SiRNA Silencing EIF4G1 Expression Influences The Growth And Invasion Of NPC

BACKGROUND & OBJECTIVENasopharyngeal carcinoma(NPC) is a common malignant epithelial tumor in Southern China.Because the primary anatomical site of tumor growth is located in a cryptic area,with slightly early symptoms,the NPC patients tend to present a more advanced stage of disease in clinic with higher metastatic potential.So finding the mechanism of the NPC tumorigenicity and metastasis is still our main research direction.Tumor formation and metastasis is a complex and continuous process that needs multi-step,multi-stage,multi-factor involving in a number of key oncogenes and tumor suppressors.In the previous study,we used cDNA microarray to detect differentially expressed genes among NPC tissues,pooled NPC cells(5-8F,6-10B and CNE2) and non-cancerous nasopharyngeal tissues.By means of the analysis of software BRB,markedly upregulated expression of eukaryotic translation initiation factors-EIF4G1 was showed in NPC tissues and NPC cells,which suggested that this gene might promote the pathogenesis of NPC.EIF4G1 serves as a scaffold for several other initiation factors to play their respective roles.Recent studies indicated that EIF4G1 not only promoted protein translation but also was closely related to cancer incidence and progress including promoting tumor angiogenesis,cell malignant transformation and inhibiting cell apoptosis..Its significantly upregulated expression has been reported in larynx cancer, lung cancer,breast cancer.In order to clarify the role of EIF4G1 in the pathogenesis of NPC,we used RNAi technology to specifically interfere EIF4G1 expression in NPC cells,and observed the alteration of the biological function of NPC cells.This investigation may provide new ideas for seaching the pathogenesis and the treament of NPCMETHODS1.Expression characterization of EIF4G1 in NPCImmunohistochemisty(IHC) method was used to detect the EIF4G1 expression in non-cancerous nasopharyngeal tissues and NPC tissues for preliminary study the relationship between EIF4G1 and the occurrence of NPC.Fluorescence quantitative PCR methods was used to detect the EIF4G1 expression in immortalized nasopharyngeal epithelial cell NP69 and NPC cells 5-8F,6-10B,C666-1,CNE1,CNE2,HNE1,HONE1,SUNE1 in the mRNA level. Compared with NP69 cells,NPC 5-8F cells with the high ablility of tumorigenesis and metastasis showed the highest mRNA expression level of EIF4G1.2.The influences on cell biology of NPC by EIF4G1 silenceConstructing EIF4G1 interference and blank lentivirus expression vectors to infect 5-8F cells.The cell clones with stably expressing anti-EIF4G1 shRNA and blank vectors were picked out.Fluorescence quantitative PCR was used to detect the interfere efficiency of each cell clone.The cell clone with highest interference efficiency would be selected as the next experiment object.Western-blot was utilized to examined the protein expression level after EIF4G gene was silenced in 5-8Fcells. MTT,flow cytometry and colony forming experiments were used to detect cells proliferation in vitro;Transwell and Boyden chamber were employed to detect cells migration and invasion in vitro;Subcutaneous xenograft experiment in nude mice was utilized to observe tumorigenicity ability in vivo.3.Statistical methodsWe use SPSS 13.0 statistical software package for statistical treatment,In ChapterⅠ,Mann-Whitney U test was used to analyze the immunohistochemistry result of protein expression between NPC and non-cancerous nasopharyngeal tissue. Kruskal Wallis Test was utilized to evaluate the relationship between EIF4G1 expression and pathological differentiation of NPC.QRT-PCR detection results was analyzed using one-way ANOVA,and multiple comparison test using SNK.In ChapterⅡ,QRT-PCR,migrate,invasion assay,fiat colony formation and cell cycle experiments were tested by one-way ANOVA,MTT results using factorial design analysis of variance,multiple comparisons test using SNK or LSD test;Transplanted subcutaneously in nude using paired samples t testRESULTS1.Expression of EIF4G1 and its clinical significance in NPC1) 16 cases of non-cancerous nasopharyngeal tissue and 40 cases of NPC specimens were collected.The results of IHC showed that EIF4G1 mainly located in cellular membrane and cytoplasm.There is significantly differentiation between NPC and non-cancerous nasopharyngeal tissues(Z=—3.971,P=0.000);No correlation was found between EIF4G1 expression and clinicopathological differentiation(χ~2 =2.508,P=0.285)2) The results of fluorescence quantitative PCR showed EIF4G1 expression with statistical significance(F=31.575,P=0.000) in immortalized nasopharyngeal epithelial NP69 cells and NPC cells 5-8F,6-10B,C666-1,CNE1,CNE2,HNE1, HONE1,SUNE1.Compared with NP69 cells,NPC 5-8F cells showed the highest expression of EIF4G1(19.5-fold) in all 8 NPC cell lines.So we chosed 5-8F cells as the interference object for further experiment study.2.The influences on cell biology of NPC by EIF4G1 silence1) The lentiviral vectors containing specific anti-EIF4G1 shRNA and negative control were transfected into 293FT cell line to produce lentivirus particles, followed by transduced into 5-8F cell line.After selected by blasticidin for 14 days,50 resistant cell clones were picked out,fluorescence quantitative PCR was used to detect the interfere efficiency for each clone,clone1 with the highest efficiency was chosen for the following study.There is markedly differentiation between positive clone and negative control clone,5-8F in EIF4G1 expression (F=11.897,P=0.000).Interference clone was named 5-8F/pshRNA-EIF4G1~-, negative control clone named 5-8F/pshRNA.western-blot results showed that reduced EIF4G1 expression for 80.1%in 5-8F/pshRNA-EIF4G1~-.2) A significantly time-dependent inhibitory proliferation was found in 5-8F /pshRNA-EIF4G1~-cells compared with 5-8F/pshRNA and 5-8F by in vitro MTT assay(F=131.448,P=0.000);Flow cytometry results showed significant differentiation among the three cells in S phase and G2/M phase change (F=22.426,P=0.002 and F=7.354,P=0.024).EIF4G1 silence effected cell cycle and prevented S phase cells into the G2/M phase,;In addition, 5-8F/pshRNA-EIF4G1~-cells had a significant impaired ability to form colonies in plates,as compared with 5-8F/pshRNA and 5-8F cells(F=54.615,P=0.000).The results indicate that EIF4G1 silence suppressed proliferation of 5-8F cells.3) 5-8F/pshRNA-EIF4G1~-cells had a significantly reduced ability of migration and invasion as compared with 5-8F/pshRNA and 5-8F cells detected by Transwell and Boyden chamber assay in vitro(F=38.329,P=0.000 )and(F=6.170,P=0.014)4) 5-8F/pshRNA- EIF4G1~-and 5-8F / pshRNA cells were inoculated with the upper limb and lower limb axillary inguinal in nude mice for 21 days.The result of tumor weight showed significant difference in two cells(P=0.032,P=0.041), EIF4G1 expression was significantly reduced in interference group xenograft compared to control group xenograft by IHC detection.CONCLUSION1) Overexpression was showed in NPC tissuse compared with non-cancerous nasopharyngeal tissues by IHC detection.2) Down-regulated EIF4G1 expression not only suppressed cells proliferation in vitro and in vivo,but also inhibited cell migration and invasion.EIF4G1 may act as an oncogene in NPC pathogenesisi.