The Experimental Research On The Effect Of MiRNA By Targeting Rac1 Gene On The Apoptosis And The Proliferation In Laryngeal Carcinoma Cell

Laryngeal carcinoma is the most common malignant disease in otolaryngology- head and neck surgery. Despite advances in diagnostic techniques and treatments such as surgery operation, radiotherapy and chemotherapy, the present curative effect in some of advanced stage, recurrence and metastasis of laryngeal carcinoma have remained poor for many years. In recent years, the tumor foundation research has obtained evident progression and gene therapy will display good perspective to the patients with tumors. RNA interference is a high effective method for gene silence.A tumor can be viewed as an aberrant organ characterized with the maladjustment between cell to cell, cell to matrix, and the disorder of cell division and cell death. It has been demonstrated that apoptosis plays an important role in normal cells conformation and the inner environment. The out of control of apoptosis and proliferation have been suggested to contribute to the formation of cancer. The weaken apoptosis has been implicated in tumorigenesis, invasion and distant metastasis. The existence of abnormally enhancement cells may be induce gene mutation and mediate the resistant of cells toxicity based on immunology. Thus the inhibition of apoptosis can markedly induce tumorigenesis and development of tumors.As a member of the Ras superfamily, Rac1 is an oncogene and has the anti-apoptosis function .It acts as a molecule switch in cell signal transduction. It has been shown to regulate a wide spectrum of cellular functions such as cytoskeleton organization, genetic transcriptional regulation and cell signal transduction control. It impacts on cell polarity, adhesion, growth and motion, correlated with proliferation, differentiation and apoptosis. Rac1 may be possible as a potential target for tumor gene therapy. Although Rac1 may play an important role in carcinogenesis and has been reported in a variety of human cancers, there are no reports on the expression in laryngeal carcinoma. Our study primarily explored the clinic pathological relationship between Rac1 and the proceeding of laryngeal squamous carcinoma. To further define Rac1 function in anti-apoptosis and to explore novel approaches of gene therapy, we employed the RNA interference technique to knock down gene expression of Rac1 in laryngeal carcinoma cell line Hep-2 to study the effect of miRNA by targeting Rac1 gene on the apoptosis and the proliferation in vitro .We suggest that specific Rac1 inhibition may have potential therapeutic value, and that Rac1 is a novel target for gene therapy of laryngeal carcinoma.1. The expression and clinic pathological significance of Rac1 in laryngeal squamouse cell carcinoma(LSCC)Immunohistochemical technique was used to examine the expression of Rac1 in 42 cases of laryngeal carcinoma samples and adjacent non-cancerous samples and 10 cases of normal laryngeal mucosa. Immunohistochemical staining showed that Rac1 was not expressed in normal laryngeal mucosa, the expression rates of Rac1 protein were respectively 69%, 12% in LSCC and the adjacent non-cancerous tissues. The expression of Rac1 protein in LSCC was up-regulated which had significantly different(both P<0.01)from that of both the adjacent non-cancerous tissues and normal laryngeal mucosa. There is no evident difference between the group of the adjacent non-cancerous tissues and normal laryngeal mucosa. Our study indicated that the expression of Rac1 had a close relationship with the proceeding of LSCC.Expression of Rac1 was highly correlated with clinical classification, lymph node metastasis and tumor differentiation(p<0.05).On the other hand, it was not correlated with gender, age or clinical type(p>0.05).In addition, experimental results have convincingly demonstrated that over expression of Rac1 is highly associated with decreased tumor differentiation, increased TNM stage, and malignant transformation, which may yield lymph node involvement. These results indicate that Rac1 might play an important role in the progression and invasion of LSCC.2.Construction of miRNA-Rac1 recombination vectorWe designed miRNAs by targeting Rac1 gene, and constructed three miRNA recombination vector , pRac1miR/GFP-494 , pRac1miR/GFP-729 and pRac1miR/GFP-733.Firstly, we got the number NM-006908 of Rac1 in the database of GenBank to design single-stranded DNA oligos.We used Invitrogen's RNAi Designer, an online tool of Co. Invitrogen to design miR RNAi, and the design rules include a proprietary algorithm. We elected three sites 494, 729 and 733 because of high scores. Annealing was operated in the single-stranded oligos to generate a ds oligo. Cloning this ds oligo into pcDNA?6.2-GW/EmGFPmiR with T4 DNA Ligase and transforming One Shot[ coincidence. We obtained recombination vectors successfully. Then we transfected it into Hep-2 cells by Lipofectimine 2000. Fluoremicroscope was applied to observe the transfectional effect. Green fluorescence could be observed in Post transfectional Hep-2 cells,which demonstrated that the transfection was successful.RT-PCR and Western blot analysis were used to detect the expression of Rac1 at mRNA and protein level. The results showed that the Hep-2 cells which have been transfected the pRac1miR/GFP-733 recombination plasmids could significantly inhibit the mRNA and protein level of Rac1 expression.3. Study on the effect of miRNA by targeting Rac1 gene on the apoptosis and the proliferation in laryngeal carcinoma cell in vitroWe ploted the growth and proliferation activity curve of Hep-2 cells which have been successfully transfected with pRac1miR/GFP-733,pLacZ-miR/GFP and Hep-2 cells. .Also we applied MTT to detect the proliferation capability of Hep-2 cells. The results suggested the growth and proliferation of Hep-2 cells transfected with the recombinant of pRac1miR/GFP-733 were suppressed significantly than those not transfected. We applied AO and FCM to analysis of apoptosis of Hep-2 cells that transfected with the recombinant of pRac1miR/GFP-733,the results showed rate of apoptosis increased in Hep-2 cells transfected with the recombinant of pRac1miR/GFP-733 than those not transfected. The constructed recombination plasmids can suppress the anti- apoptosis effect of Rac1.Innovation and significance:Immunohistochemical technique was used firstly to examine expression of Rac1 in laryngeal carcinoma, adjacent non-cancerous tissues and normal laryngeal mucosa. Our study indicated that the expression of Rac1 had a close relationship with the proceeding of LSCC. We designed miRNAs by targeting Rac1 gene and constructed three miRNA recombination plasmids,then transfected successfully it to Hep-2 cells by Lipofectimine 2000 for the first time. RT-PCR and Western blot analysis were used to detect the expression of Rac1 at mRNA and protein level. We employed the RNA interference technique to knock down gene expression of Rac1 in laryngeal carcinoma cell line Hep-2 to study the effect of miRNA by targeting Rac1 gene on the apoptosis and the proliferation in vitro .Our study suggested that specific Rac1 inhibition may have potential therapeutic value, and that Rac1 is a novel target for gene therapy of laryngeal carcinoma.