The Inhibitory Effects Of Downregulation Of Jab1 By RNA Interference On Tumour Growth Of Human Laryngeal Carcinoma

The tumourigenesis of laryngeal carcinoma is a multiple-stages process involving in multiple genes. The molecular mechanisms are very complicated. Surgery and radiotherapy are common therapies for laryngeal carcinoma, although surgical therapy has a high incidence of complications resulting in disability and radiotherapy therapy always results in serious side-effects. Therefore, it is necessary to seek for new approaches for treatment of this cancer, which have been becoming the limelight in the field of head and neck cancer.Specifically blocking and suppressing expression of tumour-associated genes at the levels of transcription, translation and post-translation can induce differentiation or/and apoptosis of tumour cells. RNA interference (RNAi) is a phenomenon of sequence-specific gene silencing in mammalian cells and is a powerful tool in post-genomic research. RNA interference silences genes with a high degree of specificity and may provide a basis for molecularly targeted anticancer therapy. Recently, RNA interference (either siRNA or shRNA), has been experimentally introduced as a cancer therapy and is expected to be developed as a nucleic acid-based medicine. Jabl (Jun-activating domain binding Protein 1), the fifth component of the COP9 signalosome complex (CSN), was originally identified as a co-activator of c-Jun. It was confirmed later as a coactivator of AP-1 transcription factor. It has recently been demonstrated that Jab1 is involved in the mediation of nuclear exportation and degradation of the tumor suppressor genes, such as p27Kip1, p53, and Smad4. Jabl/CSN5 is a potential oncogene and a regulator of progression in different cancers.p27kip1, a cyclin-dependent kinase inhibitor, normally localizes to the nucleus. Jabl interacts with p27Kip1 and may influence its location and expression. P27Kip1 is transported from the nucleus into the cytoplasm by a mechanism involving Jab1, and then is degraded. This alteration leads an uncontrolled proliferation of tumour cells. There are inverse relationships between Jab1 and p27Kip1 in a variety of tumors.In our previous studies,, we found that Jab1 was overexpressed in human laryngeal carcinoma and had an inverse relationship with the expression of p27Kip1. Using SiRNA to block Jab1 expression, we found that tumor cells'proliferation prevented or slowed in vitro. In the present project, we investigated the effect of downregulation of Jabl by RNA interference on cell proliferation of laryngeal carcinoma cell line (Hep-2) in vitro. We used a number of approaches, such as construction of short hairpin interfering RNA (shRNA) plasmid that specifically inhibiters Jabl expression and construction of a non-targeting plasmid. In order to observe the therapeutic effect of the shRNA plasmid (that specifically downregulates Jabl) in vivo, the xenograft human laryngeal carcinoma model was established by subcutaneous injection of Hep-2 tumour cells in nude mice. Following treatment with Jab1-shRNA plasmid and the control plasmid when tumour approached a reasonable size, the tumour growth were monitored. These results provided a experimental data for development of new therapeutic agent for human laryngeal carcinoma.Objective:To construct the shRNAplasmid specifically targeting the Jab1 gene, and to observe its silencing effect on Jabl expression and effect of proliferation of Hep-2 cells.Methods:The sequence of shRNA that targes Jab1 gene was designed and cloned into RNA interference vector pGenesill.1. Western blot was used for detection of Jab1 expression. MST-8 was employed for examination of cell proliferation of Hep-2 cells after transfection with Jabl-specific shRNA plasmid and non-targeting plasmid.Results:The Jab1-specific shRNA and non-targeting plasmids were constructed successfully and named as pJab1 and pKB respectively. After transfection of Hep-2 cells with pJab1, we found that Jab1 protein expression was significantly reduced compared to the pKB-transfected cells, whereas p27 protein expression was increased in pJabl-transfected Hep-2 cells as compared with pKB-transfected Hep-2 cells. The cell proliferation of Hep-2 cells, detected by MST-8, was dramatically decreased in pJabl-transfected cells compared to the control groups (p<0.001)Conclusions:Down-regulation of Jabl gene expression by our constructed pJabl plasmid results in up-regulation of p27 expression and inhibits the proliferation of human laryngeal carcinoma cell line Hep-2 cells in vitro. Objective:To determine the antitumour efficacy of the shRNA plasmid specifically targeting Jab1 gene using the human laryngeal carcinoma line Hep-2 cell transplanted in nude mice.Methods:The nude mice model bearing human laryngeal carcinoma Hep-2 cells was established by subcutaneous injection of Hep-2 cells in nude mice. The tumor growth was monitored after intratumoral injection of pJabl, pKB plasmids and PBS. The Jabl and p27 expression in tumour tissues were examined by immunohistochemistry staining (IHC), Western Blotting and RT-PCR.Results:The xenograft tumour model of human laryngeal carcinoma Hep-2 cells was established successfully in nude mice. The volume and weight of pJabl treated group were (267.60±88.19) mm3 and (0.49±0.03) g; We found that the tumor growth was significantly inhibited in the pJabl treated group compared to the pKB-treated group and PBS group (p<0.001). IHC showed that the expression of Jab1 protein was significantly reduced in the pJab1-treated group compared to the control groups, whereas the expression rate of P27 protein in the pJabl group was significantly increased compared to control groups. These results were further confirmed by Western Blotting. The downregulation of Jab1 protein by pJabl plasmid was consistent with mRNA expression confirmed by RT-PCR. The level of Jabl mRNA level in the pJabl-treated group was significantly lower than the control groups (p<0.001), however, P27 mRNA had no significant alteration.Conclusions:The pJabl plasmid that we constructed in this project results in downregulation of Jab1 in an xenograft tumour model of human laryngeal carcinoma Hep-2 cells and significantly inhibit the tumour growth in vivo. This suggests that pJAB1 plasmid that specifically targets Jabl gene expression could be an effective therapeutic for treatment of human laryngeal carcinoma.