Effects On The Biological Behaviour Of Inhibiting C-Met Expression By RNA Interference In Hep-2 Cells

Backgroud :Laryngeal carcinoma accounts for 1%～5% of the malignant tumors in human being and 13.9%of the malignant tumors on the head and neck.Squamous cell carcinoma was particularly prevalent.In some areas of China,the incidence of laryngeal carcinoma is 1.5~3.4/hundred thousands and laryngeal carcinoma harm the health of people severely.The traditional therapy for laryngeal carcinoma,including surgical treatment, radiotherapy,chemotherapy and so on,has been improved and greatly raised the five years survival rate of the patients in recent decades. But surgery often brings serious destruction of laryngeal function to the patients often lead to disabled. Radiotherapy is only effective for the patients in early stage of laryngeal carcinoma and can not take good effect on the most patients who are already in the late stage at the time of diaglosis or in relapse or metastasis.Patients always can not tolerate chemotherapy due to the severe toxic reation and side effects.So the application of these routine therapy is constrained greatly.Then to expore innovative, available treatment method with little toxicity side effects become a warm research subject of ENT researchers.The hepatocyte growth factor(HGF) is a multifunctional cytokine and its biologic activity is mediated by c-Met. C-Met receptor was a member of the receptor tyrosine kinase family,which is known to have the effects of stimulation of cell motility,dissociation of epithelial sheets,invasion of cellular matrix,and induction of angiogenesis.Abnormal expression of c-Met may lead to activation of downstream molecules and the development of tumor and metastasis.C-Met gene was found to be strongly expressed in the laryngeal carcinoma and has been shown to play an important role in proliferation and metastasis of laryngeal carcinoma. RNA interference is a new technique proved to be effective and specific for suppressing gene expression. It has become a hot spot in the tumor gene therapy field at present. In this study,a recombinant plasmid generating short hairpin RNA was construc- ted,and to transfect into human laryngeal carcinoma ce11s Hep-2 by lipofectamine.The change of the expression of c-Met between cells transfected or not transfected were observed by RT-PCR and Western-Blot method.Then Hep-2 cells proliferation, movement and invasion were studied using MTT, cell migration assay and cell invasion assay respectively to inspect the feasibility of c-Met being therapeutic target gene of laryngeal carcinoma.Objective:To construct recombinant plasmids containing short hairpin RNA that targets c-Met gene,to assay the expression of c-Met mRNA and proteinum in Hep-2 cells after transfecting with recombinant plasmids,and to elect the most inhibitive c-Met-shRNA sequence. To explore the effects of c-Met-siRNA on the proliferation, movement and invasion of laryngeal carcinoma Hep-2 cells by pSilencer2.0/c-Met-shRNA recombinant plasmid transfection.Methods:1. Three pairs of shRNAs that targets c-Met gene were designed then double-stranded RNA was derived through annealing,finally cloned into Psilencer2.0-U6 vector digested by two restricted endoenzymes according to its special orientation. Recombinant plasmid was transformed into strain DH5α, identified by using restriction enzyme and sequencing of nucleic acid.2. The recombinant plasmids pSilencer2.0/c-Met-shRNA containing short hairpin RNA targeting c-Met gene was constructed and transfected via cationic liposome Lipofectamine2000 into Hep-2 cells. The transfection efficacy was tested by RT-PCR and Western-Blot method,The difference in c-Met gene and proteinum expression levels between cells transfected or not transfected by pSilencer2.0/c-Met-shRNA was compared, then we elected the most inhibitive c-Met-siRNA sequence.3. Cell proliferation, movement and invasion were studied using MTT that time spot was taken 24h,48h,72h,96h after transfection, cell migration assay and cell invasion assay which were introducted by transwell booth, respectively.Result:1.The strain DH5αtransformed by recombinant plasmid could clony grow byam- picillin-resistant screen.2. The recombinant plasmid could be cut by BamHⅠand HindⅢ.3. The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments.4. By RT-PCR method, transfection of pSilencer2.0/c-Met-shRNA down-regulated c-Met mRNA expression in Hep-2 cells and recombinant plasmid 2 had the strongest effect(p value =0.003).5. By Western-Blot method, transfection of pSilencer2.0/c-Met-shRNA down-regul- ated c-Met protein expression in Hep-2 cells and recombinant plasmid 2 had the strongest effect(p value =0.02).6. After the pSilencer2.0/c-Met-shRNA2 recombinant plasmid transfection into laryngeal carcinoma Hep-2 cells,according to the normal control group and the negative control group,the A492-values of cells that were cultivated 24h,48h,72h,96h obviously droped (p<0.05).7. After the pSilencer2.0/c-Met-shRNA2 recombinant plasmid transfection into laryngeal carcinoma Hep-2 cells, according to the normal control group and the negative control group,the invasion cell counting is 15.00±3.61(p value =0.004, respectively)and the movement cell counting is 72.67±4.73(p value =0.001, p value =0.004).Conclusion:1. The recombinant plasmid pSilencer2.0/c-Met-shRNA1, pSilencer2.0/c-Met- shRNA2, pSilencer2.0/c-Met-shRNA3 were constructed successfully.2. The recombinant plasmid pSilencer2.0/c-Met-shRNA2 can significantly down-regulate the expression of c-Met gene mRNA and protein level in transfected on laryngeal carcinoma Hep-2 cells.3. The recombinant plasmid pSilencer2.0/c-Met-shRNA2 can markedly inhibit laryngeal carcinoma Hep-2 cell proliferation, movement and invasion.It may have the potential as a therapeutic modality to treat human laryngeal carcinoma.