Real-time Quantitative Reverse Polymerase Chain Reaction Research Articles

Regulation of Notch3 on proliferation and metastasis of pancreatic catheter adenocarcinoma cells

Objective To observe the expression of Notch3 in pancreatic ductal adenocarcinoma cells (PDAC) andcontrol of PDAC invasion and metastasis. Methods The expression of Notch3 in pancreatic ductal adenocarcinoma cells was detected by real-time quantitative reverse transcription polymerase chainreaction (RT-qPCR). Use small interfering RNA (siRNA) to transfect of PDAC. Use Western blotting to evaluate Notch3 expression in three Notch3 siRNA transfected cell lines. The impact of expression of CD44v6, E-cadherin, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), urokinase-type plasminogen activator (uPA), cyclo-oxygen-ase-2 (COX-2), extracellular regulated protein kinases1/2 (ERK1/2), phosphorylation extracellular regulated protein kinases1/2 (pERK1/2). Cell migration and invasion were assessed using Transwell cell culture chambers. Results The Transwell migration assay revealed that the number of migrating cells in BXPC-3, PANC-1 and HPDE6c7 was (186.2±3.2), (252.5±2.6) and (24.4±1.5) cells respectively (P=0.008). The transwell invasion assay revealed that the number of migrating cells in BXPC-3, PANC-1 and HPDE6c7 was (171.1±2.3), (224.7±2.4) and (24.4±1.5) cells respectively (P=0.009). After inhibition of Notch3, the transwell migration assay revealed that the number of migrating cells in BXPC-3, PANC-1 was significantly higher than the siRNA transfected cells [(140.6±1.2) cells vs. (45.4±1.5) cells; (245.4±1.6) cells vs. (70.3±1.1) cells, P=0.032]. The transwell invasion assay revealedthatthe number of migrating cells in BXPC-3, PANC-1 was significantly higher than the siRNA transfected cells [(140.6±1.2) cells vs. (45.4±1.5) cells; (245.4±1.6) cells vs. (70.3±1.1) cells, P=0.027]. Western blotting results showthat inhibition of Notch3 decreases the protein expression of CD44v6, MMP-2, MMP-9, VEGF and uPA and increase the protein expression of E-cadherin. In Notch3-inhibited PDAC cells, the expression of COX-2 and p-ERK1/2 decreased. Conclusion Notch3 maypromote the processes of migration and invasion in PDAC cells. Key words: Pancreatic ductal adenocarcinoma; Metastasis; Oncogene

Molecular cloning and expression analysis of Fem1b from oriental river prawn Macrobrachium nipponense.

Feminization-1 homolog b (Fem1b) is one of the genes essential for male development and play central roles in sex determination of Caenorhabditis elegans. In this study, we cloned and characterized the full-length Fem1b cDNA from the freshwater prawn Macrobrachium nipponense (MnFem1b) in different tissues and at different developmental stages. Real-time quantitative reverse polymerase chain reaction (RT-qPCR) showed that the MnFem1b gene was expressed in all investigated tissues, with the highest expression level found in the testes. The results revealed that the MnFem1b gene might play roles in aspects of development of the male prawn phenotype. The RT-qPCR also revealed that MnFem1b mRNA expression was significantly increased at 10 days after metamorphosis. The expression levels in all investigated tissues showed a certain degree of sexually dimorphism, the expression levels in males were significantly higher than those in females (P < 0.05). Notably, the highest expression of MnFem1b was found in the testes. The expression of MnFem1b in different tissues indicates that it plays multiple biological functions in M. nipponense.