The Function Study Of A New PGRN Interacting Protein ARID5A

PGRN is a secreted glycoprotein molecule which is also called GEP,PEPI,acrogranin and PCDGF.PGRN is an autocrine growth factor containing 593 amino acids with approximate molecular weight of 68 kDa.PGRN is composed of 7.5 repeated motif linked by cysteine,which are ordered as P-G-F-B-A-C-D-E,among them,the fragment from A to G are full motif and P is a half motif.PGRN is widely expressed in tissues,organs and systems with the enrichment of epithelial cells such as skin,kidney,spleen and reproductive system etc.PGRN has many physiological functions including promotion of cell proliferation,anti-inflammation,wound repair and it is also involved in some pathological progression such as tumor,lipid metabolic disease and neurodegenerative disease.But the exact molecular mechanism underlying the biological activities of PGRN remains unclear.The functions of protein mostly rely on its interaction with other proteins,therefore,it is of great importance to study the function of proteins interacting with PGRN for clarifying the mechanisms of biological functions mediated by PGRN.Our lab has screened out a protein named ARID5A as an interactive protein of PGRN by using yeast two-hybrid system during the early research,and the interaction between PGRN and ARID5A was further validated by CoIP and GST-pull down assays.ARID5A was firstly discovered in the nuclear extracts of undifferentiated Tera-2 and THP-1 cells,which repressed the expression of M-IE gene by binding to the modulator which was located in the upstream of human cytomegalovirus M-IE gene enhancer.Previous studies indicated that ARID5A could bind with DNA,but also it could be able to bind with receptors,transcriptional factors,even with mRNAs.It has demonstrated that ARID5A plays critical roles in many different physiological and pathological events.In this study,the influence of PGRN on ERa signal pathway mediated by ARID5A and the ARID5A's effect on HEK293 and MCF-7 cell proliferation were investigated,which would lay a foundation for clarifying the mechanism of the biological activity mediated by PGRN.There are three parts in this study,the first part is to prepare monoclonal antibody against the N terminal(the functional domain)of ARID 5A for the diagnosis of ARID5A knockout cell line and further researches on ARID5A's functions.The second part is the establishment of ARID5A knockout HEK293 and MCF-7 cell line.The third part is the research on the functions mediated by the interaction between PGRN and ARID5A,including the influence of PGRN and ARID5A interaction on ERa mediated transactivation and ARID5A's effect on the HEK293 and MCF-7 cell proliferation,which would provide a new prospect of the biological mechanism of PGRN.The specific contents of this study are as follows.1.The preparation and identification of monoclonal antibody against ARID5 A-N.At first the ARID5A-N fragment cloned by our lab was inserted into pET28a prokaryotic expression vector,then the vector was transformed into E.coli BL21(DE3)to express ARID5A-N protein,the purified protein was injected into the Balb/c mice as immunogen.Finally,8 antibodies against ARID5A-N were obtained with the fusion and selection of hybridoma,which were further diagnosed by western blot and immunofluorescence staining.2.The establishment of ARID5A knockout HEK293 and MCF-7 cell line.4 pairs of sgRNAs targeting to exons of ARID5A were designed and cloned into sgRNA expression vector constructed by our lab,then sgRNA expression vector was co-transfected with Cas9 expression vector into human HEK293.The sgRNA3 with the best biological activity was screened and used for subsequent experiments.The donor vector containing up and down arm of the targeting site used in the homologous recombination was constructed and then co-transfected with sgRNA3 expression vector and Cas9 expression vector into HEK293 or MCF-7 cells,the ARID5A-'-HEK293 and ARID5A+/-MCF-7 cell lines was established by screening and diluted cloning,which were further identified by PCR,sequencing and western blot,respectively.3.The effect of PGRN and ARID5A interaction on ERa mediated transactivation.ARID5A and PGRN expression vectors were co-transfected with the vectors relevant to dual luciferase reporter gene assay into ARID5A-/-HEK293 and wild-type HEK293 cell line respectively,then the effect of PGRN and ARID5A interaction on ERa mediated transactivation was investigated by detecting the luciferase activity in the cell lines.The results revealed that ARID5A repressed the transactivation effect of ERa and the repression could be rescued by PGRN,which suggested that the regulation of ERa mediated transactivation by PGRN was dependent on ARID5A.4.The influences of PGRN and ARID5A on cell proliferation of HEK293 and MCF-7 cells.MTT assay was used to detect the influences of PGRN and ARID5A on cell proliferation of HEK293 and MCF-7 cells using ARID5A-/-HEK293 and ARID5A+/-MCF-7 cell lines.The results indicated that over expression of ARID5A inhibited HEK293 cell proliferation and PGRN could rescue the inhibition.The results from MTT assay in ARID5A-/-HEK293 cell revealed that the absence of ARID5A promoted HEK293 cell proliferation and the promotion of HEK293 cell proliferation caused by PGRN partly depended on the presence of ARID5A.Moreover,ARID5A overexpression suppressed MCF-7 cell proliferation and the lack of ARID5A promoted cell proliferation of MCF-7.In conclusion,the following results were obtained in this study.(1)Successfully prepared 8 monoclonal antibodies against ARID5A-N,which have been proved to be an efficient tool to be used in subsequent researches by western blot and immunofluorescence staining.(2)Established ARID5A-/-HEK293 and ARID5A+/-MCF-7 cell lines by using Cas9-sgRNA system.(3)Verified the repression of ARID5A on ERa mediated transactivation and further demonstrated that PGRN could rescue the repression through interacting with ARID5A.(4)Reversion of ARID5A mediated suppression on HEK293 cell proliferation caused by PGRN partly relied on the existence of ARID5A.(5)Over expression of ARID5A inhibited the proliferation of MCF-7 cells.