| Objective: Caveolae are small (50–100 nm) flask-shaped invaginations of the plasma membrane, the main molecular feature of which is the presence of caveolin, an integral membrane protein (21–24 kDaa) , and a distinct lipid composition (enrichment of cholesterol and glycolsphingo- li-pids). Caveolae have been implicated in the sequestration of signaling molecules .Caveolins are thought to function as scaffolding proteins within membrane microdomains where it interacts with several signaling proteins . Caveolin-1 was first identified as a major tyrosine-phosphorylated protein, phosphorylated on tyrosine 14 in response to a number of growth factors such as VEGF,epidermal growth factor, and platelet-derived growth factor. However, the role of tyrosine-phosphorylated Caveolin-1 still remains largely unknown.Research showed that nearly all normal cells exhibited Caveolin-1 expression, while in most tumor cells, such as breast cancer cells and lung cancer cells, there is low or no expression.of Caveolin-1. Reexpre- ssion of Caveolin-1 can inhibit tumor cell growth and tumor cells colonies formation,whereas suppressing Caveolin-1 expression by RNAi technology enhanced the ability to transform cells, suggesting that Caveolin-1 may have anti-tumor effect. Many researchers believe that Caveolin-1 be the candidate of the tumor suppressor gene. Besides, recent studies showed that in prostate cancer and bladder cancer cells Caveolin-1 was considered to be lymphatic metastasis-related genes. Therefore, more experimental work are required to clarify the role of Caveolin-1 in different tumor cells CD147 is a highly glycosylated immunoglobulin superfamily trans- membrane protein, which a membrane spanning molecule enriched on the surface of many malignant tumor cells. Having recieved heterogeneous N-glycosylation, CD147 exists in both a highly glycosylated form,HG-CD147 ( 40-66kDaa ) and a lowly glycosylated form ,LG-CD147 (~33kDaa).But the relationship of CD147 glycosylation and tumor metastasis is unclear yet.CD147 is composed of two extracellular Ig domains, a single transmembrane domain, and a cytoplasmic domain. One Ig domain of CD147 is required for counter receptor binding activity, which is involved in MMPS induction and oligomerization.The other Ig domain of CD147 is known to associate with Caveolin-1.At present, we have found that Caveolin-1 expression was higher in the HCa-F (high of lymphatic metastasis) than in the HCa-P (low of lymphatic metastasis), No Caveolin-1 expressionin in the Hepa 1-6 (non- lymphatic metastasis), indicating that Caveolin-1 plays an important role in the lymphatic metastasis of mice liver cancer cells. Caveolin-1 expression was down-regulated specifically in Hca-F cells using an RNAi technology. Western blot analysis showed that the Caveolin-1 levels were significantly reduced in Hca-F/RNAi cells. HG-CD147 expression was obviously decreased while LG-CD147 expression showed a marked increase.It is aimed to clone mice Caveolin-1(wt), and Caveolin-1(Y14F), by building expression vectors, then to stably express the recombinant protein in Hepa1-6, and to observe the expression of CD147, building up a basis for further studies of Caveolin-1 in mice lymphatic metastasis of liver cancer cells.Methods:1. The phosphorylation site of Caveolin-1 is mutated, and cloned to PMD18-T simple vector, then subcloned to expression vector, pcDNA3.1.2. Caveolin-1(wt) is subcloned to expression vector, pcDNA3.1.3. Recombinant pcDNA3.1/Caveolin-1 (wt) and pcDNA3.1/Caveolin-1 (Y14F) were stably transferred to Hepa1-6 by Liposome, then selected by G418,and identified by RT-PCR and Western blot .4. The expression of CD147 is detected in different cells, Hepa1-6, Hepa1-6/mock, Hepa1-6/Caveolin-1(wt), and Hepa1-6/Caveolin-1(Y14F). Result:1. pcDNA3.1/Caveolin-1(wt) was obtained. And results from restrictive enzyme analysis and sequencing showed that the murine Caveolin-1 gene was successfully inserted into pcDNA3.1 expression vector. It is recorded as pcDNA3. 1/Caveolin-1(wt).2. Results from Western blot and RT-PCR showed that the recombinant vectors successfully expressed Caveolin-1(wt) and Caveolin-1(Y14F) in Hepa1-6 cells.3. In the transfected cells, Hepa1-6/Caveolin-1(wt) and Hepa1-6/Caveo- lin-1(Y14F), Compared with empty vector-transfected and non-transfected cells, LG-CD147 expression was obviously decreased and HG-CD147 expression was obviously increased.Conclusion:1. pcDNA3.1/Caveolin-1(wt) and pcDNA3.1/Caveolin-1(Y14F) were constructed successfully bu subcloning cDNA of Caveolin-1 and Caveolin-1(Y14F) into pcDNA3.1,respectively.2. Caveolin-1(wt) and Caveolin-1(Y14F) proteins expressed stably in Hepa1-6 cells by stransfecting pcDNA3.1/Caveolin-1(wt) and pcDNA3.1/Caveolin-1(Y14F), respectively.3. It was not found that the phosphorylation of Caveolin-1 enhanced the glycosylation of CD147 in murine Hepatocacinoma cells... |
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