Induction Of Chemo-sensitization In Laryngocarcinoma By Ectopic Expression Of RKIP Gene

Head and neck squamous cell carcimoma (HNSCC) is a common malignant tumor in human body, which exhibits rapid growth, strong invasiveness and metastasis and has a high rate of recurrence. Carcinogenesis is a complicate staging process. From the point of view in molecular biology, major pathogenesy is reinforcement of oncogene and metastasis gene activity or inactivation of anti-oncogene and metastasis suppressor gene in human body interacting with various extent and internal factors. Combined radiotherapy and chemotherapy with operation has become a chief method and tendency for this disease. But management of HNSCC is a puzzled question in tumor therapy right along for its low sensitivity and strong resistance to chemotherapy. Meanwhile, the side effect and injury to normal tissue caused by chemotherapy can not be ignored. Therefore, it is urgent to find chemo sensitizer to enhance tumor chemo-sensitization and decrease injury in normal tissues. Due to the advantages of gene therapy, which include uneasy to produce drug resistance and strong specificity, it offers hope in this regard. Gene therapy involves the introduction of gene DNA into cells to exert effects by vector, which will selectively kill cancer cells with no toxicity to the surrounding non-malignant cells. The key point of gene therapy is the choice of target. Through such a process we can study and choose effective, low toxic cure medicine and method for oncotherapy, it is a new direction of oncotherapy to combine orthodox chemotherapy with gene therapy.Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine binding protein family, a ubiquitously expressed and evolutionarily conserved group of proteins. In recent years, it has been reported that RKIP influence intracellular signaling cascades, cell cycle regulation, the suppression of metastasis, neuro generative processes, the modulation of emotions, and reproduction, and RKIP has been identified as a member of a novel class of molecules that suppress the metastatic spread of tumors and enhance tumor chemo-sensitization, but little is known about the role and the regulating mechanisms of RKIP in laryngocarcinoma.RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing, resulting in the corresponding gene deletion phenotype. It is a hot spot in functional genomics investigation which develops gradually to become a critical technique for defining gene function.In this study, we transfected recombinant plasmid to Hep-2 cell line with liposome to investigate the therapeutic value and possible mechanism of laryngeal carcinoma by regulating RKIP combined with DDP. Therefore, a theoretical basis and new utilization may be provided on enhancing combined therapeutic efficacy and improving prognosis of head and neck malignant tumors.Objective: (1) To construct the recombinant plasmid of RKIP short hairpin RNA and high efficiency expression and to verify their efficacy. (2) To observe the effect of RKIP expression levels on cell proliferation, cell cycle, apoptosis, invasiveness and migration in Hep-2 cells, and investigate generation pathogenesy. (3) To observe the possibility of reducing laryngocarcinoma chemotherapy resistance and enhancing laryngocarcinoma sensitivity to DDP after high efficiency expression RKIP combined with DDP demonstrated by the hyperplasia and apoptosis of laryngeal squamous carcinoma cell, and investigate generation pathogenesy.Method: (1) RKIP short hairpin RNA and high efficiency expression vector were constructed by molecular cloning technique and identified by enzyme digestion and sequencing. After transfection of Hep-2 cells with recombinant plasmid, transfection efficiency was observed and deteced by fluorescence microscope and FCM. Quantitative reverse transcriptase-polymerase chain and Western blotting were used to detect RKIP mRNA and protein expression. (2) After transfection of Hep-2 cells with recombinant plasmid, cell proliferation was determined using MTT method. Changes of cell cycle and apoptotic rates were measured by flow cytometry. Cell invasiveness was evaluated by Matrigel invasion assay. Western blot detected the effects of different RKIP levels upon MAPK pathway. (3) After transfection of Hep-2 cells with high efficiency expression vector, DDP is combined. Cell proliferation was determined using MTT method. Changes of cell cycle and apoptotic rates were measured by flow cytometry. Western blot detected the effects of combining RKIP-pIRES2-EGFP with DDP upon MAPK pathway.Result: (1) The recombinant plasmids were constructed successfully and confirmed by restiction enzyme digestion and sequencing results. After transfection with recombinant plasmid, green fluorescence were observed in Hep-2 cells under fluorescence microscope. And efficiency deteced by FCM was 81.53% at 24 hour post-transfection. Quantitative reverse transcriptase-polymerase chain and Western blotting were used to detect RKIP mRNA and protein expression. Treatment of Hep-2 with recombination plasmid resulted in a significant decrease or increase of RKIP expression at mRNA and protein level, which was more prominent in RKIP-shRNA501 group. (2) MTT assay demonstrated that cell growth was inhibited or promoted in transfected cells at 24 hour post-transfection. Cell growth decreased in high efficiency expression group, it is opposite in short hairpin RNA group, and 24 h, 48 h or 72 h growth inhibition or promotion rates of recombinant plasmid-transfection group were significantly different from that of the control group. FCM results showed that Hep-2 cells of high efficiency expression vector group increased in G1 stage, but Hep-2 cells of short hairpin RNA expression vector group decreased in G1 stage. Cell cycle was different between recombinant plasmid group and control group. Analysis of cell apoptosis showed obvious apoptotic peak in the cells transfected with high efficiency expression vector, but no obvious apoptotic peak was found in short hairpin RNA group. Matrigel invasion assay showed that cell invasiveness decreased in high efficiency expression group, it is opposite in short hairpin RNA group. Cell invasiveness was different between recombinant plasmid group and control group. Western blot detect the effects of different RKIP levels upon p-ERK protein. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP such as proliferation, cell cycle, apoptotic, invasiveness. (3) After transfection of Hep-2 cells with recombinant plasmid, DDP is combined. MTT assay demonstrated that cell growth was remarkably inhibited in RKIP-pIRES2-EGFP+DDP group. Cell growth is lower than that in any other three groups. FCM results showed cell apoptotic rate was significantly increased in RKIP-pIRES2-EGFP+DDP group. Cell apoptosis in RKIP-pIRES2- EGFP+DDP group is higher than that in any other three groups. Western blot detect the effects of combining RKIP-pIRES2-EGFP with DDP upon p-ERK protein. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP-pIRES2-EGFP combined with DDP.Conclusion:(1) RNAi to silence RKIP gene could down-regulate the express-on of RKIP and promote effect of hyperplasy and invasion level in Hep-2 cell. Applying molecular cloning technique to express RKIP, the situation is opposite. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP such as proliferation, cell cycle, apoptotic, invasiveness.(2) The high efficiency expression of RKIP could not completely inhibit the hyperplasy of tumor cells, and the apoptotic-inducing effect was limited. The high efficiency expression of RKIP could coordinate with therapeutical effect by DDP. It could enhance the inhibition effect of hyperplasy in Hep-2 cell and promoted the apoptosis of laryngeal carcinoma cells, which indicates the high efficiency expression technique targeting RKIP might become a new strategy of gene therapy in laryngocarcinoma. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP-pIRES2-EGFP combined with DDP.