Study On The Role And Its Mechanism Of Hexokinase2in Laryngeal Squamous Cell Carcinoma

Part I Clinical Significance of Hexokinase2Expression in Laryngeal Squamous Cell CarcinomaBackground and objective:Laryngeal squamous cell carcinoma （LSCC） is among the most common malignant tumor of head and neck region and is the eighth most common cause of cancer death worldwide. The incidence of LSCC is increasing in most countries. These findings indicate the need for additional information on the etiology and pathogenesis of the disease. Compared with normal cells, several aspects of cellular metabolism are altered in cancer cells, for example, Warburg effect. The altered metabolism are thought to play a key role in the proliferation, apoptosis and metastasis of tumor. The first step of glycolysis is catalyzed by hexokinases. In cancer cells, hexokinase2（HK2） is over-expressed and it is thought that the binding of HK2to the mitochondrial membrane may promote the Warburg effect, which indicate it may be a potential important target for cancer therapy. Even though the role of glycolysis and HK2has been investigated in some cancers, the role of altered glycolysis and HK2expression has not been investigated in LSCC. In this study, we investigated the expression of HK2in LSCC to explore its clinical significance. Methods:Paraffin-embedded samples were collected, including108cases of the primary tumor of LSCC,24cases of laryngeal papilloma and21cases of glottic polypus. All cases were male patitents. Immunohistochemistry was applied to test HK2expression. The staining evaluation was based on the staining intensity as well as the positive rate of tumor cells. The correlation between HK2expression and clinicopathological parameters was also investigated. All statistics was carried out by SPSS15.0statistics software.Results:The results indicated that HK2expression in primary tumor of LSCC was higher than laryngeal papilloma or glottic polypus （P<0.001）. Futher analysis showed that HK2expression was correlated with tumor location （P<0.001） and tumor stage （P≤0.001）. HK2expression was higher in supragolottis tumors than glottis tumors. Its expression was elevated in advanced staged tumors. No correlation was found between HK2exprsssion and age （P>0.05）.Conclusions:HK2expression was higher in primary tumor of LSCC than laryngeal papilloma or glottic polypus. Its expression was correlated with tumor location and tumor stages. Part Ⅱ Construction of plasmid vector for HK2shRNA and the test for interference efficiencyObjective:We aim to construct the plasmid vector for HK2shRNA and test its knock down efficiency after transient transfection.Methods:According to the principles of RNA interference sequence design, BLAST software was applied for homologization analysis. Specific targeted sequences were sought from HK2coded sequence （NM₀00189.4）. Additional bases were added to the C-and N-terminal of the targeted sequences. New sequences were ligated to the lined pGenesil-1.1plasmid to construct recombinated plasmid pGenesil-1.1-HK2-1, pGenesil-1.1-HK2-2and pGenesil-1.1-control respectively. Recombinated plasmid was subsequently transfected to human laryngeal carcinoma cell lines Hep-2cell.48h after transfection, mRNA and protein were collected to test the HK2shRNA interference efficiency.Results:The construction of plasmids pGenesil-1.1-HK2-1, pGenesil-1.1-HK2-2and pGenesil-1.1-control were identified by sequencing. Compared with untransfected Hep-2cells and Hep-2cells transfected with pGenesil-1.1-control, HK2mRNA expression were significant lower in Hep-2cells transfected with pGenesil-1.1-HK2-1and pGenesil-1.1-HK2-2by qRT-PCR detection （P<0.001）. The knock down rate of pGenesil-1.1-HK2-2（58.7±3.06%） was higher than that of pGenesil-1.1-HK2-1（45.3±3.51%）（P<0.05）. The protein analysis was in accordance with the mRNA analysis.Conclusions:Recombined plasmid pGenesil-1.1-HK2-1、pGenesil-1.1-HK2-2and pGenesil-1.1-control were successfully constructed. mRNA and protein analysis indicated HK2expression was knock down by pGenesil-1.1-HK2-1and pGenesil-1.1-HK2-2. The interference efficiency of pGenesil-1.1-HK2-2was higher and it was used for further analysis in vitro and in vivo. Part Ⅲ The effects of HK2konck down on the proliferation and apoptosis of laryngeal carcinoma cellsObjective:We aim to contruct the stable transfected cell lines of HK2shRNA and control shRNA and explore the effect of HK2knock down on the proliferation and apoptosis of Hep-2cells in vitro.Methods:After pretreated by G418for6weeks, monoclonal cells was screened for stable cell lines transfected with pGenesil-1.1-HK2-2and pGenesil-1.1-control by limiting dilution method. qRT-PCR and western blot were used to test the mRNA and protein expression of HK2. A standard glucose-6-phosphate dehydrogenase-coupled spectrophotometric assay was used to measure the HK activity. MTT assay was employed to investigate the role of HK2knock down on cancer cells proliferation. Flow cytometry analysis was also employed for the analysis of the impact of HK2knock down on the cell cycle and apoptosis.Results:qRT-PCR and western blot analysis indicated that compared with control shRNA and Hep2cells, the mRNA and protein of HK2expression of HK2shRNA group was decreased. HK activity was also downregulated in HK2shRNA group. MTT analysis indicated that knock down of HK2shRNA group could inhibit cell proliferation （P<0.05）. Cell cycle analysis showed that HK2knock down induced G0-G1arrest （P<0.001）. HK2knock down also incudced cell apoptosis （P<0.05）.Conclusions:The knock down of HK2expression by shRNA could inhibit cell proliferation and induce cell apoptosis, accompanied with G0-G1arrest, which indicated HK2may be a potential oncogene. Part IV The effect of HK2knock down on the tumogenicity of Hep-2cell in nude miceObjective:We aim to investigate the effect of HK2knock down on the tumogenicity of Hep-2cell and explore the influence on proliferation and apoptosis of transplanted tumor in nude mice.Methods:The stable transfected cell with HK2shRNA and control shRNA was employed for the subcutaneous xenograft model.2×106cells were injected subcutaneously into the right flank of each mice. The tumor diameter was measured weekly using a sliding caliper and the tumor volume was calculated. The mice were sacrificed4weeks later and tumor weight was recorded. HE staining, Ki-67staining and TUNEL test were applied to evaluate the effect on cytologic atypia, proliferation and apoptosis by HK2depletion, respectively.Results:Xenograft tumor formation was observed for all mice7-days post injection and tumor size increased during the4-week follow up period. Tumor volume and weight were significantly lower for xenografts from mice injected with HK2shRNA expressing Hep-2cells than those injected with the untransfected or shRNA control cells （P<0.01）. Compared with those made from the untransfected or those transfected with the shRNA control plasmid, xenograft tumors derived from cells expressing HK2shRNA demonstrated less cytologic atypia, lower proliferation index （P<0.001） and higher apoptosis index （P<0.001）, which were detected by HE staining, Ki-67staining and TUNEL test, respectively.Conclusions:Subcutaneous xenografts model indicated that HK2konck down could reduce the potential of tumogenicity by inhibiting cancer cell proliferation and inducing apoptosis in vivo.