The Influence Of ENKUR In Nasopharyngeal Cancer On Proliferation,Invasion And Metastasis

Nasopharyngeal carcinoma(NPC)is a malignant tumor generated from the nasopharyngeal mucosae,which is common in the southern provinces of China,especially in the Guangdong and Guangxi.Thus,NPC is usually called Cantanese cancer.lt usually had a poor prognosis because of latent tumorigenesis,high malignancy and strong metastasis.The Clinical stage Ⅰ/Ⅱ NPC 5 years of survival rate can reach more than 72%to 90%,but the stage Ⅲ/Ⅳ has decreased to 55%and 55%.Metastasis contributes mostly to the poor prognosis of NPC patients.Worsely,an ocean of patients have the poor destinies of to be and can only be diagnosed with adcanced NPC because of early metastasis and latent tumorigenesis,which also makes to the terrible survival rate.Therefore,it is imminent to make a better annd more effecteive diagnosis of NPC.However,metastasis is developed from a complex mechanism of diverse stages,varieties of routes and numerous signaling pathways.Although thanks to the rapid development of molecular biology techniques and clinical therapies,the treatment of NPC.especially the molecular targeted therapy has made great progress,we hardly had made a clearunderstanding of the pathogenesis mechanism of NPC.Fortunately,we can shed a light on the related genes and signaling pathways to explore the tumorigenesis mechanisms of NPC,because the imbalance of the activation of oncogene and inactivation of tumor supressor gene may had vital functions.Enkurin,whose relative molecular weight is29 kDa,encoded by the ENKUR in the 1 Op 12.1 chromosome,is composed of 8 exons,3378 bp long.ENKUR is discovered from a study in the sperm TRPC(transient receptor potential)activity and Ca2+channel coupling mechanism in 2004 by using yeast two hybrid screening.lt is highly expressed in testis and Vomeronasal organ and rare in other organs.ENKUR has obvious modular constructures,for there are three domains in it:The C terminal region interacts with TRP channels,Ca2+sensor combined with IQ motif,and N terminal linked SH3 domain protein rich in proline[1].The C terminal related TRP channels(TRPC)of ENKUR contain of TRPC1,TRPC2,TRPC5,but not TRPC3.TRPC,belonging to the TRP superfamily and widely expressed in nervous system,is an important ion channel of balancing the Ca2+ steady state.TRPC can be activated by a variety of chemical and physical stimuli(such as neurotxansmitters,hormones,temperature changes and mechanical stimulation,etc.)IQ motif is usually combined with GAP2,Ras,GRF1 and Myr4myosin to form a complexus,which can be connected to calmodulin.The calmodulin connected to IQ motif of Enkurin regulates the Ca2+signaling pathway and it may turn on/off the TRPC by targetting the collecting or releasing of Ca2+[1].The N-terminal of ENKUR contains about 100 residue rich in proline.In vitro studies have shown that this part covers the strongly-binded regulatory subunit of p85 kinaseor phosphatidyl inositol 3 kinase(PI3K)and the Ioose-binded SH3 domain[3]P85 regulatory subunit composes of p85α,P85β and p85γ,while p85α represents the maximum expression of these three parts.Moreover,the heterodimer combined by the p85 regulatory subunit and the p110 catalytic subunit can be transformed to be the type IA PI3K[4].PI3K is a specific kinase catalying phosphatidylinositol.In general cases,its activation products are pl(3.4)P2 and pl(3.4.5)P3,which play the role as a second messenger interacting with several intracellular targetted proteins to become a signaling cascade complexus.This complex makes can regulate some proliferation,differentiation.survival and motility.SH3 domain is an eukaryotic protein domain that can make the signaling tranduction in the form of protein complexus by binding to the tyrosine kinase phosphorylation residues strongly.SH3 domain is involved in a variety of biochemical reaction process inside the cells,including the cell movement,signal transduction and proliferation.There are three types of PI3Ks in the mammals.What attracts most is the type IA PI3K and its downstream serine/threonine kinase AKT(or PKB)signaling pathways on account of the tight connection to the tumorigenesis and progression.The abnormal activation of PI3K/AKT signaling pathways usually results in the malignization of cells.What’s more,not only does it modulate the proliferation and survival of tumor,but also the cell migrtion,adhesion.angiogenesis and the degradation of extracellular matrix[5].AKT regulates the cell proliferation and apoptosis by targetting the cyclin-dependent protein kinases(CDKs)inhibitor indirectly[6,7].The cell cycle is significantly inhibited when P15,P16,P21 upregulated.TAngiogenesis plays a key role in tumor metastasis,while PI3K shares some duties in the vascular endothelial growth factor(VEGF)mediated signaling pathway by targetting the β-catenin[8].The activation of PI3K/Akt plays key roles in the EMT of tumorigenesis,which down regulates the E-cadherin to promote tumor metastasis[9]All in all,the PI3K/AKT signaling pathway is important to keep the normal functions of cells,while the abnormal activation of it may indicate poor prognosis of tumor..The cell cycle including interphase and divison stage composes of four parts:G0/G1,S,G2 and M.Interphase can be divided into three parts:the first gap of DNA synthesis(G1 phase),synthesis(S)and the second gap of DNA synthesis(G2)[10].GO phase is supposed to be detached from the cell cycle,when the cell is regraded as still.And the GO cells such as hepatocellulars,thyroid follicular epithelial cells and myocardial cells can recycle when needed.For this respect,tuomr cells may also be considered as GO cells.The G1 phase works for the preparations for DNA synthesis.The S phase must be the most important phase becuase it stands for the synthesis of DNA in the nuclear.The protein synthesis is usually completed in the G2 or the mitosis prophase.Lastly,M stands for the mitosis.All the development of life should have the foundation of cell cycle[11].A specific serine/threonine kinase called Cyclin-dependent protein kinases(CDKs)is the key modulating point of cell cycIe.CDKs can phosphorylate the serine/threonine protein of relevant substratse to drive the cell cycle to the next.The CDKs family is strictly regulate in time phases[12];There are 10 types of known CDKs proteins.Among them,CDKl(cdc2),CDK2,CDK4 and CDK6 may play some keys roles in cell cycle.The CDKs can be classified as GI acting protein(cyclinC,D,E),G2 acting protein(cyclin A)and M acting protein(CyclinB)[14].The G1 acting protein CyclinDl can binds to CDK4 and CDK6,which helps to turn cells from G1 to S.Therefore,tumor overexpressing CyclinD1 may be more sensitive to radiation therapy on researches by Trent S et al[15].Besides,eyclin-dependent protein kinases inhibitors,such as P15,P16,P21,have draw much attentions.They have close association with tumor supression because they can inhibit the CDks and regulate cell cycle diretly simultaneously.Among them,P21 participates in the proliferaion and senility of cells by inhibiting several types of CDKs;And P15,P16 may stagnate the cells in Gl phase by blocking down CDK4 and CDK6[16].Ectomesenchymal transformation of Epithelial cells or Epithelial Mesenchymal Transitions(EMT)refers to the the progress of epithelial cells transforming into mesenchymal cells in some specific ways.EMT contributes greatly in some physiological processes,such as embryonic development,chronic inflammation,tissue reconstruction,cancer metastasis and a variety of fibrosis diseases.The main features of EMT suppose to be down regulations of some cell adhesion molecules(such as E-cadherin)and up regulations of cytoskeleton proteins(such as Vimentin),which is also the characteristics of mesenchymal cells.t[17-15].Epithelial-to-mesenchymal transition(EMT)is a morphogenetic process in which cells lose their epithelial characteristics such as cell polarity and cell-cell contact,and gain mesenchymal properties such as increased motility.EMT also endows cells with invasive properties,induces stem cell properties and prevents apoptosis and senescence.EMT is viewed as an essential early step and a critical process during tumor metastasis[16-23].It is urgent and effective to shed a light on EMT to explore the tumorigenesis and progression mechanism of NPC,which may also helps to find some exciting target of early diagnosis and therapy.ENKUR was screened out to be the research target when our resarch team studying for the possible interaction gene with NESGI by yeast two hybrid experiment.NESG1 should be a possible tumor supressor gene for it may downregulate metastasis,inhibit proliferation and prevent cell cycle turnning from G1 to S in vitro in the study of NPC.[24].Frustratingly,our team has found out that there is no interaction between ENKUR and NESG1.Nevertheless,when analyzing documents,there are few researches about ENKUR,especially in the tumor.What’s more,because of its tight connection of p85 and PI3K regulatory subunit,we deduce ENKUR may play some great roles in the tumorigenesis and progression,no matter promoting or inhibiting.In a way,in order to break for further studies,we supposed to explore the interaction between ENKUR and the metastasis or proliferation of NPC from the basic cell function invitro and then the mechanism.Research purpose1,Research the expression characteristic of ENKUR in NPC.2,Establish stable overexpression ENKUR NPC cell line.3,Explore the impact of ENKUR on proliferation in NPC.4,Probe into the interaction between ENKUR and metastasis or invasion of NPC.Content and methods1,Research the expression characteristic of ENKUR in NPCUsing RT-PCR to detect the expression of ENKUR cDNA respectively in 23 cases of nasopharyngeal carcinoma tissues and 25 cases of normal nasopharyngeal tissues,and the mRNA of ENKUR in nasopharyngeal carcinoma cell lines SUNE-1,5-8F,CNE-1 and CNE-2,HONE-1,HNE-1,6-1 OB,C666-1,and the non-NPC cell lines NP-69.2,Establish stable overexpression ENKUR NPC cell lineTransfecting NPC cell line SUNE-1 and HONE-1 with recombinant lentivirus vector containing CDS sequence of ENKUR.Western-Blot analysis were used to examine the expression of ENKUR gene in ENKUR overexpressed NPC cells.3,Explore the impact of ENKUR on proliferation in NPC(1)MTT assays were used to detect the proliferation of stably ENKUR overexpressed NPC cells in vitro.;(2)EDU assays were used to examined the proliferation and the pecentages of cells in S phase in the stably ENKUR overexpressed NPC cells in vitro;(3)Using flow cytometry to detect proportions of the ENKUR overexpressed cells in S phase after stably tansfected with lentivirus;(4)Implmenting the subcutaneous tumorigenity experiment in nude mice to exam the proliferation potiential of ENKUR stably overexpressed NPC cells in vivo;(5)Western-blot analysis to exam the changes of related gene and signaling pathways mechanisms of ENKUR overexpressed NPC cells;(6)Transfected synthetic siRNA targeting ENKUR gene into NPC cells with ENKUR overexpressed NPC cells.Using RT-PCR and Western-blot to examine the effectiveness of RNA interference.MTT and EDU are used to dectect the proliferation of interferenced ENKUR NPC cells in vitro.And Western-blot analysis are used to exam the related changes of gene and signaling pathways in the silenced ENKUR NPC cells.4,Probe into the interaction between ENKUR and metastasis or invasion of NPC(1)Transwell/boyden assays to dectect the metastasis and invasion potential of the ENKUR overexpressed NPC cells.(2)Wound healing assays to exam the migration potential of the ENKUR overexpressed NPC cells.(3)Western-blot analysis to detect the EMT-related gene and signaling pathways mechanisms of ENKUR overexpressed NPC cells.(4)Transfected synthetic siRNA targeting ENKUR gene into NPC cells with ENKUR overexpressed NPC cells and the above-mentioned assays will be used to exam the metastasis and invasion potential of the silenced ENKUR NPC cells.Statistical analysis:Using SPSS 17.0 statistical software for data analysis.With+s measurement data results.Fluorescence quantitative PCR in each cell sample 2-delta delta Ct value comparison using single factor analysis of variance(One-way AVOVA);Ratio between control group and treatment group,the number of cell membrane cell,cell spacing compares the two independent samples t-test;With P<0.05 for the difference is statistically significant.Results1.From the RT-PCR experiments,it can be concluded that ENKUR is high expressed in the normanl tissus and down expressed in the NPC tissues.The data were analyzed by One-way AVOVA and significant in statistics.2.Selected NPC cells were transfected with lentivirus carrying ENKUR.Western-blot analysis shouwn that they are successfully stably transfected,which is of vital importance to the next experiments.3.ENKUR inhibits the proliferation of NPC cells and regulates the related signaling pathays.(1)MTT assays shown that ENKUR supresseed proliferation in NPC cells significantly;(2)EDU and flow cytometry experiments have prove that ENKUR can inhibit NPC cells turning From G1 to S compared to negative control group,which leads to a down regulation in the proliferation;(3)Subcutaneous tumorigenity experiment in nude micein nude mice showed that the tumor from ENKUR group are smaller in volume and lighter in wieght compared to the negative control group,which gave strong evidences that ENKUR can supressed prolifertion of NPC cells in vivo.And the data are statistically significant.;(4)Western-blot showed that ENKUR can down regulate some key gene in the cell proliferation signaling pathways.4、ENKUR promotes cancer cell migration and invasion in nasopharyngeal carcinoma and its molecular mechanism(1)Transwell and Boyden assays proved that ENKUR might supress metastasis and invasion in the ENKUR overexpressed NPC cells compared to the negative control group;(2)Wound healing assays strenghtened the opinion that ENKUR overexpressed NPC cells gained a weakened ability of migration and metastasis compared to the negative control group;(3)Western-blot assays show that ENKUR may modulate some EMT-related signaling pathways to inhibit metastasis and invasion,such as Wnt/β-cantenin signaling pathway.conclusionFrom the aboved-mentioned experiments,we can conclude that ENKUR can supress the proliferation,metastasis and invasion potential of NPC cells in vivo and in vitro.ENKUR may regulate the proliferation of NPC cells by targetting the PI3K/AKT signaling pathway,while it can modulate the metastasis and invasion by targetting the EMT-related Wnt/p-catenin signaling pathway.In this way,ENKUR can be regarded as a tumor supressor gene in the NPC and this may become a highlighted therapy target.