Experimental Study On The Downregulation Of EIF4E Expression Via Survivin Promoter-mediated RNA Interference In Laryngeal Squamous Cell Carcinoma

Objective Eukaryotic initiation factor eIF4E, an important regulator of translation, is overexpressed in a variety of human head and neck squamous cell carcinoma(HNSCC) cells and tissues, while the high levels of eIF4E expression is inversely correlated with prognosis. Morever, the overexpression of this gene is associated with tumorigenicity, development and chemoresistance of laryngeal carcinoma. To tesitify the tumor specificity and high efficacy of survivin promoter-mediated RNA interference system, survivin promoter-mediated RNAi expression vector targeting EGFP gene was transfected into various head and neck squamous cell carcinoma cell lines and normal blood vessel endothelium cell line, respectively. Secondly, survivin promoter-mediated siRNA eukaryotic expression vector targeting eIF4E was constructed and then transfected into laryngeal carcinoma cells(Hep-2). To explore the possibility of eIF4E as an therapeutic target for the treatment of laryngeal carcinomas, the effects of eIF4E downregulation on in vitro and in vivo proliferation, cell cycle, apoptosis and chemosensitivity of laryngeal carcinoma cells were evaluated.Methods (1) The survivin promoter gene was cloned from pGL3-surP vector by RT-PCR method, then sequenced and analyzed in Genbank. Then, the survivin promoter gene and in vitro-synthesized mpA sequence were inserted into pSUPER.retro.puro vector, respectively and confirmed by the digestion analysis of restriction endonuclease. (2) siRNA oligonucleotide sequence was designed and synthesized accoding to the EGFP cDNA sequence. The siRNA oligonucleotide was annealed and inserted into the downstream of survivin promoter. Then, the cmvP-EGFP-polyA expression frame was coloned from pEGFP-C1 vector by PCR and then inserted between EcoRI and NheI restricted enzyme sites of pSUPER.retro-pur vector. The reconstructed vector was named pS-surP-shEGFP-mpA-cmvP-EGFP-polyA. The vector was transfected into human nasopharyngeal carcinoma cell line(CNE-2), laryngeal squamous cell carcinoma cell line(Hep-2),thyroid squamous cell carcinoma cell line(SW579) and normal vascular endothelial cell(ECV304). 72h after transfection, the cells were photographed by using a fluorescence microscope. RT-PCR and flow cytometry assays were performed to detect the levels of EGFP mRNA and the levels of EGFP fluorescence using fluorescence-activated cell sorting. (3) Two siRNA olignucleotides were designed and in vitro synthesized, and the target sequence homology with other genes was analyzed by BLAST search. Then, those olignucleotides were annealed and inserted into pS-surP-shRNA-mpA vector, respectively. Followingly, the siRNA expression vectors were transfected into Hep-2 cells using Lipofectamine 2000. Stable cell lines were selected with G418 (600μg/mL)48h later after transfection, and individual clones were isolated and maintained in a medium containing G418(100μg/mL). RT-PCR and Western blotting assays were performed to detect the levels of eIF4E mRNA and protein expression, respectively. The stable transfectant showing downregulated-eIF4E expression of was chose for further assays. (4) Western blotting was performed to detect the changes of VEGF, FGF-2 and cyclinD1 protein expression in stable transfectants. MTT and colony formation assays were performed to evaluate effects of eIF4E/shRNA2 on the in vitro cell proliferation of Hep-2 cells. Flow cytomatery and TUNEL staining assays were performed to detect the changes of cell cycle and apoptosis. The activity of caspase-3 was determined and the possible mechanism of apoptosis was explored. Tumorigenicity and tumor inhibition assays were performed to detect in vivo proliferation. In vitro and in vivo chemotherapy assays were performed to evaluate the effect of eIF4E downregulation on the chemosensitivity of Hep-2 cells to cisplatin.Results (1) The recombinant vector pS-surP-shRNA-mpA including 980bp survivin promoter gene and interrupted signal mpA were successfully constructed. (2) 72h after transfection, RT-PCR results showed that the levels of EGFP mRNA expression in CNE-2, Hep-2 and SW579 cells were significanty inhibited compared with that in ECV304 cells. (3) The levels of eIF4E mRNA and protein expression in Hep-2-s2 cells were decreased by 68.5% and 59.6%, respectively, but there were no obvious difference in Hep-2-s1. Hep-2-s2 cells were chose for further assays. (4) The levels of VEGF, FGF-2 and cyclinD1 protein expression were significantly inhibited in Hep-2-s2 cells. The proliferation of Hep-2-s2 cells was also obviously decreased and the highest inhibitory rate was 48.2±1.6% on day 7(P<0.01). The colony numbers of Hep-2-s2 cells were significantly reduced (averaged number=342, P<0.01). The percentage of Hep-2-s2 cells in the G0/G1 phase was increased and that in the S phase was decreased. The activity of Caspase-3 showed obvious enhancement. The high levels of c-IAP1, c-IAP2 and c-myc protein expression but not Bcl-2 family protein expression were involved in the process of apoptosis. The tumor volume developed from Hep-2-s2 cells(averaged size=233.5mm3)was obviously smaller than that developed from control cells. Moreover, pS-SP-eIF4E/shRNA2 could significantly inhibit the proliferation of in vivo tumor. eIF4E/shRNA2 also could lead to in vitro and in vivo chemosensitivity enhancement in Hep-2 cells.Conclusions (1) Survivin promoter-mediated RNA interference system could silence endogenous gene expression with tumor specificity and high efficacy. (2) The downregulation of eIF4E expression could reduce the levels of downstream target gene expression and lead to proliferation inhibition both in vitro and in vivo. The Hep-2 cells stably expressing eIF4E/shRNA2 showed cell arrest in the G0/G1 phase and obvious apoptosis induction. The downregulation of this gene could also enhance chemosensitivity of Hep-2 cells to cisplatin. (3) Combination use of survivin promoter and RNA interference provides a novel strategy for the targeted therapy of human laryngeal carcinoma. Specific silencing of eIF4E gene provides a new approach for exploiting novel therapeutic target and reversing chemoresistance phenotype of laryngeal carcinoma.