Macrophage Migration Inhibitory Factor (MIF) Transfected Cervical Cancer Cells SiHa And Its Effect On Angiogenesis Of Cells

Cervical cancer is the second largest female malignant tumors, second only to breast cancer.In recent years, it present obvious younger trendency, which severely threaten women’s health and the quality of life.Macrophage migration inhibitory factor(MIF) is a pleiotropic cytokine,which plays a key role in inflammation, immune response and the development process of tumor. MIF is associated with many kinds of tumor’s invasion and metastasis, clinical stages and prognosis. Our previous studies in vitro showed that the expression of MIF in cell lines SiHa was lower than in Caski, which may promote the invasion and metastasis of cervical cancer through some mechanism, for example, promoting cell proliferation, transfer, angiogenesis and inhibiting cell apoptosis.Objective:To construct eukaryotic expression vector pEGFP-N1/MIF that expressed green fluorescent,and studied the effects of MIF over-expression on the expression of interleukin-8(IL-8), martix metalloproteinase9(MMP9), vascular endothelial growth factor (VEGF) and proliferation and invasion of human cervical cancer cells SiHa, researched the mechanism of angiogenesis in cervical cancer cells furtherly, initially explored the development mechanism of MIF via NF-κB signal way in cervical cancer.Methods:1. Chemical synthesis MIF cDNA gene, designed primer sequence including XhoI and BamHI enzyme sites, MIF gene was amplified by polymerase chain reaction (PCR). constructed eukaryotic expression vector pEGFP-Nl/MIF and transfected into SiHa cells using lipofectamine, observed transfection efficiency under fluorescence microscope; the expression of MIF in supernatant fluid was detected by ELISA, the expression of MIF mRNA and protein in cells were detected by real-time fluorescence quantitative-PCR and immunocytochemistry respectively.2. The ability of proliferation in transfected and untransfected cells was detected by MTT; the expression of NF-κB, IL-8, MMP9, VEGF mRNA and protein were detected by real-time fluorescence quantitative-PCR and immunocytochemistry respectively.3. Intervened the pEGFP-Nl/MIF/SiHa by NF-κB inhibitors PDTC, the ability of proliferation was detected by MTT; the expression of MIF, NF-κB, IL-8, MMP9, VEGF protein were detected by immunocytochemistry.Results:1. By XhoI and BamHI double enzyme digestion and sequencing identification proved the eukaryotic expression vector pEGFP-N1/MIF was successfully constructed, and cells green fluorescence can be observed under fluorescence microscope after transfecting SiHa, when transfected48hours, transfection efficiency to (61±2.0)%; ELISA result showed that the expression of MIF protein in supernatant fluid transfected with pEGFP-N1/MIF were significantly increased (P＜0.05), real-time fluorescence quantitative-PCR and immunocytochemistry results showed that the expression of MIF mRNA and protein in SiHa cells transfected with pEGFP-N1/MIF were significantly increased (P<0.05).2. The ability of proliferation in pEGFP-Nl/MIF/SiHa was obviously increased (P＜0.05), and the expression of NF-κB, IL-8, MMP9,VEGF mRNA and protein in SiHa cells transfected with pEGFP-N1/MIF were significantly increased (P<0.05).3. The ability of proliferation was obviously inhibited by NF-κB inhibitor PDTC (P＜0.05), and the expression of NF-κB, IL-8, MMP9, VEGF protein was inhibited by NF-κB inhibitor PDTC (P<0.05);4. Pearson correlation analize showed that there were positive correlation among MIF and NF-κB, IL-8, MMP9,VEGF; NF-κB and IL-8, MMP9, VEGF; IL-8and MMP9, VEGF (P＜0.05) Conclusion:1. The eukaryotic expression vector pEGFP-Nl/MIF was successfully constructed,and the expression of MIF was obviously upregulated.2. MIF could improved the ability of proliferation of SiHa, and promoted the expression of NF-κB, IL-8, MMP9, VEGF in cervical cancer cells obviously.3. The ability of proliferation of SiHa and the expression of NF-κB, IL-8, MMP9, VEGF in cervical cancer cells SiHa could be obviously inhibited by NF-κB inhibitor PDTC.4. There were positive correlation among MIF and IL-8, NF-κB, MMP9,VEGF; NF-κB and IL-8, MMP9, VEGF; IL-8and MMP9,VEGF,which showed that MIF may via promoting human cervical cancer proliferation, angiogenesis, and promoting the development of cervical cancer, the mechanism may be associated with NF-κB pathway. So explored the angiogenesis mechanism of MIF via NF-κB in cervical cancer may contributed to diagnosis and treatment in cervical cancer.