The Effect Of Human Larygneal Carcinoma Cells' Proliferation By Inhibiting The Expression Of Jab1 Gene

Background and purposeLaryngeal carcinoma is a common cancer of the head and neck. The disease incidence has increased significantly in rencent years. The therapeutics of LSCC such as surgery, radiotherapy and chemotherapy have many seriously complications, and the life level of the patients is bad. Therefor, to find novel, effective methods with little poisonous side effects for the treatment, have become a serious concern.RNA interference (RNAi), or gene silenceing, is a technique for down-regulating the expression of a specific gene in living cells by introducing a double-stranded RNA (dsRNA) with homology to the gene of interest. The RNAi technique has become the focus of gene therapy worldwide. RNAi technique can make the target gene's mRNA to silence or terminate the expression of purpous oncogene, making the tumor cells to divide toward maturity or inducing the cells apoptosis, so as to get the aim of treatment.With more deepening study on laryngeal squamous cell carcinoma, people understand that the development of laryngeal squamous cell carcinoma is a multistage evolution with more than one factor involved, and the blocked the excessive cell proliferation and apoptosis is the basis of their occurrence. According to reports, Jab1 gene plays an important role in the development of cancer. Jab1 (C-Jun activation domain-binding protein 1) is the fifth subunit of COP9 signalsome regulatory complex. The protein Jab1 is a ubiquitous soluble protein in cells, the gene of Jab1 is highly conserved in creatures. The cyclin-dependent kinase inhibitor (p27^kip1) is a negative regulator of G₁-S transition . The study indicates that the regulation of p27^kip1 is happening after gene transcription, and p27^kip1 makes its effect on nucleus. The mutation of p27^kip1 is rare.There is a few reports of the expression of Jab1 gene in the LCSS in our country. We study the inhibitory effect of RNA interference(RNA i) on Jab1 gene expression in the Hep-2 cells line of human beings. Hep-2 cells were transfected with synthetic Jab1 sequence-specitic siRNA by Lipofectamrne2000. The effects of siRNA targeting Jab1 in reducing the target gene expression and cell growth affecting in Hep-2 cell lines we observed.Methods1. Immunohistochemical method was used to examine the expressions of Jab1 and p27^kip1 proteins in 50 cases laryngeal squamous cell carcinomaes and 10 cases normal laryngeal tissues next laryngeal squamous cell carcinoma.2. Iimmunofluorescence method was used to examine the expressions of Jab1 and p27^kip1 proteins in Hep-2 cells.3. Hep-2cells were transfected with synthetic Jab1 sequence-specitic siRNA by Lipofectamrne2000, MTT method was used to measure the gorwth of the cells at different times and different concentrations atfer trnasefction, and to calculate the inhibition ratio of each group.4 . RT-PCR method was adopted to examine the mRNA expression of the Jab1 gene in Hep-2 cells which was treated with Jab1 siRNA at different time points and different concentration. The same method was adopted to examine the mRNA expression of the p27^kip1 gene in the same groups.5. Apoptosis of the Jab1 siRNA II (100nm/L) transfected cells was examined by flow cyometry after24, 48, 72 hours. The same method was adopted to examine the apoptosis of the comtrol groups.6. SPSS13.0 statistical software was applied to statistics the data. Statistical evaluation was performed using Chi-Square test and Fisher's Exact test. One-factor analysis of variance was used to analyse. The significant level was a = 0.05. Results1. The results of immunohistochemical showed: clearly brown staining restricted to nucleus was considered as positive expression of Jab1 and p27^kip1 protein. The expression rate of Jab1 protein in laryngeal squamous cell carcinoma is significantly higher than that of normal laryngeal mucosa, and the expression rate of protein p27^kip1 in laryngeal squamous cell carcinoma is significantly lower than that of normal laryngeal mucosa, suggesting that these two kinds of protein closely related to the occurrence and development of laryngeal squamous cell carcinoma.2. The results of immunofluorescence showed: the expressions of Jab1 and p27^kip1 proteins can be detecded in Hep-2 cells.3. The results of RT-PCR showed: the Hep-2 cells was transfected by Jab1 siRNA II, the expression of Jab1 mRNA was suppressed markedly. The expression of p27^kip1 mRNA had no change. As the reaction time increasing, the expression of Jab1 mRNA of Hep-2 cells decresed significantly. Jab1 siRNA II of the same concentration performed higher inhibition effect than Jab1 siRNA I and Jab1 siRNA III on Hep-2 cells.4. The results of MTT showed: the gorwth of the Hep-2 cells was restraintded after the transfecting by Jab1 siRNA II.5. The results of flow cyometry showed: After 24h, 48h, 72h of the transfection of Jab1 siRNA II (100nm/L), the rate of cells' apoptosis increased obviously(P< 0.05).Conclusion1. The expression rate of Jab 1protein in laryngeal squamous cell carcinoma is significantly higher than that of normal laryngeal mucosa, and the expression rate of protein p27^kip1 in laryngeal squamous cell carcinoma is significantly lower than that of normal laryngeal mucosa. Jab1 and p27^kip1 protein have negative correlation in laryngeal squamous cell carcinoma, suggesting that these two kinds of protein closely related to the occurrence and development of laryngeal squamous cell carcinoma. 2. Jab1 protein and p27^kip1 protein were expressed in the Hep-2 cells.3. The expression of Jab1 mRNA in Hep-2 cells transfected with siRNA II is markedly inhibited. RNAi efficiency depended on time and concentrations to some extent. And may be associated with the induction of apoptosis.4. Jab1 might be a suitable target for new therapeutic strategies using siRNA in laryngeal squamous cell carcinoma.