The Expression Of MIF Gene In Bladder Cancer And Study On Biological Effects Of MIF Antisense Vector In Bladder Cancer Cells

Human Bladder Transitional Cell Carcinoma(BTCC)is ranked first in terms of the number of malignant tumor of the urinary system with the characteristics of high recurrence.The biological behavior and malignant potential of BTCC is variable.At first presentation,the disease appears to be superficial in 70%-80%of cases.However, despite the treatment of tumor local resection,approximately 50%to 70%of these tumors recur after operation with 10%to 30%showing grade and stage progression.On the other hand,of patients with BTCC 20%-30%initially present with muscle invasive or metastatic disease,of whom 50%die within 2 to 3 years after diagnosis despite aggressive local therapy(see the references 1 of chapter I).Therefore, Recurrence,Invasion and Metastases have been the key factors that influence the efects of treatment and prognosis of bladder cancer.But the mechanism of cancer recurrence and development is still unclear.Macrophage migration inhibitory factor(MIF)was found and named by Bloom and Bennett in 1966,Although MIF was found earlier,its function and constitution was investigated preferably until its cDNA was cloned in 1989.Until now,MIF was identified as a tumorigenesis cytokine,it can directly not only regulate cell division and oncogenes induced malignant transformation but also inhibit gene P53 and encourage tumor angiogenesis(see the references 1 of chapter I).MIF has alreadly become a hot spot in tumor recently,but there is not reports about the study on MIF gene in BTCC.According to this mentioned above,The present study will explore the role of MIF in the development of BTCC and its potential clinical significance.Chapterâ… The expression of MIF and MMP9 in bladder transitional cell carcinomaObjective:To study the clinical value of the expression of MIF in the bladder transitional cell carcinoma(BTCC)and to evaluate the possible relation the expression of MIF and matrix metalloproteinase-9(MMP9).Methods:MIF was examined by immunohistochemically and RT-PCR in 43 cases of BTCC and 10 cases of normal bladder tissue.The correlations between the expression of MIF and tumor grade,stage,survival rate and MMP9 were closely studied.Results:immunohistochemically manifestation:(1)The expression of MIF-protein was abnormal in BTCC but absent in normal bladder tissue.(2)The expression of MIF-protein increased with tumor stage(P＜0.01)and had nothing to do with grade(p＞0.05).(3)High expression of MIF-protein was associated with poor prognosis of BTCC.(4)The expression of MMP9-protein was abnormal in BTCC but absent in normal bladder tissue.RT-PCR manifestation:(1)The expression of MIF-mRNA was abnormal in BTCC and absent in differentiated normal bladder tissue.(2)The expression of MIF-protein increased with tumor stage(P＜0.01)and had nothing to do with grade(p＞0.05).(3)The expression of MMP9-mRNA was abnormal in BTCC and absent in differentiated normal bladder tissue.(4)The MIF-mRNA expression had correlation with the MMP9-mRNA;MIF might induce the augmentation of MMP9-mRNA expression.Conclusion:According to this mentioned above,The abnormal expression MIF is common in BTCC.MIF plays an important role in the invasion and metastasis of BTCC,and would be a useful predictor of prognosis in BTCC.The abnormal expression MMP9 is common in BTCC.The MIF expression had correlation with the MMP9 in BTCC,high expression of MIF might increase expression of MMP9 in the level of transcription.Chapterâ…¡Identification of eukaryotic expressing vector for antisense MIF and surpression of MIF gene expression by using antisense RNA in BIU-87 cell lineObjective:To establish and identify BTCC cell line that could stably express antisense MIF in order to study its efects on MIF gene expression and the biological behavior of BTCC cell line. Method:Five diferent plasmids including pSecTag-MIF,pcDNA₃-antiMIF, empty pSecTag,empty pcDNA₃ and PN3-EGFP were prepared by transformation of bacterium,amplification and purification of plasmids.Then pcDNA₃-antiMIF plasmids and empty plasmids pcDNA₃ were reseparately transferred into BTCC cell line BIU-87 cultured in vitro by using lipfectamine.After transfection,cells were selected by G418 and the positive cell clones were chosen and expanded culture.The expression of gene MIF on the levels of mRNA and protein were reseparately determined by RT-PCR,western blot and immunocytochemical staining.Result:The clones of cell transfected by pcDNA₃-antiMIF plasmids were smaller and grew slowly.All of the two cell lines transfected by corresponding plasmid acquired resistance to G418;The expression of gene MIF on the levels of mRNA and protein was detected by RT-PCR,western blot and immunocytochemical staining in cell lines transfected by pcDNA₃-antiMIF plasmids,whereas,it did not show in cell lines un-transfected or transfected by an empty vector.Conclusion:We successfully established BTCC cells that could stably express the gene antisense MIF,or empty plasmid pcDNA₃ respectively.The expression of MIF protein and mRNA was weak in transfected pcDNA₃-antiMIF plasmids BIU-87 cell lines.It provid a foundation for post experimentation.Chapterâ…¢The invitro growth influence of human BTCC cell line BIU-87 by transfer of pcDNA₃-antiMIF plasmidsObjective:To investigate whether the pcDNA₃-antiMIF plasmids transfer could inhibit the invitro growth and apotosis of BTCC cell lines and to explore the possible mechanism of this effect.Methods:Two BTCC cell lines we constructed by transferring respectively by the pcDNA₃-antiMIF plasmids,empty plasmids pcDNA₃ and un-transfected BIU-87 cell line were taken as our object.Cell growth curve was obtained by cell counter and cell survival rate was measured by MTT assay.Flow cytometry was used to analyze the cell cycle and cell apoptosis.Results:Compared with other two groups,cell line that transferred by pcDNA₃-antiMIF grew more slowly,had a lower survival rate,a higher proportion of cells in phase whereas cells in G0/G1 phase and had an in the of apoptosis diference are of significance(p＜0.05),however other two groups had no diference.Conclusions:pcDNA₃-antiMIF transfer could inhibit the in vitro growth of BTCC cell line BIU-87 and this elect was possibly atributed to inducement of cell cycle arrest at G1 phase and inducement of cell apoptosisChapterâ…£The invitro motility and invasion influence of human BTCC cell line BIU-87 by transfer of pcDNA₃-antiMIF plasmidsObjective:To investigate whether the pcDNA₃-antiMIF plasmids transfer could inhibit the invitro motility and invasion of BTCC cell lines and to explore the possible mechanism of this effect.Methods:Two BTCC cell lines we constructed by transferring respectively by the pcDNA₃-antiMIF plasmids,empty plasmids pcDNA₃ and un-transfected BIU-87 cell line were taken as our object,the invitro motility and invasion ability was obtained by cell adhere assay,scratch assay,matrigel invasion assay,RT-PCR was used to analyze expression of MMP9mRNA in three groups.Results:The motility and invasion in BIU-87 cells cell line that transferred by pcDNA₃-antiMIF were decreased comparing with other two groups(P＜0.05),The expression of MMP9mRNA was weak in transfected pcDNA₃-antiMIF plasmids BIU-87 cell lines.Conclusions:pcDNA₃-antiMIF transfer could inhibit the in vitro motility and invasion of BTCC cell line BIU-87 and this efect was possibly atributed to supression of expression of MMP9mRNA.