Study Of Heat Shock Protein 27 And Head And Neck Squamous Cell Carcinoma Metastasis

Head and neck squamous cell carcinoma (HNSCC) is the 6th most prevalent cancer types worldwide with an incidence of more than 500,000 cases annually and a high mortality rate, making it the fifth leading cause of cancer related death. In the United States, it accounts for 6% of all cancer diagnoses and results in an estimated 14,000 deaths annually. Despite the advanced therapeutic regimens used in treating HNSCC, oral cavity cancer survival during 1996-2003 was less than 50% on average according to the data from American Cancer Society. Furthermore, patients with recurrent or metastatic HNSCC have median survival of approximately 6 months. The major contributing factors for low survival include local-regional relapse, lymph node or distant metastatic spread of the primary tumor. The small Heat Shock Protein 27(Hsp27) belongs to heat shock protein family, and acts as a molecular chaperone. Expression of Hsp27 can be induced by environmental stress, such as heat shock, heavy metals, oxidants, infection, inflammation, ischemia. Hsp27 has been shown to have various cellular functions to promote differentiation, proliferation, cell growth, and motility. In addition, it has been reported that Hsp27 is associated with various carcinomas. RNA interference(RNAi) is a potential approach for gene therapy, thanks to its high efficience in silencing target sequence-specific gene, it has been used as a useful method in molecular biology study. And Lentivirus vector has become a common vector to expressing specific gene, as it could be transfected into separated/unseparated cells with larger gene fragment without the host immune response induced. To establish a head and neck squamous cell cancer model with stable expression of luciferase for in vivo imaging is a rising method to detect the tumor progression,metastasis and drug sensitivity. Especially, the left intracardic injection has been used as a better way to set up the in vivo imaging mice model, compared with tail vein injection,in site injection and other methods, it induces tumor quicker, easier to practice and has a higher survival rate for mice. To figure out how Hsp27 expression regulate head and neck squamous cell carcinoma metastasis behavior and find out a noval target for HNSCC treatment, in the present study, we silenced Hsp27 gene expression in head and neck squamous cell cancer cells UM-SC-22B which shows a high potential of metastasis by using temporary transfection of Hsp27 siRNA and stable transfection of Lentivirus with shRNA Hsp27. At the same time, we overexpressed Hsp27 expression in UM-SC-22A with lower metastasis potentcy by re-constructuring a Lentivirus vector expressing Hsp27 gene. And we used MTS assay to detect the proliferation changes of cells, wound-healing motility assay and Matrigel invasion assay to detect the metastasis ability changes before and after silencing or overexpressing Hsp27 gene in vitro. Otherwise, we set up a metastasis in vivo imaging mice model to detect the tumor cells metastasis ability in vivo.The whole dissertation consists of four parts:Part 1 Biology behaviors and Hsp27 expression in HNSCC cell lines with different metastasis potential1. Biology behaviors in HNSCC cell lines with different metastasis potentialProliferation of the primary and metastatic HNSCC cell lines was evaluated using the MTS proliferation assay. Metastatic behavior was assessed using migration and invasion assays in vitro. Set up imaging mice model to detect the metastasis behavior in vivo. MTS assays showed that the primary (UM-SCC-22A) and metastatic (UM-SCC-22B) HNSCC have similar proliferation rates after cultured for 24/48h(p>0.05). However, UM-SCC-22B derived from the metastasis showed 2.3 to 3.6-fold higher migration ability and 2-fold higher invasion ability than UM-SCC-22A. And UM-SCC-22B shows high rate of metastasis of kidney and thing-bone in vivo.2. Hsp27 expression in HNSCC cell lines with different metastasis potentialThe expression of Hsp27 in primary and metastatic cell lines derived from the primary HNSCC and a synchronous lymph node metastasis in the same patient was determined using real-time PCR and western blotting. Real-time PCR demonstrated that Hsp27 mRNA is 22.4-fold higher in metastatic UM-SCC-22B than primary UM-SCC-22A. Similarly, Western blotting showed that Hsp27 is rarely detectable in UM-SCC-22A whereas UM-SCC-22B expresses a higher level of Hsp27 protein.These data indicate higher expression of Hsp27 shows in UM-SCC-22B with higher metastasis potential, and Hsp27 may regulate metastatic potential of HNSCC cancer cells.Part 2 Hsp27 expression and biology behaviors changes in vitro after silencing Hsp27 by transfected with siRNA Hsp27 in UM-SCC-22B1. Hsp27 expression changes in vitro after silencing Hsp27 by transfected with siRNA Hsp27 in UM-SCC-22BDesigned a specific siRNA sequence to knocking down Hsp27 in the highly migratory metastatic HNSCC cell line UM-SCC-22B. The expression of Hsp27 in UM-SCC-22B before and after silencing Hsp27 was determined using real-time PCR and western blotting. Compared to blank control group, after transfected with siRNA Hsp27 by lipofectamine 2000, the mRNA level of Hsp27 was reduced by 93%.And at 48h after transfection, the protein level of Hsp27 was dramatically decreased in consistent.2. biology behaviors changes in vitro after silencing Hsp27 by transfected with siRNA Hsp27 in UM-SCC-22BFurthermore, proliferation of UM-SCC-22B before and after silencing Hsp27 was evaluated using the MTS proliferation assay. Metastatic behavior was assessed using migration and invasion assays in vitro. MTS assays showed that the UM-SCC-22B before and after silencing Hsp27 have similar proliferation rates after cultured for 24/48h (p>0.05). However, compared to blank control group,siRNA knockdown of Hsp27 decreased metastatic behaviors of UM-SCC-22B by 3 to 4-fold in migration and 2-fold in cell invasion reducing cell invasion and migration.These data indicate temporarily silencing Hsp27 expression by siRNA may decrease metastasis in head and neck squamous cell cancer cells.Part 3 Bilology behaviors and Hsp27 expression changes after knocking down Hsp27 by tranfected with Lentivirus-shRNA-Hsp27 in UM-SCC-22B 1. Hsp27 expression changes after knocking down Hsp27 by tranfected with Lentivirus-shRNA- Hsp27 in UM-SCC-22BPhurcase the Lentivirus vector plasmid with shRNA Hsp27 from Open Biosystem. After temperarily transfected into UM-SCC-22B, using real-time PCR to detect the mRNA of Hsp27 expression. The result shows that compared with blank control group, after 24/48h, the mRNA level of Hsp27 reduced to 42%/24%. Then produce lentivirus particals with shRNA Hsp27,stably transfected into UM-SCC-22B,using real-time PCR and western blotting assay to detect the mRNA and protein level changes of Hsp27 expression. The data shows mRNA level of Hsp27 was decreased by 94%,comparing to blank control group, meanwhile, the protein level was also significantly reduced in consistent.2. Bilology behaviors changes after knocking down Hsp27 by tranfected with Lentivirus-shRNA- Hsp27 in UM-SCC-22BUsing MTS assay to detect the proliferation changes of UM-SCC-22B before and after stable transfected with lentivirus particals pLenti-shRNA-Hsp27 with shRNA Hsp27. Metastatic behavior was assessed using migration and invasion assays in vitro. MTS assays showed that the UM-SCC-22B before and after knocking down Hsp27 have similar proliferation rates after cultured for 24/48h (p>0.05). However, compared to blank control group,shRNA knockdown of Hsp27 decreased metastatic behaviors of UM-SCC-22B by 4.38-fold in migration and 2.03-fold in cell invasion reducing cell invasion and migration. Besides, UM-SCC-22B with shRNA Hsp27 shows lower rate of metastasis of kidney and thing-bone in vivo.These data indicate stably knocking down Hsp27 expression by shRNA may decrease metastasis in head and neck squamous cell cancer cells.Part 4 Bilology behaviors and Hsp27 expression changes after over-expressing Hsp27 by tranfected with Lentivirus- Hsp27 in UM-SCC-22A1.Re-constructured Lentivirus vector expressiong Hsp27 and Hsp27 expression changes after over-expressing Hsp27 by tranfected with Lentivirus- Hsp27 in UM-SCC-22AThe full length cDNA pOTB7-Hsp27 plasmid was got from Invitrogen, design specific primers with BamHI, Xbal restriction enzyme sites,PCR was used to get the fragment of Hsp27 sequence with BamHI, Xbal. The fragment of Hsp27 and the pLentiLox-RSV lentivirus vector were ligated by T4 DNA ligase.After transferring them into the DH5-alpha competent cells, restracted the recombined plamides DNA.The recombinant clones were identified by PCR with 'the primers and digested it by restriction enzyme sites BamHI, Xbal for further identification. Then the positice recombinant pLenti-RSV-Hsp27 plasmid was sequenced. Packaging and producing pLenti-RSV-Hsp27 were processed.Stably transfected UM-SCC-22A with lentivirus particals pLenti-RSV-Hsp27,using real-time PCR and western blotting assay to detect the Hsp27 expression changes. The data shows mRNA level of Hsp27 was increased by 15.07-fold,and protein level of Hsp27 was also induced dramatically.2. Bilology behaviors changes after over-expressing Hsp27 by tranfected with Lentivirus-Hsp27 in UM-SCC-22AUsing MTS assay to detect the proliferation changes of UM-SCC-22A before and after stable transfected with lentivirus particals pLenti-RSV-Hsp27 with expressing Hsp27. Metastatic behavior was assessed using migration and invasion assays in vitro. MTS assays showed that the UM-SCC-22A before and after overexpressing Hsp27 have similar proliferation rates after cultured for 24/48h (p>0.05). However, compared to blank control group, overexprssing Hsp27 increased metastatic behaviors of UM-SCC-22A by 2.56-fold in migration and 2.01-fold in cell invasion reducing cell invasion and migration. Besides, UM-SCC-22A with overexpressing Hsp27 shows higher rate of metastasis of kidney and thing-bone in vivo.These data indicate overexpressing Hsp27 expression by stably transfected lentivirus particals pLenti-RSV-Hsp27 may increase metastasis in head and neck squamous cell cancer cells.Conclusion:1.Successfully set up a head and neck squamous cell cancer metastasis cell model in vitro and imaging in vivo mice model.2.Hsp27 could regulate the metastasis behavior of HNSCC, and it show higher expression in HNSCC cells with higher metastasis ability. 3.Temperarily transfected with siRNA Hsp27 could efficiently decrease Hsp27 expression on mRNA an protein levels in UM-SCC-22B.4.silencing Hsp27 expression by siRNA could reduce the metastasis ability of UM-SCC-22B.5.Stably transfected with lentivirus -shRNA-Hsp27 could efficiently decrease Hsp27 expression on mRNA an protein levels in UM-SCC-22B.6. Stably transfected with lentivirus -shRNA-Hsp27 could reduce the metastasis ability of UM-SCC-22B.7. Successfully re-constructured Lentivirus vector expressiong Hsp27. Stably transfected with lentivirus -RSV-Hsp27 could efficiently increase Hsp27 expression on mRNA an protein levels in UM-SCC-22A.8. Stably transfected with lentivirus -RSV-Hsp27 could induce the metastasis ability of UM-SCC-22A.