The Effect Of Migration And Invasion Inhibitory Protein (MIIP) On Tumor Angiogenesis And Its Molecular Mechanism

Angiogenesis is a complex process in our body,which is required for many normal physiological processes,such as wound healing,embryonic development and the female physiological cycles.However,abnormal angiogenesis is associated with many diseases,including ischemic diseases,chronic inflammation and malignant tumor.The balance between angiogenesis factor and anti-angiogenesis factor is very important.Angiogenesis is the key to tumor growth,a new strategy that inhibits tumor angiogenesis(Anti-angiogenesis)has become way to cure tumor.Looking for an ideal target of the tumor vascular for inhibiting tumor cell invasion and migration and the promotion of the normalization of tumor is one of the hot issues of current cancer research.The migration and invasion inhibitory protein(MIIP),also known as invasion inhibitory protein 45(?p45),which was found to be a tumor suppressor gene by the screen of a human fetal brain cDNA library using mature IGFBP2 as the bait molecule.MIIP regulates cell migration and invasion and the mitosis checkpoint,thus resulting in the inhibition of tumor development by the interaction with other proteins.Recent study found that MIIP could bind histone deacetylase enzyme 6 off(HDAC6),heat shock protein 90(HSP90),which affected its regulation on acetylation of microtubule cytoskeleton system.As a result,MIIP is likely to become the important molecular targets of anti-tumor therapy in the future.In our study,we carried out some experiments both in vivo and in vitro for clarifying the inhibitory effect of MIIP on tumor angiogenesis and exploring the molecular mechanisms of MIIP for inhibiting tumor angiogenesis.We hope that MIIP can become a reliable antitumor molecular.This topic was performed from the following several aspects,and the results were obtained as follows.1)The effect of MIIP on HUVEC migration,invasion and tube formation.To study the contribution of endothelial MIIP to angiogenesis,HUVEC with the MIIP over-expression was constructed successfully.Wound Healing assay and transwell chamber experiment were used for testing HUVEC cell migration and invasion in vitro;Tube Formation experiment for testing the effect of MIIP expression level on the ability of cells in vitro tube formation.EdU experiment for testing cell proliferation and MTT for detecting growth curve.Finally,HUVEC + Matrigel angiogenesis is used in vivo model for detecting whether MIIP inhibits the angiogenesis of vascular endothelial cells.Results showed that MIIP can significantly inhibit human umbilical vein endothelial cell migration,invasion,and tube formation and angiogenesis in vitro.2)The effects of MIIP on tumor angiogenesis.In order to further study,the breast cancer cell line with the MIIP over-expression or knockdown was constructed successfully,then conditioned medium comtaining MIIP from breast can cell line with the MIIP over-expression or knockdown was used in Rat aortic vascular ring experiment and Chick Chorioallantoic assay.The results demonstrated that MIIP over-expression could significantly inhibit the blood vessel formation of tumor.Western blotting was used to detect the effect of MIIP on the expression level of VEGF-A,MMP2 and MMP9 in conditioned medium of breast cancer cells.The MIIP over-expression could decrease the expression level of VEGF,MMP2 and MMP9 in conditioned medium with MIIP over-expressing vector;All results above are opposite with MIIP knockdown vector in breast cancer cells.MIIP might inhibits tumor angiogenesis by inhibiting tumor angiogenesis foctors.3)The effect of MIIP on the expression level of CD31 in transplantation of nude mice:The breast cancer cells with the over-expression of MIIP was innculated into nude mice,30 days later,tumor tissues were removed for detecting CD31 by immunohistochemical staining.The result proved that the tumor tissue in MIIP over-expression of MDA-MB-231 could obvioulsly inhibit the expression level of CD31.The experiment proved that the MIIP significantly inhibited tumor angiogenesis.4)The possible mechanisms of anti-tumor angiogenesis of MIIP.Western blotting was used to detect the effct of over-expression of MIIP on the expression level of VEGF-R2,Erkl/2 and phosphorylation of ErK,FAK and phosphorylation of FAK in HUVEC.The results demonstrated that MIIP could decrease the expression level of VEGFR2,Erk1/2 and phosphorylation of Erk1/2?phosphorylation of FAKY-397 and FAKY-925 in HUVEC.At the same time,over-expression of MIIP could inhibit the expression level of VEGF-A and HIF1-? in breast cancer cells.Finally,MIIP have obvious effect on inhibiting angiogenesis via FAK and Erkl/2 signal pathway in HUVEC.The result proved that MIIP inhibits the expression level of HIF1-? in breast cancer cells to decrease the expression level of VEGF-A.Conclusion:MIIP was found to inhibit the migration,invasion and tube formation of human umbilical vein endothelial cell(HUVEC).The conditioned medium containing MIIP from breast can cell line with the MIIP over-expression or knockdown was used in vitro experiment,the results indicated that MIIP could significantly inhibit the blood vessel formation of tumor.The experiment of MIIP could obviously inhibit VEGF-A,MMP2 and MMP9 angiogenic factors by tumor cells.The result proved that the tumor tissue in over-expression of MIIP in breast cancer cells could obvioulsly inhibit the expression level of CD31.The study of the molecular mechanisms of MIIP for inhibiting tumor angiogenesis indicated that MIIP could decrease the expression level of VEGF-R2 by Erk1/2 and FAK signal pathway in HUVEC.The expression level of HIF1-? and VEGF-A in breast cancer cells decrease to inhibit tumor angiogenesis.