The Role Of AIMP2-DX2 In The Migration And Invasion Of Nasopharyngeal Carcinoma Cells

Background Nasopharyngeal carcinoma(NPC)is a kind of head and neck malignant tumors derived from nasopharyngeal epithelium,most of which are poorly differentiated squamous cell carcinoma with high degree of malignancy and ease to transfer.C hina is a country with high incidence of NPC,especially in the southern China.The treatment of NPC are radiotherapy and chemotherapy,and the early diagnosis is an effective way to save patients' lives and to improve the living quality.NPC occults and can be easily transferred,early metastasis and easy to relapse,75% of the patients are diagnosed with advanced NPC in the first check.The 5-year survival rate of patients with NPC is around 70%.Thus the elucidation of molecular mechanisms of carcinogenesis,recurrence and metastasis of NPC,the selection of biomarker of NPC and the identification target of NPC show extremely important clinical significance.Aminoacyl-t RNA synthetase interacting multifunctional proteins(AIMPs)are the scaffold proteins for human t RN A synthetase complex and the core proteins of acylt RNA ligase complexes.There are three isoforms of AIMPs: AIMP1,AIMP2 and AIMP3.AIMP2(also p38/JTV-1)is one of the AIMPs,participating in a variety of biological functions,such as cell differentiation and apoptosis.Recent studies showed that AIMP2-DX2,one of the transcripts of AIMP2 that lacks the exon2,is also involved in cancer development,but functions diversely.AIMP2-DX2 can be detected in a lot of cancer cells and tissues.Like AIMP2,AIMP2-DX2 can competitively bind to FBP,TRAF2 and P53 to block the function of AIMP2,showing the character of carcinogenesis factor.The increased ratio of AIMP2-DX2/AIMP2 is positively associated with the severity of lung cancer and associated with the chemoresistance of ovarian cancer.Inhibition of AIMP2-DX2 can suppress the differentiation of lung cancer cells and the tumor formation in nude mice.In all,AIMP2-DX2 is a potential target for the treatment of cancer.Matrix metalloproteinases(MMPs)are a group of zinc dependent protein enzymes,which are responsible for the degradation and remodeling of extracellular matrix proteins.The expression and activity of MMPs in adult tissues were relatively low,but increased significantly in tissues with abnormal patho logical conditions,such as inflammatory diseases,tumor growth and metastasis.In recent years,MMPs is a hot spot in the research of anti-tumor invasion and migration mechanism.Among the various MMPs members,MMP-2 and MMP-9 are closely related to the tumor invasion and migration phenotype,The main substrates are IV type collagen,gelatin,elastin,etc.they are the main components of basement membrane and ECM.The degradation of basement membrane means the invasion and migration of tumor.The activities of MMP-2 and MMP-9 in NPC tissues were significantly higher than those in normal nasopharyngeal tissues,and were closely related to the clinical stage of NPC.The results showed that inhibition of MMP-2 and MMP-9 could inhibit the invasion migration of nasopharyngeal carcinoma cells.Therefore,MMP-2 and MMP-9 play an important role in the invasion and migration of nasopharyngeal carcinoma cells,which are the key targets to inhibit the invasion and migration of NPC cells.Till now,the role of AIMP2-DX2 in NPC and the detailed mechanisms of AIMP2-DX2 regulated cancer metastasis remain obscure.In the current study,in a highly aggressive NPC cell line CNE-1,by molecular cloning,transfection,q PCR and RTPCR,western blot,flow cytometry,MTT and transwell approaches,and in nude mice,we aim to determine the impact of AIMP2-DX2 on cell growth,migration and invasion of NPC cells,to detect the levels of MMPs and determine the relationship of AIMP2-DX2 and MMPs,thus to elucidate the related mechanisms of AIMP2-DX2 involved NPC development.Objective(1)To determine the correlation of AIMP2-DX2 and NPC cells migration and invasion in cell model and nude mice model;(2)To determine the level changes of MMP-2/9 with different AIMP2-DX2 treatment,trying to elucidate the related mechanism.Methods(1)By IHC,western blot and q RT-PCR,we evaluated the levels of AIMP2-DX2 and AIMP2-F(ulllength)in normal nasopharyngeal and NPC tissues;(2)By western blot,RT-PCR and transwell assay,we compare the migration and invasion ability of NPC cells with normal nasopharyngeal cells;By constructing AIMP2-DX2 overexpression plasmids,to elucidate the role of AIMP2-DX2 overexpression on NPC cell growth,migration and invasion and apoptosis;(3)By constructing sh AIMP2-DX2 lentivirus and CNE-1 stable cell line,to determine the effect of AIMP2-DX2 knockdown on on NPC cell growth,migration and invasion;(4)By nude mice model,we determine the impact of AIMP2-DX2 knockdown on NPC cell growth in vivo,with sh AIMP2-DX2 CNE-1 cell injection;(5)All statistical analyses were performed using the SPSS 19.0 statistical software package.Data are expressed as the mean ± standard deviation.Statistical significance was determined by an analysis of variance.P?values of <0.05 were considered to indicate a statistically significant difference.Results(1)Compared with normal nasopharyngeal tissue,NPC tissues showed higher levels of AIMP2-DX2,there are significant difference between normal nasopharyngeal tissue and NPC tissues(<0.05);with no changes of AIMP2-F;The 5 year survival rate of AIMP2-DX2 low expression group was higher than that of AIMP2-DX2 high expression group,and the difference between them was statistically significant(P=0.018),suggesting that the expression of AIMP2-DX2 was related to the prognosis of NPC patients;(2)The results from transwell assay showed that the ability of cell invasion and migration is associated with AIMP2-DX2,by AIMP2-DX2 overexpression and knockdown experiments;(3)Compared with Immortalized nasopharyngeal epithelial cells line,The nasopharyngeal carcinoma cells line of m RN A and protein levels of AIMP2-DX2 were significantly increased,along with the increased levels of MMP-2/9,increased ability of migration and invasion,there are significant difference between Immortalized nasopharyngeal epithelial cells line and nasopharyngeal carcinoma cells line(<0.05);(4)qRT-PCR and WB results showed that sh-AIMP2-DX2 lentivirus suppressed the expression of AIMP2-DX2 and inhibited the CNE-1 growth,migration and invasion,and inhibited the levels of MMP-2/9,there are significant difference(<0.05);(5)In nude mice model,the tumor formation of CNE-1 cells was suppressed by sh-AIMP2-DX2.Conclusion(1)AIMP2-DX2 is significantly increased in NPC patients and is correlated the prognosis;(2)AIMP2-DX2 is necessary and sufficient for the growth,migration and invasion of NPC cells;(3)Inhibition of AIMP2-DX2 suppresses the level of MMP-2/9,indicating that MMP-2/9 functions downstream of AIMP2-DX2;(4)Inhibition of AIMP2-DX2 suppresses the tumor growth in nude mice model.