| Purpose:Lung cancer is one of the most common malignant tumor all over the world,and the new cases and death cases every year are at the forefront of the various types of malignant tumor.In recent 20 years,its mortality is growing fastest.For the treatmet of lung cancer,there are several methods in clinical before,including surgery,radiation and chemotherapy,immune therapy and so on.But nowadays,personalized treatment according to the pathological type,tumor size,invasion range and comprehensive treatment including molecular targeted therapy are highly rewarded and respected.Although treatment achieved significant improvement,the overall prognosis of lung cancer is still not optimistic,and the 5 years survival rate is only 15%on average.Angiogenesis is of vital importance in tumor development.A variety of angiogenesis induced factors involved in this process,including HIF,VEGF,MMP-2,MMP-9.Hypoxia is an important feature of tumor microenvironment.Hypoxia-inducible factor?HIF?as a transcription factor existing in humen or mammals in hypoxia environment.Not only can enhance the adaptability of the tumor cells in hypoxia environment,but also can increase the ability of tumor cell invasion and drug resistance to chemotherapy and radiotherapy.Under the anaerobic environment,the expression of HIF-1?is upregulated and which activates the downstream gene such as VEGF,finally resulted to the proliferation of endothelial cells in tumor,which provide more nutrition to tumor cells and promote tumor growth.Therefore,anti-angiogenesis is very important for tumor controlling.miRNAs participate in multiple cell biological functions,including proliferation,differentiation,apoptosis and so on,and play an important role in the process of normal biological development and disease.There are reports that nearly half of miRNAs gene is located in the tumor related genomes area or fragile site,which suggests that miRNAs may play an important role in the occurrence and development of human tumor.Recent years,there are reports about miR-214 expresstion disorder in many kinds of tumors,and has the potential to be new tumor marker or new targets for the treatment of tumor.However,its expression and function in lung cancer is still unclear.What‘s more,there is also no report to disscus the relationship between miR-214 and HIF or angiogenesis.Inhibitor of growth family?ING4?,as a new candidate tumor suppressor gene,is widely involved in tumor occurrence and development,including tumor angiogenesis,cell adaptation of hypoxia state,the loss of contact inhibition between cells,and inhibition tumor cells invasion and metastasis,cell apoptosis and cell cycle adjustment,etc.The mechanism includes P53 activation and NF-?B inhibition.At present,a lot of scholars focus on the relationship of decreasing of ING4 expression with tumorigenesis,and the relationship between over-expression of ING4 and tumor growth and drug resistance.In the aspect of ING4 inhibiting tumor angiogenesis,they also only related to angiogenesis related factors secreting by tumor cells and microvascular density in tumor tissue.However,the direct regulation function on endothelial cells by ING4 and the regulating function on ING4 by miR-214 have not been reported yet.This study uses technologies such as microRNA mimics and inhibitor transfection,Real time PCR,western blot,to explor the expression of miR-214 in lung cancer cells and its biological funtion;and make it clear that whether the function of miR-214mentioned above is due to the inhibition of ING4 and activation of HIF signaling pathway;the effect of ING4 on the growth and biological funtion of endothelial cells and its regulating function on HIF-1?.In order to reveal the relationship between miR-214 and ING4,ING4 and angiogenesis,and make it clear that whether miR-214could regulate the expression of ING4,regulate tumor angiogenesis and moreover promote malignant biological behavior of tumor.Methods:1.Cell culture:HUVECs;HUVECs treated with for 12h to simulated hypoxia environment.2.Cell culture:A549 and H1299.3.Construction of ING4 plasmid and siRNA and transfect to HUVECs,A549 and H1299.4.Construction of miR-214 mimic and inhibitor and transfect to A549 and H1299.5.MTT assay and colony forming assay to detect cell proliferation.6.Transwell assay to detect cell migration.7.Wound healing assay to detect cell invasion.8.Luciferase reporter gene assay to detect the regulatory effect of miR-214 on ING4 expression9.Real time PCR and western blot to check the mRNA and protein expression of ING4,VEGF,MMP-2,MMP-9,HIF-1?and its target gene AK3.Results:1.Effects of ING4 on the proliferation,migration,and angiogenesis related factors of HUVECsMTT assay shows that after the transfection of ING4 plasmid,the proliferation of HUVECs is decreased,while transfection of ING4 siRNA and promote the proliferation.HUVECs treated with CoCl2 for 12h,the result of MTT assay is the same with often oxygen state,but the effects decreased.Transwell assay shows that,the migration ability of HUVECs is decreased by transfection of ING4 plasmid,but enhanced by transfection of ING4 siRNA.Real time PCR and western blot shows that,transfection of ING4 plasmid can inhibit the the mRNA and protein expression of VEGF,MMP-2,MMP-9.2.Reguation effect of ING4 on HIF-1?Real time PCR and western blot shows that,HUVECs treated with CoCl2 for 12h,the mRNA and protein expression of HIF-1?and its target AK3 are increased,while after transfection of ING4 plasmid,the expression is inhibited,and after transfection of ING4 siRNA,the expression is enhanced.3.Effects of miR-214 on the proliferation,migration,invasion and angiogenesis related factors of A549 and H1299Colony forming experiment shows that,after transfection of miR-214 mimics,number of colony forming is apparently increased appaerd with control group.Transwell assay shows that,after transfection of miR-214 mimics,number of cells through the well is apparently increased.Wound healing test shows that,after transfection of miR-214 mimics,the speed of wound healing is faster than control group.Real time PCR and western blot shows that,transfection of miR-214 mimics can up-regulate the mRNA and protein expression of VEGF,MMP-2,HIF-1?and its target AK3.While transfection of miR-214 inhibitor can show the opposite results.4.Reguation effect of miR-214 on ING4Real time PCR and western blot shows that,transfection of miR-214 mimics can down-regulate the mRNA and protein expression of ING4.Luciferase reporter gene assay results showed that luciferase activity decreased when co-transfection of miR-214 mimic and plasmid with wild-type ING4 gene.At the same time,transfection of miR-214 mimics can up-regulate the expression of VEGF,MMP-2,HIF-1?and its target AK3,while co-transfection of miR-214 mimic and ING4 plasmid can reverse the result above,and co-transfection of miR-214 mimic and ING4 siRNA can enhance the result above.The expression of VEGF,MMP-2 and HIF-1?was down-regulated when trasfection with miR-214 inhibitor,and co-transfection of miR-214 inhibitor and ING4siRNA can reverse the result.Conclusions:1.ING4 is able to inhibit the proliferation and migration of HUVECs,down-regulate the expression of angiogenesis related factors such as VEGF,MMP-2,MMP-9,and then inhibit tumor angiogenesis.The mechanism may be related to the ability of ING4 to down-regulate the expression of HIF-1?and its target gene AK3 in hypoxia environment.2.miR-214 promotes the proliferation,invasion and migration of A549 cells,and enhances tumor angiogenesis through up-regulating the expression of angiogenesis related factors such as VEGF,MMP-2,HIF-1?and its target gene AK3,which may come to the conclusion that miR-214 may act as a oncogene in lung tumor cells.3.ING4 may be a target gene of miR-214,while miR-214 may activate HIF-1?and up-regulate the expression of angiogenesis related factors such as VEGF,MMP-2through ING4 inhibition.So we speculate that miR-214 may promote malignant biological behavior of lung tumor cells through ING4 inhibition and HIF signaling pathway activation. |
PDF Full Text Request