In this study,immunoprecipitation was used to verify the interaction between them,and the bimolecular fluorescence complementation system was used to further identify the interaction between the interacting proteins and CYGB verified by the co-immunoprecipitation system.At the same time,in order to find more CYGB-related genes and discover more biological information,this study compares human hepatocyte transcriptome before and after knockout,over-expression CYGB,and then conducts bioinformatics analysis to discover more biological information.In the first part of this study,the frist chapter,the coding sequence of the target protein was amplified by PCR.After digestion and ligation of the vector pENTER and the positive clones were identified by PCR and sequencing,and the eukaryotic expression vector containing the target fragment was correctly constructed.Co-immunoprecipitation experiments:The experimental group plasmid and the control plasmid were transfected into HEK293T cells,and the cell lysate was mixed with ANTI-MYC affinity agarose gel(treated),and then the supernatant was removed and the precipitate was washed with phosphate buffer,and then go with protein electrophoresis and Western blot experiments.The results showed that the eukaryotic expression vector pENTER-LSM8-Flag and pENTER-LARP7-Flag containing the target fragment were successfully constructed by PCR and sequencing.The eukaryotic expression vector with the remaining target protein was synthesized by the technology company and kept in the laboratory.Western blot analysis of cell lysates in control and experimental groups:Target protein bands appeared in both control and experimental groups,mean that CYGB and interacting proteins can be expressed normally in HEK293T cells;Western blot results of co-immunoprecipitation:In the experimental group,only the western blotting experiments of pcDNA3.1-TUBB4B-Flag,pcDNA3.1-PCNA-Flag and pENTER-RNF2-Flag-His could detect two target protein bands,and the rest only detected CYGB protein.It indicates that TUBB4B,PCNA and RNF2 interact with CYGB in HEK293T cells.In the first part of this study,the second chapter,the bimolecular fluorescence complementation experiment:the target fragment was amplified by PCR,and the positive clone was identified by restriction enzyme digestion.The intermediate vector pENTR was ligated with the target fragment,and the positive clone was identified by PCR and sequencing;The intermediate vector with the target fragment was ligated with the target vector-BiFc vector by LR ligation,and the positive clone was identified by sequencing;the HEK293T cell line stably overexpressing cYFP-CYGB was constructed by lentivirus-mediated manner,and the positive clone was transfected into the cell which overexpressing cYFP-CYGB.The fluorescence of the cells was observed under a fluorescence microscope after 16 h.After PCR and sequencing,the results showed that pENTR-PCNA,pENTR-TUBB4B,pENTR-RNF2 and pENTR-CYGB intermediate vectors were successfully constructed.The target vector-BiFC vector,include NC-CYGB,NVN-PCNA,NVN-TUBB4B,NVN-RNF2 eukaryotic expression vectors,was constructed by LR ligation.The HEK293T cell line stably overexpressing cYFP-CYGB was successfully constructed.Under the fluorescence microscope,the cells transfected with NVN-PCNA,NVN-TUBB4B and NVN-RNF2 eukaryotic expression vectors were saw all fluoresced.The cells stained with NVN-PCNA had stronger fluorescence.In the first part of this study,the third chapter,the changes of PCNA expression in kidney tissue before and after recombinant CYGB treatment of diabetic mice,and the effect of overexpression of CYGB on the proliferation rate of HEK293T cells:PCNA was performed on kidney tissues before and after treatment of diabetic mice with recombinant CYGB by Histochemical analysis;the effect of stable expression of CYGB on the proliferation rate of HEK293T cells was examined using the CCK8 kit.The results showed that the expression of PCNA in kidney tissue in the recombinant CYGB treatment group was significantly higher than that in the model group.The result of CCK8 kit test mean that the proliferation rate of HEK293T cells stably expressing CYGB was faster(p<0.05).In the second part of this study,the frist chapter,the CYGB encoding gene was amplified by PCR,and the lentiviral recombinant expression vector Plenti-N-GFP-CYGB was constructed by Gateway cloning technology.After identified by digestion and sequencing,HEK293T cells were transfected with the plasmid,virus-containing supernatant was collected and concentrated,virus particles were obtained,LO-2 cells were infected,and stable cell lines were screened by flow cytometry.The expression was detected by Western blot.The results showed that the lentiviral recombinant expression vector’s(Plenti-N-GFP-CYGB)double enzyme digestion and gene sequence alignment were correct.After the three plasmids were co-transfected into HEK293T cells,most of the cells showed green fluorescence under fluorescence microscope.After collecting the virus particles and infecting LO-2 cells,the LO-2 cells stably expressing CYGB were screened by flow cytometry.It was shown that the CYGB expression level of the LO-2 stable strain group was significantly increased relative to the control group by Western blot.In the second part of this study,the second chapter,the online software was used to design CRISPR/Cas9 sgRNA,select the sgRNA with higher score,then linearize the PX458 vector,sgRNA were phosphorylation and annealing,and finally clone the sgRNA into PX458 by rapid ligase.The positive clones were identified by sequencing.The positive cloning vector was transfected into LO-2 cells by lipofection,and the cells expressing green fluorescent protein were sorted into 96-well plates,and monoclonal cells were picked,and genomic DNA was extracted to amplify sgRNA-binding sequence for sequencing identification.The results showed that the sequencing results showed that the knockout plasmid PX458-sgRNA was successfully constructed.After the positive clones were picked and transfected into LO-2 cells,9 monoclonal cells were picked and sequenced,and 1C2 monoclonal was obtained.The cells are homozygous mutant and have a base insertion in the knockout region,resulting in a frameshift mutation that prematurely terminates protein translation of CYGB.The CYGB knockout cell line was successfully established.In the second part of this study,the third chapter,the CYGB knockout group and the control group,the CYGB stable strain group and its control group were placed in a 10 cm culture dish for RNA extraction and RNA quality detection,database construction,and transcriptome Sequencing.After bioinformatics analysis,differential genes and cellular pathways associated with CYGB were screened.The results showed that 1348 differential genes were screened out from the bioinformatics analysis of transcriptome sequencing in CYGB knockout group and empty control group,of which 751 were up-regulated and 597 were down-regulated;The sequencing results showed that 1198 differential genes were screened in over-expressed CYGB experimental group and control group transcriptome,of which 720 were up-regulated and 478 were down-regulated.The most important differential genes involved in the redox pathway and cancer-related pathways.The most significant statistical differences genes involved in cholesterol biosynthesis and glycogen metabolism.Providing relevant data for further exploration of CYGB-related pathways.The conclusions drawn from the above results:1.The eukaryotic expression vector pENTER-LSM8-Flag and pENTER-LARP7-Flag were successfully constructed,and the interaction between CYGB and PCNA/TUBB4B/RNF2 was verified by Co-immunoprecipitation.The eukaryotic expression vector NC-CYGB and NVN-PCNA,NVN-TUBB4B and NVN-RNF2 of BIFC,were successfully constructed.The interaction between CYGB and PCNA was further screened and verified by BiFC system.These three interacting proteins are involved in cell proliferation,DNA damage repair,and oxidative stress,which is close to the related functions of CYGB and deserves important research.2.The expression of PCNA in kidney tissue was increased after reconstituted CYGB treatment in diabetic mice.The proliferation rate of HEK293T cells stably expressing CYGB increased.It is suggested that CYGB may increase the expression and activity of PCNA to promote the proliferation of renal cells through interaction with PCNA,and reduce the damage of diabetic nephropathy in mice.3.Plasmid PX458-sgRNA and Plenti-N-GFP-CYGB were successfully constructed;Human hepatocyte CYGB knockout strain and CYGB stable overexpressing strain were successfully established.The results showed that 1348 differential genes were screened out from the bioinformatics analysis of transcriptome sequencing in CYGB knockout group and empty control group,of which 751 were up-regulated and 597 were down-regulated;The sequencing results showed that 1198 differential genes were screened in over-expressed CYGB experimental group and control group transcriptome,of which 720 were up-regulated and 478 were down-regulated.Among them,the differentially expressed genes involved in the redox pathway and cancer-related pathway were the most significant,andThe most significant statistical differences genes involved in cholesterol biosynthesis and glycogen metabolism.The above experimental results laid a foundation for further exploration of CYGB treatment mechanism for related diseases.