A Preliminary Study Of Ovarian Tumor Associated Antigen TM4SF1 Affect The Biological Function Of Cancer Cells

Part1. The expression of TM4SF1protein in epithelial ovarian cancerand its clinical valueObjective To investigate the expression of TM4SF1protein in humanovarian tissues and lymph code metastasis and its relationship withclinicopathologic factors.Methods Immunohistochemical SP method was used to detect theexpression of TM4SF1protein in16normal ovarian epithelial tissues,23benignovarian tumors,55EOCs and30lymph code metastasis. The relationship ofTM4SF1expression with clinicopathologic factors and patient prognosis of theEOCs was analyzed.Result (1)The positive rate of TM4SF1protein expression in EOCs（90.90%） was higher than that in benign ovarian tumors（65.22%） andnormal ovarian epithelial tissues（31.25%）,(P<0.05). The positive rate ofTM4SF1protein expression in benign ovarian tumors was higher than that innormal ovarian epithelial tissues,(P<0.05).TM4SF1showed positive expressionin all lymph node metastasis (100%).(2)The TM4SF1protein was mostlypredominant at the basal surface and other parts of the epithelial cell membranesas well as cytoplasts in normal ovarian epithelial tissues while the main positionin benign ovarian tumors was at the basal surface and other parts of the tumorcell membranes. But in EOCs and lymph node metastasis the protein mainlydisclosed a cytoplasmic staining.（3）The positive rate of TM4SF1proteinexpression in tissues of patients with stage Ⅲ～Ⅳ was higher than that in patients with stage Ⅰ～Ⅱ,(P<0.05). The positive rate of TM4SF1proteinexpression in tissues of EOCs patients with grade2-3was higher than that inpatients with grade1,(P<0.05).(4)The positive rate of TM4SF1proteinexpression had no significant association with histological type and amount ofascites,(P>0.05).(5)Cox multivariate analysis showed that positive expressionof TM4SF1protein was not an independent factor for patient prognosis.Conclusion TM4SF1protein was overexpressed in EOC and lymph nodemetastasis cells. It may be associated with the oncogenesis, development anddistant metastasis of EOC. Part2. The effect of knocking-down TM4SF1on the biologicalfunction of epithelial ovarian cancer cells in vitro experimentObjective To explore the effect of TM4SF1knockdown on the biologicalfunction of HO8910PM by RNA interfere technology.Methods Detected TM4SF1gene expression in different ovarian cancersand filtered out the HO8910PM with a high TM4SF1expression using RT-PCR.We designed3small interfering RNA(siRNA) and transfected them intoHO8910PM using lipofectin method. Filtered out the best siRNA silencefragment and inserted into pGPU6/GFP/Neo plasmid. Transfected the complexinto HO8910PM and adopted G418to screen the stable cell line. Real-timePCR(QRT-PCR) analyses and Western blot were performed to measure TM4SF1mRNA and protein levels respectively. Cell’s growth, proliferation, cell cycleprogression, migration and invasion were detected by MTT, flow cytometryassays, Transwell, metrigel.Result SKOV3,HO8910, especially HO8910PM expressed high amountsof TM4SF1while A2780presented an absence. QRT-PCR showed thatsi-TM4SF1-813presented the best gene silencing effection; After constructedthe pGPU6/GFP/Neo-TM4SF1-813using the DNA recombinant technology, wetransfected the recombinant into HO8910PM and got the stable cell line byG418selection. QRT-PCR result showed that TM4SF1gene expression could bereduced by about85%in pGPU6/GFP/Neo-TM4SF1-813cells and Westernanalisis further presented the significant decline of TM4SF1proteinexpression(50%). Migration tests showed that the number ofpGPU6/GFP/Neo-TM4SF1-813-HO8910PM (37.67±3.79) that cross the membrane was much smallar than that of pGPU6/GFP/Neo-TM4SF1-control(107.31±5.13)(P<0.05). Invasion tests showed the same result inpGPU6/GFP/Neo-TM4SF1-813-HO8910PM and pGPU6/GFP/Neo-TM4SF1-control, with the cell number (32.33±3.05) and (88.01±2.646), respectively(P<0.05). There were no significant differences in3groups when compared thecell’s growth, proliferation or cycle progression (P>0.05).Conclusion TM4SF1gene downregulation expression could inhibitinvasion and migration but not affect growth, cell cycle progression orproliferation of HO8910PM. Part3. The effect of over-express TM4SF1on the biologicalfunction of epithelial ovarian cancer cells in vitro experimentObjective To construct the recombinant lentiviral vector(LV) that carryingTM4SF1gene and explor the biological impact of TM4SF1upregulation onepithelial ovarian cancer cell in vitro.Methods Design primers to amplify the full length of TM4SF1gene.Inserted the TM4FS1gene into LV expression vector and got the recombinant.The there plasmids of LV vectors: pWPI、pCMV-dR8.74、pMD2.G werecotransfected into293T cells to produce the LV vectors which could expressTM4SF1gene. Infected host cells A2780with the produced LV vector and thensorted the purposed cells. The results of TM4FS1upregulation were obtained byQRT-PCR and Western blot. Cell’s growth and proliferation, cell cycleprogression, migration and invasion were detected by MTT assays, colonyformation assays, flow cytometry assays, migration and invasion assaysrespectively.Result The relative expression of TM4SF1in TM4SF1-pWPI-A2780measured by QRT-PCR was (242.200±9.385) which was much higher than thatin pWPI-A2780(1.040±0.104) and A2780(1.794±0.147)(P<0.05). Westernblot also showed the same tendency. Migration tests showed that the number ofTM4SF1-pWPI-A2780(132.71±10.21) that cross the membrane was muchlarger than that of pWPI-A2780(69.67±9.05) and A2780(64.00±7.94)(P<0.05). Invasion tests showed the same result in TM4SF1-pWPI-A2780with the crossed cell number (100.01±11.14) compared with pWPI-A2780(47.33±7.51) and A2780(52.02±7.55)(P<0.05). There were no differences in cell invasion and migration abilities when compared with pWPI-A2780andA2780(P>0.05). And there were no significant differences in3groups whencompared the cell growth, proliferation or cycle progression(P>0.05).Conclusion TM4SF1gene upregulation could increase the invasion andmigration of A2780but not affect the growth, cell cycle progression orproliferation.