S: RA RO AS DW DMM. Performed the experiments: RA DMM. Analyzed the data: RA DMM REE. Contributed reagents/materials/analysis tools: RA DMM REE RO. Wrote the paper: RA DMM.
CTGF is a cysteine-rich, matrix-associated, heparin-binding protein, and is widely expressed in variety human tissues and organs, such as connective tissue, pancreas, placenta, and lung. Its expression has been associated with tumor cell proliferation, adhesion, and angiogenesis [1], [2] and serves as a prognostic marker in many types of human cancer [3?]. Interestingly, CTGF plays different roles in different types of cancer. In pancreatic cancer, prostate cancer, liver cancer, breast cancer, and sarcoma, CTGF has been shown to be an oncogenic factor promoting tumor progression [1], [6?]. Conversely, CTGF functions as a tumor suppressor in lung cancer, ovarian cancer, and oral squamous cell cancer [5], [10], [11]. The expression pattern and functional mechanisms of CTGF in NPC have not been Title Loaded From File established.Nasopharyngeal carcinoma (NPC) is a tumor arising from the epithelial cells that cover the surface and line the nasopharynx. Its highest incidence worldwide occurs in Southern China, with an age-standardized incidence rate varying from 20 to 50 cases per 100,000 people. Typical cervical lymph node metastases frequently occur in early stages. Synergetic 16985061 effects of viral infections, genetic alterations, and environmental factors are thought to drive abnormal gene expression, which contributes to the initiation and development of NPC [12?5]. In a previous study, cDNA microarray was utilized to examine differentially expressed genes between NPC tissues and non-cancerous nasopharyngeal tissues. Through BRB-array tool analysis, the expression of connective tissue growth factor (CTGF), a member of CCN family, was found to be notably downregulated in NPC tissues, suggesting a potential role in suppressing the pathogenesis of NPC [12].CTGF in NPCIn order to further clarify the role of CTGF in the pathogenesis of NPC, we investigated its expression and correlation with clinicopathologic features in NPC patients, as well as its effects on cell growth, cell cycle, migration, and invasion in cell lines. Our studies demonstrated that reduced CTGF expression stimulates cell proliferation, migration, invasion and cell cycle progression via FAK/PI3K/AKT signaling, EMT and MMP pathways.Table 2. SiRNA sequences of CTGF.No 1 Sense Antisense 2 Sense AntisenseSequence 59GCACCAGCAUGAAGACAUA dTdT 39 39dTdT CGUGGUCGUACUUCUGUAU 59 59CCAGACCCAACUAUGAUUA dTdT 39 39 dTdT GGUCUGGGUUGAUACUAAU59 59GUGCAUCCGUACUCCCAAA dTdT 39 39dTdT CACGUAGGCAUGAGGGUUUMaterials and Methods Cell Culture and Sample CollectionEight NPC cell lines 5?F, 6?0B, CNE2, CNE1, C666?, HONE1, HNE1 and SUNE1 were obtained from Cancer Research Institute of Southern Medical University. All cell lines were maintained in RPMI 1640 medium supplemented with 10 newborn calf serum (NBCS) (PAA Laboratories, Inc, Pasching, Austria). NP69, an immortalized human nasopharyngeal epithelial cell line, was grown in defined-KSFM medium supplemented with epidermal growth factor (EGF) (Invitrogen, Carlsbad, USA). All cell lines were incubated in a humidified chamber with 5 CO2 at 37uC. 20 fresh primary NPC tissues, 11 fresh NP tissuses, 92 paraffin-embedded undifferentiated primary NPC specimens and 25 paraffin-embedded NP specimens were obtained at the time of diagnosis before any therapy from People’s Hospital in Title Loaded From File Zhongshan City (Guangdong, China).S: RA RO AS DW DMM. Performed the experiments: RA DMM. Analyzed the data: RA DMM REE. Contributed reagents/materials/analysis tools: RA DMM REE RO. Wrote the paper: RA DMM.
CTGF is a cysteine-rich, matrix-associated, heparin-binding protein, and is widely expressed in variety human tissues and organs, such as connective tissue, pancreas, placenta, and lung. Its expression has been associated with tumor cell proliferation, adhesion, and angiogenesis [1], [2] and serves as a prognostic marker in many types of human cancer [3?]. Interestingly, CTGF plays different roles in different types of cancer. In pancreatic cancer, prostate cancer, liver cancer, breast cancer, and sarcoma, CTGF has been shown to be an oncogenic factor promoting tumor progression [1], [6?]. Conversely, CTGF functions as a tumor suppressor in lung cancer, ovarian cancer, and oral squamous cell cancer [5], [10], [11]. The expression pattern and functional mechanisms of CTGF in NPC have not been established.Nasopharyngeal carcinoma (NPC) is a tumor arising from the epithelial cells that cover the surface and line the nasopharynx. Its highest incidence worldwide occurs in Southern China, with an age-standardized incidence rate varying from 20 to 50 cases per 100,000 people. Typical cervical lymph node metastases frequently occur in early stages. Synergetic 16985061 effects of viral infections, genetic alterations, and environmental factors are thought to drive abnormal gene expression, which contributes to the initiation and development of NPC [12?5]. In a previous study, cDNA microarray was utilized to examine differentially expressed genes between NPC tissues and non-cancerous nasopharyngeal tissues. Through BRB-array tool analysis, the expression of connective tissue growth factor (CTGF), a member of CCN family, was found to be notably downregulated in NPC tissues, suggesting a potential role in suppressing the pathogenesis of NPC [12].CTGF in NPCIn order to further clarify the role of CTGF in the pathogenesis of NPC, we investigated its expression and correlation with clinicopathologic features in NPC patients, as well as its effects on cell growth, cell cycle, migration, and invasion in cell lines. Our studies demonstrated that reduced CTGF expression stimulates cell proliferation, migration, invasion and cell cycle progression via FAK/PI3K/AKT signaling, EMT and MMP pathways.Table 2. SiRNA sequences of CTGF.No 1 Sense Antisense 2 Sense AntisenseSequence 59GCACCAGCAUGAAGACAUA dTdT 39 39dTdT CGUGGUCGUACUUCUGUAU 59 59CCAGACCCAACUAUGAUUA dTdT 39 39 dTdT GGUCUGGGUUGAUACUAAU59 59GUGCAUCCGUACUCCCAAA dTdT 39 39dTdT CACGUAGGCAUGAGGGUUUMaterials and Methods Cell Culture and Sample CollectionEight NPC cell lines 5?F, 6?0B, CNE2, CNE1, C666?, HONE1, HNE1 and SUNE1 were obtained from Cancer Research Institute of Southern Medical University. All cell lines were maintained in RPMI 1640 medium supplemented with 10 newborn calf serum (NBCS) (PAA Laboratories, Inc, Pasching, Austria). NP69, an immortalized human nasopharyngeal epithelial cell line, was grown in defined-KSFM medium supplemented with epidermal growth factor (EGF) (Invitrogen, Carlsbad, USA). All cell lines were incubated in a humidified chamber with 5 CO2 at 37uC. 20 fresh primary NPC tissues, 11 fresh NP tissuses, 92 paraffin-embedded undifferentiated primary NPC specimens and 25 paraffin-embedded NP specimens were obtained at the time of diagnosis before any therapy from People’s Hospital in Zhongshan City (Guangdong, China).
