AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was ...AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was performed using the vector (pGPU6)-based small interference RNA (siRNA) plasmid gene silence system to specifically knock down MMP-2 expression in pancreatic cancer cell line,BxPC-3. Four groups of different specific target sequence in coding region of MMP-2 and one non-specific sequence were chosen to construct four experimental siRNA plasmids of pGPU6-1,pGPU6-2,pGPU6-3 and pGPU6-4,and one negative control siRNA plasmid of pGPU6 (-). MMP-2 expression was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation and apoptosis were examined by methyl thiazolyl tetrazolium (MTT) and flow cytometry,respectively. The abilities of adhesion and invasion were detected by cell adhesion assay and cell invasion assay using Transwell chambers.RESULTS:The expression of MMP-2 was inhibited and the inhibitory effects of different sequence varied. pGPU6-1 group had the most efficient inhibitory effect,followed by pGPU6-2 and pGPU6-3 groups.Invasiveness and adhesion were more significantly reduced in pGPU6-1,pGPU6-2 and pGPU6-3 groups as compared with pGPU6 (-) and blank control groups. However,no difference concerning cell proliferation and apoptosis was observed after transfection between experiment groups and control groups.CONCLUSION:RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the pancreatic cancer cell line,BxPC-3,leading to a potent suppression of tumor cell adhesion and invasion without affecting cell proliferation and apoptosis. These findings suggest that the RNAi approach towards MMP-2 may be an effective therapeutic strategy for the clinical management of pancreatic tumor.展开更多
AIM: To explore the effects of siRNA silencing of PIK3CA on proliferation, migration and invasion of gastric cancer cells and to investigate the underlying mechanisms. METHODS: The mutation of PIK3CA in exons 9 and 20...AIM: To explore the effects of siRNA silencing of PIK3CA on proliferation, migration and invasion of gastric cancer cells and to investigate the underlying mechanisms. METHODS: The mutation of PIK3CA in exons 9 and 20 of gastric cancer cell lines HGC-27, SGC-7901, BGC-823, MGC-803 and MKN-45 was screened by polymerase chain reaction (PCR) followed by sequencing. BGC-823 cells harboring no mutations in either of the exons, and HGC-27 cells containing PIK3CA mutations were employed in the current study. siRNA targeting PIK3CA was chemically synthesized and was transfect- ed into these two cell lines in vitro . mRNA and protein expression of PIK3CA were detected by real-time PCR and Western blotting, respectively. We also measured phosphorylation of a serine/threonine protein kinase (Akt) using Western blotting. The proliferation, migration and invasion of these cells were examined separately by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT), wound healing and Transwell chambers assay. RESULTS: The siRNA directed against PIK3CA effectively led to inhibition of both endogenous mRNA and protein expression of PIK3CA, and thus significantly down-regulated phosphorylation of Akt (P < 0.05). Furthermore, simultaneous silencing of PIK3CA resulted in an obvious reduction in tumor cell proliferation activity, migration and invasion potential (P < 0.01). Intriguing, mutant HGC-27 cells exhibited stronger invasion ability than that shown by wild-type BGC-823 cells. Knockdown of PIK3CA in mutant HGC-27 cells contributed to a reduction in cell invasion to a greater extent than in non-mutant BGC-823 cells. CONCLUSION: siRNA mediated targeting of PIK3CA may specifically knockdown the expression of PIK3CA in gastric cancer cells, providing a potential implication for therapy of gastric cancer.展开更多
Chaetomium globosum is one of the most common fungi in nature. It is best known for producing chaetoglobosins; however, the molecular basis of chaetoglobosin biosynthesis is poorly understood in this fungus. In this s...Chaetomium globosum is one of the most common fungi in nature. It is best known for producing chaetoglobosins; however, the molecular basis of chaetoglobosin biosynthesis is poorly understood in this fungus. In this study, we utilized RNA inter- ference （RNAi） to characterize a polyketide synthase gene, pks-1, in C. globosum that is involved in the production of chaeto- globosin A. When pks-1 was knocked down by RNAi, the production of chaetoglobosin A dramatically decreased. Knock-down mutants also displayed a pigment-deficient phenotype. These results suggest that the two polyketides, melanin and chaetoglobosin, are likely to share common biosynthetic steps. Most importantly, we found that pks-I also plays a critical role in sporulation. The silenced mutants ofpks-1 lost the ability to produce spores. We propose that polyketides may modulate cellular development via an unidentified action. We also suggest that C. globosum pks-1 is unique because of its triple role in melanin formation, chaetoglobosin biosynthesis and sporulation. This work may shed light on chaetoglobosin biosynthesis and indicates a relationship between secondary metabolism and fungal morphogenesis.展开更多
基金Supported by Tiantan Hospital Scientific Project Grant Fund
文摘AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was performed using the vector (pGPU6)-based small interference RNA (siRNA) plasmid gene silence system to specifically knock down MMP-2 expression in pancreatic cancer cell line,BxPC-3. Four groups of different specific target sequence in coding region of MMP-2 and one non-specific sequence were chosen to construct four experimental siRNA plasmids of pGPU6-1,pGPU6-2,pGPU6-3 and pGPU6-4,and one negative control siRNA plasmid of pGPU6 (-). MMP-2 expression was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation and apoptosis were examined by methyl thiazolyl tetrazolium (MTT) and flow cytometry,respectively. The abilities of adhesion and invasion were detected by cell adhesion assay and cell invasion assay using Transwell chambers.RESULTS:The expression of MMP-2 was inhibited and the inhibitory effects of different sequence varied. pGPU6-1 group had the most efficient inhibitory effect,followed by pGPU6-2 and pGPU6-3 groups.Invasiveness and adhesion were more significantly reduced in pGPU6-1,pGPU6-2 and pGPU6-3 groups as compared with pGPU6 (-) and blank control groups. However,no difference concerning cell proliferation and apoptosis was observed after transfection between experiment groups and control groups.CONCLUSION:RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the pancreatic cancer cell line,BxPC-3,leading to a potent suppression of tumor cell adhesion and invasion without affecting cell proliferation and apoptosis. These findings suggest that the RNAi approach towards MMP-2 may be an effective therapeutic strategy for the clinical management of pancreatic tumor.
基金Supported by Natural Science Foundation of Hunan Province, No.09JJ3060Health Bureau Fund of Guangzhou, No.201102A213006Education Bureau Fund of Guangzhou, No.10A186
文摘AIM: To explore the effects of siRNA silencing of PIK3CA on proliferation, migration and invasion of gastric cancer cells and to investigate the underlying mechanisms. METHODS: The mutation of PIK3CA in exons 9 and 20 of gastric cancer cell lines HGC-27, SGC-7901, BGC-823, MGC-803 and MKN-45 was screened by polymerase chain reaction (PCR) followed by sequencing. BGC-823 cells harboring no mutations in either of the exons, and HGC-27 cells containing PIK3CA mutations were employed in the current study. siRNA targeting PIK3CA was chemically synthesized and was transfect- ed into these two cell lines in vitro . mRNA and protein expression of PIK3CA were detected by real-time PCR and Western blotting, respectively. We also measured phosphorylation of a serine/threonine protein kinase (Akt) using Western blotting. The proliferation, migration and invasion of these cells were examined separately by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT), wound healing and Transwell chambers assay. RESULTS: The siRNA directed against PIK3CA effectively led to inhibition of both endogenous mRNA and protein expression of PIK3CA, and thus significantly down-regulated phosphorylation of Akt (P < 0.05). Furthermore, simultaneous silencing of PIK3CA resulted in an obvious reduction in tumor cell proliferation activity, migration and invasion potential (P < 0.01). Intriguing, mutant HGC-27 cells exhibited stronger invasion ability than that shown by wild-type BGC-823 cells. Knockdown of PIK3CA in mutant HGC-27 cells contributed to a reduction in cell invasion to a greater extent than in non-mutant BGC-823 cells. CONCLUSION: siRNA mediated targeting of PIK3CA may specifically knockdown the expression of PIK3CA in gastric cancer cells, providing a potential implication for therapy of gastric cancer.
基金the National Natural Science Foundation of China (Grant No. 30970084)the National Basic Research Program of China (Grant No. 2007CB707801)
文摘Chaetomium globosum is one of the most common fungi in nature. It is best known for producing chaetoglobosins; however, the molecular basis of chaetoglobosin biosynthesis is poorly understood in this fungus. In this study, we utilized RNA inter- ference （RNAi） to characterize a polyketide synthase gene, pks-1, in C. globosum that is involved in the production of chaeto- globosin A. When pks-1 was knocked down by RNAi, the production of chaetoglobosin A dramatically decreased. Knock-down mutants also displayed a pigment-deficient phenotype. These results suggest that the two polyketides, melanin and chaetoglobosin, are likely to share common biosynthetic steps. Most importantly, we found that pks-I also plays a critical role in sporulation. The silenced mutants ofpks-1 lost the ability to produce spores. We propose that polyketides may modulate cellular development via an unidentified action. We also suggest that C. globosum pks-1 is unique because of its triple role in melanin formation, chaetoglobosin biosynthesis and sporulation. This work may shed light on chaetoglobosin biosynthesis and indicates a relationship between secondary metabolism and fungal morphogenesis.
