Screening And Identification Of A New Molecule Interacting With PGRN

As a secreted glycoprotein,progranulin(PGRN)contains a signal peptide with 17 amino acids and seven and one half granulin-like domains which are named as paragranulin,granulin G,F,B,A,C,D and E,respectively.Human PGRN is widely expressed in different cells,tissues and organs,especially in those comprised of many epithelial cells such as spleen,reproductive system,lung,kidney and skin.And it was also found highly expressed in certain neurons and most cancer cells.According to recent pulished data,PGRN has been implicated in many physiological and pathological events,including neurodevelopment,inflammation,wound repair,tumorigenesis and tumor progression.But the mechanisms of its actions in a variety of physiological and pathological activities have not been explained quite clearly.Because the biological functions of PGRN depend on the interactions between PGRN and many other proteins,it is meaningful to look for those proteins interacting with PGRN for elucidating the mechanisms of PGRN functions.In this study,PGRN was firstly used as bait protein aiming to screen proteins interecting with it by yeast two-hybrid system(Y2H system)and we found a possible molecule named as SLC39A6(ZIP6/LIV-1).Then we also did further analysis to validate their interaction through Y2H system and GST-Pull down assays.Finally,LIV-1-knockdown HepG2 cell line and control cell line were constructed using lentiviral vectors carrying LIV-1 shRNA or control shRNA,and then they were treated with PGRN siRNA to reduce the expression of PGRN.After that,the mRNA expression level of EMT related proteins among LIV-1-knockdown HepG2,PGRN-knockdown HepG2 and LIV-1/PGRN double-knockdown HepG2 cells were detected by Real-time PCR.The contents of this study includes following aspects:(1)Screening of proteins interacting with PGRN by Y2H system:Firstly,AH109 was transformed with yeast expression vector pGBKT7/PGRN no signal peptide which was constructed by our lab and then the expression of recombined protein GAL4-BD-PGRN was detected by western blot.Next,GAL4-BD-PGRN was used as a bait to screen molecules which could interact with PGRN from Human leukocyte Matchmaker cDNA Library by Y2H system.Through sequencing analyses,a new PGRN interacting protein LIV-1,was identified through sequencing analyses.(2)Identification of the binding sites between PGRN and LIV-1:According to the characteristics of LIV-1 sequence and structure,we speculated that the longest ectodomain and intracellular domain of LIV-1 that were histidine rich could probably bind to PGRN.Therefore,these two longest domains of LIV-1 were cloned by PCR from Mega ManTM cDNA Library,and then two yeast expression vectors,pGADT7/LIV-1 ectodomain stop and pGADT7/LIV-1 intracellular domain stop,were constructed.Next,these two yeast expression vectors were respectively trasformed into AH 109 together with pGBKT7/PGRN no signal peptide to verify the interactions between two LIV-1 domains and PGRN.Subsequently,the constructed trunctions of PGRN or two domains of LIV-1 were used to identify the precise binding sites between them by Y2H system.Then we have found that the binding sites of PGRN include GrnB,GrnA and GrnC,whereas the histidine-rich regions of two LIV-1 domains are necessary for their interactions with PGRN.Finally,the interactions between PGRN and the longest ectodomain or intracellular domain of LIV-1 were further identified by GST pull-down assay in vitro.(3)Preliminary study on the role of LIV-1 in invasion and metastasis of hepatocellular carcinoma cells induced by PGRN:It has been reported that LIV-1 and PGRN are both highly expressed in hepatocellular carcinoma cells and related to metastasis of these cells.In order to explore the function of LIV-1 in hepatocellular carcinoma cell metastasis induced by PGRN,lentiviral vectors carrying different LIV-1 shRNAs or control shRNA were constructed.After testing the interfering effect of LIV-1 shRNAs,the lentiviral vector carrying the most effiency LIV-1 shRNA was chose to establish the LIV-1-knockdown HepG2 cell line as well as lenti-control shRNA HepG2 cell line.To reduce the mRNA expression level of PGRN,two established cell lines above were transfected with PGRN siRNAs for 48h.Consequently,those cells of which PGRN mRNA expression level decreased significantly were selected to detect the mRNA expression of EMT(Epithelial-to-mesenchymal transition)associated genes.The results showed that the mRNA expression levels of Snail,MMP9 and N-cadherin were significantly reduced in LI V-1-knockdown HepG2,PGRN-knockdown HepG2 and LIV-1/PGRN double-knockdown HepG2 cells compared to control cell.Interestingly,except for Snail,the expression of MMP9 and N-cadherin were still higher in LIV-1-knockdown HepG2 cell than that in PGRN-knockdown HepG2 and LIV-1/PGRN double-knockdownHepG2 cells.All these results suggested that PGRN and LIV-1 could both promote the EMT process of hepatocellular carcinoma cells through same pathway(such as Snail)or part of same pathway(such as MMP9 and N-cadherin),and PGRN possiblely plays a dominant role in this process.In summary,results of this study are as follows:(1)a novel protein LIV-1 which could interact with PGRN were screened by Y2H system;(2)the precise binding sites of PGRN interacting with two LIV-1 domains were found to reside within GrnB,GrnA and GrnC,while in the longest LIV-1 etcodomain and intracellular domain,those sites were found to be present in histidine-rich regions.What's more,the interactions between PGRN and two LIV-1 domains were further validated by GST-Pull assays;(3)LIV-1-knockdown HepG2 cell line and lenti-control shRNA HepG2 cell line were established.After treating those cell lines with PGRN siRNAs,we have also obtained PGRN-knockdown HepG2 and LIV-1/PGRN double-knockdown HepG2 cells.Having tested the mRNA expression levels of EMT-associated molecules by Real-time PCR,we suggested that PGRN and LIV-1 could promote epithelial-to-mesenchymal transition of hepatocellular carcinoma cells through same pathway or part of same pathway,in which PGRN could probably play an important role.In all,our findings provided a new prospect for the research of the mechanism of PGRN promoting fuction in the malignant transformation of liver cancer cells.