NF90 Regulates The Angiogenesis Of Cervical Cancer Through PI3K/AKT Signaling Pathway

Background:Cervical cancer is the fourth most common cancers in developing countries.530,000 women were diagnosed with cervical cancer,and266,000 were died from it in 2012.In 1989,the German virologist Harald zur Hausen first proposed that HPV is the etiology of cervical cancer and opened a new era in the etiology of cervical cancer.Since then,a variety of experiments and epidemiological investigations have demonstrated that 90%of cervical cancer cases are closely related to high-risk HPV persistent infection.The establishment of HPV etiology reveals the relationship between HPV infection and the tumorigenesis tand recurrence of cervical cancer,and promotes the development of cervical cancer primary prevention（HPV vaccine）and secondary prevention（HPV screening）for the prevention and early diagnosis of cervical cancer.However,the current HPV vaccine can not prevent all of the genotype of the HPV virus,the protective duration is unclear,and it can not play the protective role in women with previous HPV transfection,and there is no evidence that the HPV vaccine can completely prevent the cervix The occurrence and development of cancer.Meantime,the combined therapies effect of advanced cervical cancer patients is still not satisfactory.Therefore,exploring the new direction of cancer treatment to improve the efficacy and prognosis of patients with advanced stage is still the direction of cervical cancer treatment.Tumor growth,invasion,and metastasis are rely on angiogenesis to prove essential oxygen,nutrients,and metabolic functions.Meantime,revascularization also provides structural support for the distant metastasis of tumors.The indispensable correlation between tumor growth and neovascularization has led to the study of antiangiogenic therapy.Pro-angiogenic signals and receptors are targets for anti-angiogenic therapies（AATs）,bevacizumbab（anti-VEGF-A）and sorafenib（anti-VEGF-R2）are well established and achieved certain results.It is of limited benefits using AATs for cervical cancer treatment of.Therefore,multi-target anti-angiogenesis therapy may provide a new idea for the combined treatment of cervical cancer.NF90 and NF45 combine to form NF90/NF45 heterodimer complexes and involved in the regulation of DNA metabolism,transcription,translation,RNA export,mRNA stability,and other cellular metabolic pathways.NF90is upregulated in a variety of milignancies,including nasopharyngeal carcinoma,hepatocellular carcinoma,non-small-cell lung cancer,and ovarian cancer.NF90 accelerates the cell cycle,and promotes proliferation of hepatocellular carcinoma.Moreover,it also maintains the sustained expression of u PA,or increase the VEGF-A mRNA stability,promotes angiogenesis of breast cancer.Although the regulatory function of NF90 has been widely studied,its role in the development of cervical cancer remains to be further studied.The present study is to explore the relationship between NF90 and VEGF-A expression and angiogenensis in cervical cancer,and find the possible signaling pathway.The role of NF90 in a variety of malignancies has been extensively studied,and it has also led us to explore its relationship with angiogenesis in cervical cancer and potential molecular mechanisms in order to provide new ideas for multi-target anti-angiogenesis.There are three parts of our study.Part 1 NF90 participates in the expression of VEGF-A induced by hypoxia and angiogenesisChapter 1 ILF3 is upregulated in human cervical cancer specimensObjective:To determine whether the expression of ILF3 is related to cervical cancer.Methods:The ILF3 mRNA and protein expressions in cervical cancer specimens were retrieved from the Oncomine（www.oncomine.org）and Human Protein Atlas（www.proteinatlas.org）.Results:The mRNA and protein expressions of ILF3 are upregulated in cervical cancer specimens.Conclusion:ILF3 may be related to the tumorigenesis of cervical cancer.Chapter 2 NF90 participates in the expression of VEGF-A induced by hypoxia and angiogenesisObjective:To determine whether NF90 promotes the expression of VEGF-A,and induce the angiogenesis in vitro.Methods:1.Hypoxia（2%O₂）and CoCl₂ were used to simulate the hypoxic stress of cervical cancer cells,and the protein expressions of HIF-1α、VEGF-A、NF90、NF110 and NF45 were determined.2.HeLa and Si Ha cells were transfected with lenti-virus knockdown or ectopic overexpression fo NF90.The mRNA and protein expressions of HIF-1αand VEGF-A were investigated by PCR、Western blot、immunofluorescence and ELISA.3.The tube formation and cell migration of HUVECs were also been determined.Results:1.The protein expressions of NF90、VEGF-A、HIF-1αof HeLa cells are most at 12 h in hypoxia.While,the expression of NF90 and VEGF-A is most at 12 h,and 24 h for HIF-1αin hypoxia.The protein expression of NF110 can’t be stimulated by hypoxia.2.Knockdown of NF90 reduces the expression of NF90 and VEGF-A,while overexpression of NF90 restore it.3.Depletion of NF90 inhibits the tube formation and cell migration of HUVECs.Conclusion:Hypoxia induced the expression of NF90,HIF-1αand VEGF-A protein in the time-dependent manner.Among them,the increasing expression of NF90 and VEGF-A protein was consistent,suggesting that NF90 might be related to VEGF-A expression.Knockdown or overexpression of NF90 can significantly affect the expression of HIF-1αand VEGF-A,as well as the in vitro angiogenesis of cervical cancer,suggesting that NF90 may be one of the targets of multi-target anti-angiogenic therapy.Part 2 NF90 induces the expression of VEGF-A through PI3K/Akt signaling pathwayObjective:To explore the potential molecular mechanism.Methods:The PI3K/Akt signaling pathway inhibitors LY294002,Wortmannin and MK-2206 were used,and the pho-Akt was detected by Western blot.Results:The mRNA and protein expressions of VEGF-A induced by NF90 can be blocked abolished by HIF-1αinhibitor（PX-478）,PI3K inhibitor（LY294002,Wortmannin）and Akt inhibitor（MK-2206）.Conclusion:The PI3K/Akt signaling pathway participates in the progress.Part 3 Knockdown of NF90 inhibits the angiogenesis and tumor growth in mice cervical caner xenografts.Objective:To determine whether depletion of NF90 inhibit angiogenesis and tumor growth in vivo.Methods:Subcutaneous injection of HeLa-Ctrolsh and HeLa-sh3 to establish the cervical cancer xenografts.The xenografts were constructed by subcutaneous injection of HeLa/NF90-shRNA3,tumor volume and weight were measured,the neovascularization was observed in vivo by photoacoustic imaging,the expressions of Ki-67 and VEGF-A were detected by immunofluorescence.Results:The tumor volume,tumor weight,photoacoustic signals,the protein expressions of Ki-67 and VEGF-A were decreased in NF90knockdown group.Conclusion:Suppression of NF90 in cervical cancer cell lines can decrease VEGF-A expression,inhibit angiogenesis,and reduce tumorigenic capacity in vivo.