Exogenous Collagen I Induced TM4SF1 And DDR1 To Regulates Migration And Invasion Of Ovarian Cancer Cells

Part I Expression and Clinical Significance of Discoid Domain Receptor 1 in Ovarian Cancer TissuesObjective To investigate the expression and clinical significance of discoidin receptor 1(DDR1)protein in human ovarian cancer.Methods The expression of DDR1 protein were detected by immunohistochemical method in 33 normal ovarian tissue,43 benign ovarian tumors and 94 ovarian cancer tissues.The positive expression rate of DDR1 protein was compared between the ovarian cancer patients with different clinicopathological features.The relation of positive expression of DDR1 protein with clinical pathological features and the prognosis of ovarian cancer patients were analyzed.Results The positive rate of DDR1 protein in ovarian cancer [55.32%(52/94)] was higher than those in normal ovarian tissues [0(0/33)] and benign ovarian tumors [55.32%(52/94)] [11.63%(5/43)](all P<0.05),but there was no significant difference between the positive rate of normal ovarian tissue and benign ovarian tumor(P>0.05).The positive expression rates of DDR1 protein were higer in the patients with FIGO stage III-IV patients,poor differentiation and peritoneal metastasis[60.98%(50/82),62.16%(46/74),66.67%(42/63)]compared to the patients with FIGO stage I-II,moder ate/well differentiated,and without peritoneal metastasis respectively [16.67%(2/12),30%(6/20),32.26(10/31)](all P<0.05).The positive expressio n of DDR1 protein and peritoneal metastasis were the risk factors for death in the ovarian cancer patients(P>0.05).Conclusion DDR1 protein is overexpressed in ovarian cancer tissues,is probably participated in the occurrence,progression and distal metastasis of ovarian cancer,and may be taken as an important factor for evaluating the prognosis.Part II Effects of Exogenous Collagen I induced TM4SF1 on Biological Behavior of Highly Metastatic Ovarian Cancer Cell Strain HO8910 PM In VitroObjective To study the effect of exogenous Collagen I induced TM4SF1 on biological behavior of highly metastatic ovarian cancer cell strain HO8910 PM.METHODS RNAi was used to inhibit the expression of TM4SF1 in HO8910 PM.The expression of TM4SF1 protein in two stable cell strains were detected by RT-PCR and Western Blotting.Cellular immunofluorescence was used to detect the localization of TM4SF1 and DDR1 in HO8910 PM in the presence or absence of exogenous Collagen I.Transwell migratio n assays and invasion assays were used to detect the changes in cell mig ration and invasion.Results Lentiviral vectors targeting to silence TM4SF1 and expressing GFP fluorescence were successfully constructed.The stable expression cell str ain LV-TM4SF1-RNAi-GFP-HO8910 PM and the negative control LV-CONTM4SF1-GFP-HO8910 PM were infected and screened.RT-PCR experiments showed that the silencing efficiency of TM4SF1 gene at mRNA level was about 65%,and the silencing efficiency of TM4SF1 protein detected by Western Blot was about 70%.The outcome of immunofluorescence sho wed that both TM4SF1 and DDR1 were cell membrane proteins.When exogenous Collagen I was present,TM4SF1 clustered DDR1 on the cell m embrane,and concomitant recruitment of TM4SF1.Transwell migration experiments showed that the number of cells passing through the polycarbon ate membrane in the LV-RNAi-TM4SF1-GFP-HO8910 PM group was 94.67±9.018,which was significantly lower than that in the HO8910 PM group(257.67±17.898)and the LV-CON-TM4SF1-GFP-HO8910 PM group(261.67±29.023)(all P<0.05).In the presence of 50 μg/ml Collagen I,the number of cells passing through the polycarbonate membrane in LV-RNAi-TM4 S F1-GFP-HO8910 PM was 150.67±7.371,which was significantly lower than that in the HO8910PM+50 μg/ml Collagen I group(370.00±16.523)and LV-CON-TM4SF1-GFP-HO8910PM+50μg/ml Collagen I group(377.33±15.948)(all P<0.05).In the presence of exogenous Collagen I,the cell numbers of HO8910 PM,LV-RNAi-TM4SF1-GFP-HO8910 PM,and LV-CON-TM4SF1-GFP-HO8910 PM cells that passed through the polycarbonate membrane were all significantly higher than those in the absence of exogenous Collagen I(all P<0.05).Transwell invasion assay showed that the number of cells passing through the Matrigel-coated Transwell chamber in LV-RNAiTM4SF1-GFP-HO8910 PM was 57.00±5.568,which was significantly lower than that of the HO8910 PM group(134.33±6.658)and LV-CON-TM4SF1-GFP-HO8910 PM group(141.33±7.638)(all P<0.05).In the presence of exogenous Collagen I,RNAi-TM4SF1-GFP-HO8910 PM cells passed through Matrigel-coated Transwell chamber was 109.00±4.359,which is lower than HO8910PM+50 μg/ml Collagen I group(216.33±16.921)and LV-CON-T M4SF1-GFP-HO8910PM+50μg/ml Collagen I group(226.67±4.509)(all P<0.05).In the presence of exogenous Collagen I,the cell numbers of HO8910 PM,LV-RNAi-TM4SF1-GFP-HO8910 PM,and LV-CON-TM4SF1-GFP-H O8910PM cells that passed through the Matrigel-coated Transwell chamber s were all significantly higher than those in the absence of exogenous Collagen I(all P<0.05).Conclusion TM4SF1 and DDR1 were cell membrane proteins.TM4SF1 can aggregate DDR1 on the cell membrane in the presence of exogenous Collagen I,and concomitant recruitment of TM4SF1.Down-regulation of T M4SF1 expression can inhibit the migration and invasion of ovarian cancer HO8910 PM cells.The presence of exogenous Collagen I can promote the migration and invasion of ovarian cancer HO8910 PM cells.Part III Effects of Exogenous Collagen I induced DDR1 on Biological Behavior of Highly Metastatic Ovarian Cancer Cell Strain HO8910 PM In VitroObjective To investigate the effect of DDR1 on biological behavior of highly metastatic ovarian cancer cell strain HO8910 PM in the presence or a bsence of exogenous Collagen I.Methods Lentivirus targeted to silence DDR1 was infected with HO8910 PM.The interference efficiency was detected by RT-PCR and Western Blot to select LV-DDR1-RNAi-mcherry-HO8910 PM cell strain with the best in terference effect.Transwell migration and invasion assays were used to detect changes in cell migration and invasion in the presence or absence of exogenous Collagen I.Results RT-PCR and Western results showed that lentiviral vector named LV-DDR1-RNAi-mcherry-2 was the best for DDR1 silencing.RT-PCR exp eriments showed that the efficiency of DDR1 gene silencing at m RNA level was about 85%,and the efficiency of DDR1 protein silencing detected by Western Blot was about 95%.Transwell migration experiments show ed that the number of cells passing through the Transwell polycarbonate membrane in the LV-RNAi-DDR1-mcherry-HO8910 PM group was 99.00±5.292,which was significantly lower than that in the HO8910 PM group(160.33±6.028)and the LV-CON-DDR1-mcherry-HO8910 PM group(161.00±4.583)(all P<0.05).The number of LV-DDR1-RNAi-mcherry-HO8910 PM cells in the presence of 50 μg/ml Collagen I passing through the Transwell chamber was 150.67±5.508,compared with HO8910PM+50 μg/ml Collagen I group(239.00±10.583)and LV-CON-DDR1-mcherry-HO8910PM+50μg/ml Collagen I group(237.67±8.386)was significantly decreased(all P<0.05).In the presence of exogenous Collagen I,the cell numbers of HO8910 PM,LV-RNAi-DDR1-mcherry-HO8910 PM,and LV-CON-DDR1-mcherry-H O8910 PM cells that passed through the polycarbonate membrane were all significantly higher than those in the absence of exogenous Collagen I(all P<0.05).Transwell invasion assay showed that the number of LV-RNAi-DDR1-mcherry-HO8910 PM cells passing through the Matrigel-coated Trans well chamber was 66.00±6.557,which was significantly lower than that of HO8910 PM group(126.67±6.506)and LV-CON-DDR1-mcherry-HO8910 PM group(133.67±7.572)(all P<0.05).The number of LV-DDR1-mcherry-HO8910 PM cells in the presence of 50 μg/ml Collagen I passing through the Matrigel-coated Transwell chamber was 117.00±7.211,and HO8910PM+50 μg/ml Collagen I group(180.00±2.646)and LV-CON-DDR1-Mcherry-H O8910PM+50μg/ml Collagen I group(190.00±9.849)was significantly decreased(all P<0.05).In the presence of exogenous Collagen I,the numbers of HO8910 PM,LV-RNAi-DDR1-mcherry-HO8910 PM,and LV-CON-DDR1-mcherry-HO8910 PM cells that passed through the Matrigel-coated Trans well chambers were all significantly higher than those in the absence of exogenous Collagen I(all P<0.05).Conclusion Down-regulation of DDR1 expression can inhibit the migration and invasion of ovarian cancer cell line HO8910 PM.The presence of exogenous Collagen I can promote the abilities of migration and invasion in ovarian cancer HO8910 PM cells.