| Tumor metastasis is a process complicated and multiple steps were involved. Recently many studies have shown that epithelial-mesenchymal transition (EMT) is close related to epithelial-derived tumor in the process of metastasis, many researches have also shown that TGF-β1-mediated signaling pathway in cancer played a very important role in EMT. If we could find an important gene which regulates TGF-(31 signaling pathway and prevents the formation of EMT, it will be effective in controlling cancer metastasis. To explore the gene on regulation of metastasis in the SCCHN, a series of preliminary studies were conducted to search for the candidate genes, and we found that caveolin-1 played an important role in the metastasis of the the SCCHN.To investigate the role of caveolin-1 in the metastasis of SCCHN。We prepared the lentivirus-mediated caveolin-1 expression and caveolin-1shRNA plasmid at first; then the lentivirus-mediated caveolin-1shRNA was used to infect human head and neck squamous cell carcinoma cell lines Tu686 to get a cell line which had a stable downexpression of caveolin-1, and we studied the changes in cells'phenotype and proliferation. We tried to further explore the molecular mechanism of Caveolin-1 on EMT and metastasis in squamous cell carcinoma of the head and neck and how the Caveolin-1 regulated the TGF-β1 sigalling pathway. At last, we hope to provide experimental evidence in the mechanisms of metastasis in squamous cell carcinoma of the head and neck. And the study is divided into three parts listed below. Objectives To construct the lentivirus-mediated Caveolin-1 over-exppress and Caveolin-1 shRNA lentivirusMethods Got the Caveolin-1 cDNA through reverse transcription method and synthesized target genes for caveolin-1 interference sequence shRNA online; they were connected with the respective lentviral vector and then were transformed into E. coli and identification of positive clones to confirm the vectors successfully constructed. The over-expression cDNA sequences and interference sequence were packaged into the 293T cells with the lentiviral packaging kit. The recombinant lentivirus that contained caveolin-1 over-expression cDNA sequences and interference sequence were made after producing and purifying.Results Sequencing results showed that lentiviral vectors containing over-expression cDNA and interference sequences were successfully constructed, the recombinant lentivirus were got after producing and purifying.Objectives To study the effect of caveolin-1 on EMT, migration and invasiveness in SCCHN.Methods Tu686 cells were infected with lentivirus-mediated caveolin-1 shRNA, puromycin was used to select a stable clone, and caveolin-1 mRNA and protein expression were detected by semi-quantitative PCR and Western-blot. Then, The cell growth was detected by CCK8 and flow cytometry was used to assay the cell cyele and apoptosis.And the cells were observed the change of phenotype under the microscope.The expression of E-cadherin and Vimentin were examined by Western-blot, the invasiveness ability was tested by wound-healing assay and migration ability was performed by a matrigel invasiveness assay in transwell culture chambers.Results The infection efficiency of lentivirus-mediated caveolin-1shRNA was more than 90%, and a stable clone was successfully selected, which caveolin-1 mRNA and protein expression were significantly down-regulated compared with the control group. There were no different between the experimental group and the control group by CCK8 and flow cytometry to detected cell growth and apoptosis (P> 0.05).The experimental group looked like mesenchymal cells under microscope. Western-blot results showed that of the experimental group epithelial marker E-cadherin significantly decreased and mesenchymal markers Vimentin significantly up-regulated compared to control group. The result of wound-healing assay showed that the invasiveness capability of the experimental was signifieantly enhanced compared with control group, the healing rate were (55.50±2.3)% and (24.20±1.5)% at 24h (P<0.05).And the result of matrigel invasiveness assay in transwell culture chambers showed that the capability of experimental group invasiveness was significantly increased, compared with the control group, the cells getting through the basement membrane were 103±15 and 34±4 at 48h (P＜0.05)Objectives To study the effect of Caveolin-1 on TGF-β1 mediated signal pathway and its impact on the invasiveness and migration of SCCHN.Methods Cellular immunofluorescence was applied to detect the intracellular location of both TGF-β1R I and Caveolin-1 in Tu686 cells; Western-blot was applied to detect the expressions of downstream of TGF-β1: Smad2 and its phosphorylated product p-Smad2 in both control and Caveolin-1 interventional groups. The cells that were treated with TGF-β1 for 48 hours were infected by Caveolin-1 over-expression lentivirus. And the protein levels of Smad2 and p-Smad2 were detected by Western-blot; and the invasiveness of the cells after infection was performed by a matrigel invasiveness assay in transwell culture chambers.Results Cellular immunofluorescence results showed that TGF-β1R I and Caveolin-1 were co-located in the cells'membrane. Western-blot results showed that the protein level of overall Smad2 had no significant change, but the protein level p-Smad2 was up-regulated in experimental group compared with the control group (P<0.05). With TGF-J31 dose increased, the protein level of caveolin-1 was gradually decreased (P<0.05), and the protein level of p-Smad2 was gradually increased (P<0.05), but the protein level of overall Smad2 had no significant change (P>0.05). After the cells were infected by Caveolin-1 over-expression lentivirus, the protein level of p-Smad2 was decreased (P<0.05), the invasiveness capability was significantly reduced (P <0.05), but the protein level of overall Smad2 had no significant change (P >0.05).Conclusions:1. Caveolin-1 has no significant effect on the cells proliferation and apoptosis in experimental group compare with the control group in SCCHN.2. Caveolin-1 can induce EMT, and can significantly enhance the capabilities of invasiveness and migration in SCCHN.3. TGF-β1 can induce EMT in SCCHN, and Caveolin-1 plays an important role in the EMT, migration and invasiveness in SCCHN by regulating the TGF-β1 mediated signaling pathway.
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