Experimental Study Of Tumor Suppressor Gene WWOX Regulating The Migration And Invasion Of Gallbladder Cancer Cells Through HIF-1α/VEGFA

Objectives:To study the effects of WW domain containing oxidoreductase(WWOX)gene on the migration and invasion of GBC-SD gallbladder cancer cells.To study the effect of overexpression of WWOX on the expression levels of HIF-1α and VEGFA and the effect of overexpression of WWOX on promoting angiogenesis of gallbladder cancer cells.Explore the role of WWOX in the migration and invasion of gallbladder cancer cells and put forward a preliminary discussion on its possible mechanism.Methods:Mix WWOX overexpression plasmid,no-load plasmid and packaging plasmid respectively to form DNA-EndoFectin transfection complex,and then co-transfect them into 293T cells to obtain WWOX overexpression lentiviral particles and no-load lentiviral particles.GBC-SD gallbladder cancer cells infected with WWOX overexpression lentivirus were used as overexpression group(WWOX group),GBC-SD cells infected with no-load lentivirus were used as negative control group(NC group),and uninfected GBC-SD cells were used as blank control group(GBC-SD group).Puromycin was used for the selection of stable transfected cell lines.The expression of WWOX mRNA and protein were detected by qPCR,Westemblot,and immunofluorescence staining methods respectively in each group of GBC-SD gallbladder cancer cells.The changes in the migration ability of GBC-SD gallbladder cancer cells in each group were detected by the cell scratch test and the Transwell cell migration test.The changes in the invasion ability of GBC-SD gallbladder cancer cells in each group were detected by the Transwell cell invasion experiment.The mRNA and protein expression levels of HIF-1α and VEGFA in GBC-SD gallbladder cancer cells of each group were detected by qPCR and Westernblot.ELISA was used to detect the extracellular secretion levels of HIF-1α and VEGFA in the supernatant culture medium of GBC-SD gallbladder cancer cells in each group after infection of GBC-SD cells for 48 hours.After infection of GBC-SD cells for 48 hours,the supernatant culture medium of gallbladder cancer cells in each group was collected and made into conditioned medium for culturing HUVEC vascular endothelial cells to observe the formation of tubules,Which to evaluate the effect of WWOX on the promoting angiogenesis effect of gallbladder cancer cells.Results:1.Successfully package WWOX overexpressed lentiviral particles and collect them for subsequent in vitro cell experiments.2.The results of qPCR,Westernblot,and immunofluorescence staining experiments displayed that the mRNA and protein expression levels of WWOX in GBC-SD gallbladder cancer cells in the WWOX overexpression group were obviously increased by comparision with the negative control group and the blank control group,suggesting that WWOX overexpression lentivirus successfully infected GBC-SD cells.3.The cell scratch test results displayed that the scratch healing rate of GBC-SD gallbladder cancer cells in the WWOX overexpression group was sharply slower by comparision with the negative control group and the blank control group.The results of the Transwell cell migration experiment manifested that the number of migrated GBC-SD gallbladder cancer cells in the WWOX overexpression group was distinctly reduced by comparision with the negative control group and the blank control group.The results of the Transwell cell invasion experiment suggested that the number of GBC-SD gallbladder cancer cells passing through matrigel in the WWOX overexpression group was dramatically reduced by comparision with the negative control group and the blank control group.4.The results of qPCR and Westernblot experiments displayed that the mRNA and protein expression levels of HIF-1α and VEGFA in GBC-SD gallbladder cancer cells in the WWOX overexpression group were distinctly down-regulated by comparision with the negative control group and the blank control group.The results of ELISA experiment showed that the extracellular secretion of HIF-1α and VEGFA in the supernatant culture of GBC-SD gallbladder cancer cells in the WWOX overexpression group was distinctly reduced by comparision with the negative control group and the blank control group.5.The results of tubule formation experiment displayed that the number of nodes,number of intersections,total length,and branch length of tubules formed by HUVECs endothelial cells in the conditioned medium derived from the supernatant of GBC-SD gallbladder cancer cells in the WWOX overexpression group were distinctly reduced by comparision with the negative control group and the blank control group.WWOX overexpression can inhibit the promoting effect of GBC-SD gallbladder cancer cells on vascular endothelial cell tubule formation.Conclusions:Overexpression of WWOX gene can significantly inhibit the migration and invasion of GBC-SD gallbladder cancer cells.The possible mechanism is overexpression of WWOX can down-regulate the expression levels of HIF-1α and VEGFA mRNA and protein in GBC-SD gallbladder cancer cells and extracellular secretion,and inhibit the angiogenesis effect of gallbladder cancer cells.