Verification Of Rev-erb? Interacting Molecules And Research On Rev-erb? Function

Rev-erb? is a member of the Rev-erbs nuclear receptor family,which is expressed in many tissues and cells,especially in skeletal muscle,brain,kidney,liver and adipose tissue.It is an important transcriptional factor,which is widely involved in many physiological and pathological processes,such as circadian rhythm,metabolism,inflammation and cancer.Recently,it has been found that the Rev-erb? expression is significantly higher than Rev-erba's in many cancer cell lines.These studies suggested that Rev-erbp may play an important role in the development and progression of tumors,but the function and molecular mechanisms of Rev-erbp have been still unclear.PGRN is a growth factor with many functions which has been studied in our laboratory for many years,and it is also widely involved in physiological and pathological processes such as metabolism,inflammation and tumor.In the early stage of the yeast two hybrid screen library in our lab,Rev-erb? was screened for interaction with PGRN.Therefore,in order to further verify the Rev-erb? and PGRN really has the interaction and to explore the molecular mechanism of their interaction in mediating metabolism,inflammation and tumor processes.The research contents of the subject are as follows:(1)Identification and preparation of the monoclonal antibodies against human Rev-erb?.Due to the shortcomings of the current commercial antibodies against Rev-erb?,such as low titer,poor specificity,difficulty in testing the endogenous Rev-erb? and using in IP and ChIP experimental studies ect.,we successfully prepared five monoclonal antibodies against different domains of Rev-erb beta by monoclonal antibody preparation technology in this study.Through a series of analysis and verification experiment,the five strains of monoclonal antibody not only can identify specific domains of Rev-erb beta,but also be used for the detect endogenous Rev-erb beta protein,Western blot,immunofluorescen cytochemistry,immunoprecipitation(IP)and ChIP experiments etc.(2)Verification of the interaction between Rev-erbp and PGRN.On the basis of previous studies(Human PGRN was used as bait protein to screen out Rev-erb beta,which might be a candidate molecule interacting with PGRN),PGRN was identified a molecule that directly interacts with Rev-erb? by means of the yeast two hybrid technology,immunoprecipitation and GST-pull down binding assay.The colocalization of Rev-erb? and PGRN in CHO cells was studied by confocal microscopy.The result showed that Rev-erb? could mediate the transfer of PGRN from cytoplasm to nucleus,and Rev-erb? and PGRN formed a granular co-localization in nucleus and cytoplasm.(3)Study on the molecular mechanism of PGRN mediated Rev-erb? in regulation of target gene expression.As the nuclear receptor Rev-erb? is an important transcription factor,in order to verify the effect of PGRN on the transcription of Rev-erbp,we constructed GAL4-BD-Rev-erb?-E fusion protein vector and GAL4-UAS-CMV-Luci reporter gene detection system in this study.PGRN and Rev-erb? double knockout HEK293 cell line was also constructed using CRISPR/Cas9 genome editing technology,which was used to detect the effect of the PGRN and Rev-erbp interaction on the regulation of Rev-erb?'s target genes.The plasmid expressing PGRN and the two plasmids including GAL4-BD-Rev-erb?-E and GAL4-UAS-CMV-Luciferase derived from the luciferase reporter assay system were co-transfected into HEK29 3cells,and it was found that PGRN could enhance the inhibitory effect of GAL4-BD-Rev-erb? on the activity of GAL4-UAS-CMV promoter.PGRN was also found to significantly enhance the transcriptional regulation of Rev-erb? on the activities of the the foreign promoters(hBmal1/hApoC?/Srebp-1c)of target genes in PGRN and Rev-erb? double knockout HEK293 cell lines.At the same time,it was also confirmed that the binding of PGRN and Rev-erb? could lead to a significant decrease mRNA expression level of the endogenous Bmal1(Rev-erb? target gene).(4)Preliminary study on biological function of Rev-erb? in hepatocellular carcinoma HepG2 cells.Owing to the high expression of PGRN that interacts with Rev-erb? in tumor cells,and it can promote the proliferation,migration and invasion of tumor cells.The study of the roles of Rev-erb? in the occurrence and development of tumor was carried out in HepG2 cell lines with the knockout of Rev-erb? gene or the over-expression of Rev-erb?.In summary,the results were obtained from the study as follows:(1)Five monoclonal antibodies against different domains of Rev-erb? were successfully prepared.(2)The direct interaction between PGRN and Rev-erb? was validated by the yeast two hybrid technology,immunoprecipitation and GST-pull down binding assay.It also has been confirmed that the interaction binding sites of Rev-erb? and PGRN are the BAC region of PGRN and the Rev-erb?-E domain.(3)The effect of PGRN on transcription regulation of Rev-erb? target gene was investigated that the interaction of PGRN with Rev-erb? can cooperate with Rev-erbp to regulate the transcription of Rev-erb?'s target genes.(4)Biological function studies of Rev-erbp showed that the Rev-erb? can not only inhibit fat formation in HepG2 cells,but also inhibit the proliferation,migration and invasion of HepG2 cells.In conclusion,these results provide a basis for exploring the possible molecular mechanisms and signal pathways regulated by Rev-erb? and PGRN in tumor cells.