Charaterization Of Sema4A In Breast Cancer

Breast cancer(BCa),accounting for 25%of cancer case and 15%of death rate related cancer,is one of the most common cancer and a malignancy affecting women.BCa is a heterogeneous disease with complex Cytological type,which is a great challenge for diagnosis and treatment.The semaphorins family has large number of secreted protein and membrane-binding protein.The initial studies showed they were related with the guidance of axonal growth and neurodevelopment.Among the eight subtypes of semaphorins,type III-VII is found in vertebrate,hypotype III is secretory protein,the semaphorins IV-VI is transmembrane protein and the subtype VII of semaphorins is connected with GPI.The followed-up studies showed the relationships to bone differentiation,development of cardiovascular system and regulating immune responses.Semaphorin4A(Sema4A)exists in transmembrane form as a family member of semaphorins.Sema4A expressed as a soluble ligand outside the cell and its extracellular morphology could be dissociated.Sema4A is related with development of tissue and organ and expression in brain,lung,kidney,testis and spleen.Studies showes it is engaged in the formation of various lesions by affecting T cell and macrophage activity,such as Viral bronchiolitis,asthma and degenerative diseases of the retina.Increased levels of Sema4A were detected in synovial tissues and fluids.And LPS could regulate its up-regulation expression.Exogenous Sema4A could potentiate production of inflammatory cytokines intracellular expression and secretion by stimulating synoviocyte and macrophage.Moreover,the exerting of this effect depends on its receptor--PlexinB 1.In addition,interactions of Sema4A and NF-kappaB could create a positive feedback loop to inflame synoviocyte cells.The cancer researches show Sema4A could promote the epithelial-mesenchymal transition to accelerate invasion and migration of hepatoma cell induced by AZM.At the moment,Sema4A had seven known receptors,including Tim-2,NRP-1,Plexin B1,Plexin B2,Plexin B3,Plexin D1 and ILT-4.The initial studies showed that Plexin molecules expressed on non-immunocytes,however,the expression level of Tim-2 and ILT-4 were restricted to activated CD4+T cell sourced of mouse and homo species.And NRP-1 molecules has verified in mouse Treg cells.Then,it turned out that Plexin B1 and/or D1 could express in DC cells,T cells and Treg cells of the immune system.Semaphorins' primary receptors involved Plexins receptor(Plexin A1-A4,Plexin B1-3,Plexin C1 and Plexin D1)and neuropilins receptor(Nrpl and Nrp2).Sema4A combined with Plexin D1 to support endothelial cells mediated by vascular endothelial growth factor(VEGF)migration and proliferation.However,Sema4A also could change cell morphology through receptor Plexin B1,B2 and Plexin B3.In addition,in treg cell,it was necessary for Sema4A regulating function and stability of treg cell to combined with neupilin-1.Each receptor(Plexin Bs,Plexin D1,Nrp1 and Tim-2)produced different functions by binding Sema4A.In a mouse model,Sema4A need to combined with Nrpl,a receptor expressed by Treg cells,in order to regulate its function and stability.Increased levels of Nrpl were detected in lymphoid tissue as compared to expression in Peripheral Blood(PB).NRP+Treg was found in synovia of patients with Rheumatoid arthritis and Muscular inflammation.In some cancer patients,NRP1 is up-regulated in Treg cells and the expression level of Tim-2 were restricted to activated T cells.Since Sema4A binding induces tyrosine phosphorylation in the cytoplasmic tail of tim-2,tim-2 transduces Sema4A signaling,and the phenotype of tim-2-is similar to that of Sema4A deficient mice,suggesting that tim-2 is a functional receptor for Sema4A on activated T cells.Taken together,the pathogenic networks involved in the regulation of Sema4A and its receptors are complex and have cell,tissue and disease specificity.There is a strong link between Sema4A and tumors.Germline variation of Sema4A gene may lead to the development of familial colorectal cancer type X.Sema4A also promotes drug resistance in liver cancer.In BCa,Sema4A could regulate BCa cell migration by interacting with receptors.However,the exact role of Sema4A and its pathological mechanism in BCa are not clear.To provide data support for enriching the research of Sema4A family members and oncology,in this project,we take BCa as a disaster model to explore its potential role and related mechanisms in the progression of the disease.In addition,hypoxia plays an important role through hypoxia induction factor which can lead to disease progression by regulating the expression of multiple target genes as a transcription factor in the pathological process of breast cancer.In this study,the regulation of Sema4A on breast cancer cell activity was clarified.In addition,this project will detect whether Sema4A is involved in the regulation of the activity of breast cancer cells in hypoxic conditions and the relationship between Sema4A and hypoxia.Research Objectives1.Analyze the expression and regulation of Sema4A in breast cancer.2.Investigate the effect of Sema4A on breast cancer cells.3.Explore the effect of Sema4A on the activity of BCa cells under hypoxic conditions.Innovation and research significance1.This study is the first time to prove that Sema4A expression in BCa cells is regulated by hypoxia and is dependent on hypoxia inducer 1 alpha(HIF-1 alpha).2.Sema4A can regulate the proliferation and apoptosis of BCa cells under hypoxic conditions.3.Exogenous Sema4A can protect BCa cells from apoptosis induced by hypoxia.4.Sema4A could regulate the activity of multiple signaling pathways in BCa cells.5.Sema4A can affect the progression of BCa as a pathogenic molecule.Materials and Methods1.Explore the expression characteristics of Sema4A in BCa tissues and cells.1.1 The expression,of Sema4A in breast cancer tissues was detected by real-time fluorescence quantitative PCR and Western blot.Collect BCa tissues and peripheral normal tissues and detect their expression by real-time fluorescence quantitative PCR and Western blot.1.2 Sema4A was detected in serum of BCa patients and normal control group by ELISA.2.Regulation and potential mechanism of hypoxia on Sema4A expression.2.1 Treatment breast cancer cells with hypoxia in vitro.The expression of Sema4A in breast cancer cells was detected by real-time fluorescence quantitative PCR and Western blot.The expression of Sema4A in the supernatant of breast cancer cell culture was detected by ELISA.2.2 Regulation of Sema4A expression by HIF-1 alphaUnder hypoxic conditions,the expression of HIF-1 alpha and HIF-2 alpha was silenced by siRNA method,and the expression of Sema4A in breast cancer cells was detected by fluorescence quantitative PCR.2.3 Detect whether HIF-1 alpha binds to the Sema4A promoter region by ChIP.3.Sema4A regulates the expression of hypoxic-related molecules in breast cancer cells.Expression of silent Sema4A in hypoxic condition.3.1 Detect the secretion of VEGF in the supernatant of breast cancer cell culture by ELISA.3.2 Detect the changes in the expression of MAPK(Mitogen-activated protein kinase),Akt(Protein kinase B)and STAT3(signal transducer and activator of transcription)pathway total proteins and phosphorylated proteins in breast cancer cells by Western blot.4.Effect of Sema4A on the activity of BCa cells under hypoxic conditions.4.1 Under hypoxic conditions,silence the expression of Sema4A in breast cancer cells and MTS method to detect cell proliferation activity and Apoptosis was detected by Annexin V assay.4.2 Under hypoxic conditions,exogenous Sema4A treated BCa cells and MTS method to detect cell proliferation activity and Apoptosis was detected by Annexin ? assay.Results1.Expression of Sema4A was significantly increased in breast cancer tissues.By detecting the expression level of Sema4A in breast cancer tissues and adjacent non-cancerous tissues,it was found that the expression level of Sema4A in breast cancer tissues was higher than that in the corresponding normal tissues at mRNA level and protein level.Sema4A expression in serum of breast cancer patients was detcted by ELISA.The results showed that the expression of Sema4A in serum of breast cancer patients was significantly higher than that of healthy control group.2.Hypoxia induces Sema4A expression in breast cancer cells through HIF-1 alpha dependent pathway.The expression of Sema4A in breast cancer cells was up-regulated by hypoxia(1%and 0.2%O2).Sema4A levels in cell supernatant fluid were also up-regulated after hypoxia stimulation.Then we analyzed the effect on Sema4A expression with knockout HIF-1 alpha and HIF-2 alpha in breast cancer cells.Silencing HIF-1 alpha could inhibit basal Sema4A expression in mda-mb-231 and McF-7 cells,rather than HIF-2 alpha.Notably,Sema4A produced by breast cancer cells induced by hypoxia(1%O2)was attenuated by si-HIF-1 alpha,but not by si-HIF-2 alpha.We also analyzed the expression of Sema4A in the supernatant fluid of breast cancer cells.Consistent with the changes of endogenous Sema4A expression in breast cancer cells,soluble Sema4A was significantly increased after hypoxia(1%O2)stimulation,while HIF-1 alpha silencing could antagonize the up-regulation of hypoxia-induced expression.To further verify the effect of HIF-1 alpha on Sema4A,we used Jaspar software to predict the potential binding of HIF-1 alpha to Sema4A promoter.Secondly,chip-qpcr was performed on MDA-MB-231 and McF-7 cells.The result shows HIF-1 alpha could bind to-697 bp--70 6 bp,Sema4A transcription initiation point.Importantly,HIF-1 alpha binding was significantly increased in this region after hypoxia stimulation.3.Hypoxia activity of breast cancer cells requires Sema4A.Next,we determined the effect of Sema4A on hypoxia activity.Data showed that the production of VEGF and phosphorylation of MAPK,Akt and STAT 3 pathways significantly increased in mda-mb-231 and McF-7 cells due to hypoxic treatment.However,silencing Sema4A could mitigate the increase.Then we examined the effect of rh-Sema4A on VEGF expression under hypoxia.It was found that rhSema4A combined with hypoxia increased the production of VEGF and the phosphorylation of ERK(extracellular regulated kinases),Akt and STAT3 pathways as compared with hypoxia alone.Then we determined the effect of Sema4A on apoptosis under hypoxia(0.2%O2).Under hypoxic conditions,silencing Sema4A inhibited tumor cell proliferation and induced tumor cell apoptosis.In other words,we treated breast cancer cells silenced by Sema4A with hypoxia stimulation and it was found that silencing Sema4A expression could significantly inhibit the proliferation activity of breast cancer cells,which was more obvious under hypoxia conditions.Annexin V data showed that silence of Sema4A had no significant effect on apoptosis under normal conditions.However,in the case of hypoxia,apoptosis was significantly increased when the Sema4A gene was knocked out.4.RhSema4A could protect apoptosis of breast cancer cells induced by hypoxia.In order to investigate the effect of soluble Sema4A on breast cancer cells,we applied rhSema4A to detect the effect of soluble Sema4A on the biological activity of breast cancer cells.The results showed that rhSema4A could promote the proliferation of breast cancer cells and further promote the proliferation of breast cancer cells under the stimulation of 1%O2.We also used severe hypoxia(0.2%O2)to induce apoptosis and observed the effect of rhSema4A on apoptosis.The results showed that rhSema4A could resist the apoptosis induced by hypoxia.Conclusion1.Sema4A is regulated by the expression of hypoxic HIF1 alpha in BCa.2.Sema4A can regulate the activity of BCa cells under hypoxic conditions.3.Exogenous Sema4A can protect BCa cells from cell damage induced by hypoxia.4.This study suggests that Sema4A can promote the progress of breast cancer under hypoxia conditions and it may hold potential for treatment target for BCa.