Study Of The Functions Of PC Cell-derived Growth Factor In Laryngeal Squamous Cell Carcinoma And Precancerous Lesions

Part I Protein and mRNA expression of PCDGF gene in laryngeal squamous cell carcinoma and precancerous lesionsObjective: PCDGF is novel growth factor for tumor formation and progression. However no comprehensive literature concerning PCDGF expression status and its biological function in squamous cell carcinoma, especially in larynx, is available. The target of this study is to evaluate the clinical significance of PCDGF.Methods: 146 primary laryngeal cancer, 108 adult laryngeal papilloma, 41 laryngeal leukoplakia samples, and 10 normal larynx tissues were detected. The PCDGF RNA level was examined by Real-time PCR, and protein localization by immunohistochemistry.Results: the PCDGF protein levels and RNA levels of the LSCCs were significantly higher than those of normal laryngeal tissues (P<0.001). Simultaneously the difference was significant between those of laryngeal precancerous lesions (papilloma/leukoplakia) and those of normal tissues at the levels of RNA and protein (P<0.05, P<0.05), while those of laryngeal precancerous lesions (papilloma/leukoplakia) were significantly lower than those of LSCCs. Strong PCDGF expression was associated with lymph node metastases in LSCC (P<0.05). There is no significant difference of PCDGF expression level among the samples of different histological grade or those of different T stages (P>0.05).Conclusions: PCDGF is pivotal autocrine growth factor in tumorigenesis of LSCC. Our findings also indicate PCDGF to be a logical and potential target for early diagnosis, specific therapy and prognosis of LSCC.Part IIThe effect of PCDGF hairpin siRNA expression vectors on expression and biological roles of PCDGF gene in Hep-2 cell linesObjective: Recently, RNAi has emerged as a powerful tool for investigating gene function and as a plat-form for therapeutic development in mammalian cells. The target of this study is to evaluate the potential of siRNA-induced genetic silencing of PCDGF as a supplementary therapeutic way for the LSCC.Methods: The PCDGF biological function was assessed by transfection ofsiRNA PCDGF construction. We stably transfected vectors, combined with twosequences of SiRNA target to PCDGF, to Hep-2 cells by using lipofectamine2000; calculated inhibition efficiency with Western-Blot; analyzed the ability of cell growth and anchorage-independent cell growth through protracting growth curve and using colony formation in soft agar respectively; estimated occurrence of apoptosis applying FACS with double staining labeled Annexin-V-FITC and PI; and detected tumorigenicity.Results: PCDGF expression was 73.7% lower in the siRNA clone than in the control vector cells. Only the siRNA transfectants had a dramatic decrease in cell number by 51.9% and 75.3% with or without serum, respectively (P < 0.001). SiRNA PCDGF transfectants showed impaired clonogenicity in semisolid agar compared either with control-transfected/G418-selected cells or with untransfected cells (p<0.001). SiRNA PCDGF transfectants developed smaller tumors only in 2 of 5 mice examined (40% incidence), whereas the control vector transfectants developed larger tumors in all of the 5 mice tested (100% incidence). The tumor weight of the siRNA group was significantly decreased by 87% compared with the control vector group (0.46±0.6 and 2.2±0.5 g, respectively; P< 0.01). After transfection siRNA PCDGF, the proportion of apoptosis cells was increased from 21.40% to 71.72% (n=3; p<0.001).Conclusions: our functional tests revealed that PCDGF positively modulated the cell proliferation, colony-forming ability in anchorage-independent environment, tumorigenicity in nude mice, and resistance to anoikis. For a tumor progress, invasive cells must escape anoikis without attachment to their normal substrate and actively proliferate in the newly encountered matrix environment. PCDGF may involve in these discrete reciprocal scenarios of laryngeal tumorigenesis. Our work suggests that siRNA-inducing genetic silencing of PCDGF, might be a potential supplementary therapeutic way for the LSCC treatment.Part IIIEstablishment of system for primary culturing of human laryngeal epithelial cells and fibroblastsObjective: Cells of short term culture are more reliable for study than cell lines because the relationship between primary cells and cell lines is uncertain. The aim of this study is to detail a rapid and reliable method for culturing of epithelial cells and fibroblasts.Methods: The technique of collagenase digestion was improved by our study. Overcoming constrains such as bacterial infection, cell type purity, cell activity and passage difficulty, we received many progress in culturing of epithelial cells and fibroblasts. We observed morphology and structure under light microscope and electron microscope. By immunochemical staining, we identified cell type.Results: Under light microscope, epithelial cells like pavement. The squamous epithelial phenotype was confirmed by ultrastructural presence and immunopositivity. With respect to culture fibroblasts, positive staining was found with Vimentin, but negative staining with cytokeratin. In comparison, epithelial cells carried positive staining with cytokeratin.Conclusions: The technique is simple and reliable for primary culture. Performing this method, we can provide useful cell model for investigating human squamous cell carcinoma of head and neck.Part IVProtein and mRNA expression of PCDGF gene in fibroblasts from laryngeal neoplasitc and normal tissuesObjective: PCDGF is a novel growth factor for tumor formation and progression. Recently, increasing evidence suggests that the stromal cells of the carcinogenic lesion are more positive "inducers" instead of passive "enablers" in tumorigenesis However, no literature, concerning expression status and possible role of PCDGF in fibroblasts, in squamous cell carcinoma and normal tissues, especially in larynx, is available. The target of this study is to evaluate the PCDGF expression level of fibroblasts in laryngeal squamous cell cancer (LSCC), in tumor adjacent noncancerous region, in precancerous lesions and in normal larynx.Methods: we detected the RNA and the protein level of PCDGF on the fibroblasts from 10 samples of LSCC, compared with the fibroblasts from tumor adjacent noncancerous region, normal larynx and precancerous lesions (papilloma). And based on primary cell culture, we investigated the PCDGF expression in the sequential passages of these fibroblasts.Results: Both the RNA expression and protein secretion levels of PCDGF were significantly higher in CAFs versus fibroblasts adjacent noncancerous region (P<0.01) and CAFs versus papilloma-associated fibroblasts (P<0.05) and CAFs versus laryngeal normal mucosa fibroblasts (P<0.001). The difference was also significant in papilloma-associated fibroblasts versus laryngeal normal mucosa fibroblasts (P<0.05), whereas between tumor adjacent noncancerous region fibroblasts and laryngeal normal mucosa fibroblasts, there was no significant difference in RNA and protein level (P>0.05). These CAFs and papilloma-associated fibroblasts present the elevated expression of PCDGF in vitro. Moreover, the RNA and protein levels of both types of fibroblasts in passage 8 were significantly higher than those in passage 3 (p<0.01, p <0.01; respectively). On the contrary, there was no significant difference between passage 8 and passage 3 in the fibroblasts from the normal laryngeal mucosa and tumor adjacent noncancerous region (P>0.05). PCDGF expression, in stroma of LSCC and papilloma tissues, was confirmed by Immunohistochemistry.Conclusions: we have demonstrated that PCDGF is overexpressed in CAFs and papilloma-associated fibroblasts in epithelial-independent fashion. This study has implicated that PCDGF in fibroblasts is involved in carcinogenesis of human laryngeal squamous cell cancer.