Effects Of Human Vasorin On The Proliferation And Migration Of HepG2 And MB231 Cells

Human Vasorin(VASN)protein belongs to type I transmembrane glycoprotein.Its relative molecular weight is about 72 KDa.It was reported for the first time in 2004,as one TGF-?-binding protein in vascular smooth muscle to participate in the signaling pathway of TGF-?,and regulate the injury response of the aorta during the repairing of damaged vessels.Recent years,Vasorin has been reported highly expressed in some tumors,including hepatocellular carcinoma,breast cancer and glioma.Vasorin expression was significantly induced under the condition of TNF-?treatment or hypoxia,suggesting that the protein has the abilities of resisting hypoxia-induced apoptosis and promoting tumor progression.The results of our previous work showed that Vasorin was expressed higher in HepG2 cells than in normal liver cells,and that ethanol treatment could increase the expression of Vasorin in HepG2.Meanwhile,exosomal Vasorin protein from HepG2 cells could be transported into HUVEC cells and promote the migration of HUVEC cells,suggesting that Vasorin was related to the occurrence and development of liver cancer.In our laboratory,we preliminarily established the Vasorin stable-knockout HepG2 cell line by using CRISPR/Cas9 technology.In this study,the constructed HepG2 cells were screened to obtain the cells with stable knockout of human Vasorin gene,and the ability of cell proliferation and migration were analyzed by CCK8 and Transwell asasys.The results showed that the proliferation and migration ability of HepG2 cells was significantly lower than that of wild type cells,suggesting that Vasorin in HepG2 cells had important biological functions.It was reported that Vasorin protein was highly expressed in breast cancer cells,and was related to the invasion ability and poor prognosis of Triple Negative Breast Cancer cells(TNBC).However,the biological function of Vasorin in breast cancer cells has been rarely reported.To explore the biological functions of Vasorin in MB231 cells,the expression of Vasorin was knocked down by siRNA,and the results of CCK-8 and Transwell assays showed that the proliferation and migration of Vasorin-knock down MB231 cells was significantly decreased,which was consistentwith those of HepG2 cells.As is known,ethanol is an important inducer of breast cancer.Previous work in our laboratory showed that ethanol could promote the expression of Vasorin in HepG2 cells.To validate whether ethanol had similar effects on MB231 cells,we treated MB231 cells with 25,50,100 mM ethanol for 24 h individually.The expressions of Vasorin mRNA and protein were detected by qRTPCR and Western Blotting.The results showed that both of the expressions of Vasorin mRNA and protein were significantly increased and positively correlated with the concentration of ethanol.Furthermore,we try to explore the molecular mechanism of ethanol upregulation of Vasorin expression.We found that after MB231 cells were stimulated with 25,50,100 m M ethanol for 24 h,HIF1-? level was increased.So was that of Vasorin level.When the HIF1-? level in MB231 cells was knocked down by its specific si RNA,the Vasorin level was downregulated corresponding to decrease of HIF1-?,indicating that the expression of Vasorin was related to HIF1-?.In our laboratory's previous work,several mouse monoclonal antibodies against Vasorin were produced.In this work,phage peptide library was bioscreened against antibody V20 to obtain the epitope.After three rounds of biopanning,eight positive clones were isolated.After DNA sequencing and sequence alignment analysis,seven sequences of binding peptides were obtained and two mimic peptides V20P1 and V20P2 were selected for chemical synthesis.The results of ELISA experiments showed that the two mimic peptides could specifically bind antibody V20.These results suggested that both V20P1 and V20P2 are mimotopes of Vasorin,which laid a foundation for further function researches of the corresponding domain.