ABSTRACT
Objective:To investigate epithelial-mesenchymal transition and to explore the effects of mitochondrial dysfunction and increased expression of TGF-β1 pathway on epithelial-mesenchymal transition (EMT) in pancreatic adenocarcinoma after X-ray irradiation.Methods:Split-dose irradiations of total 40 Gy (2 Gy × 20 and 4 Gy × 10) of 6 MV X-rays were performed on PATU1 988 t cells. The migration of the cells were examined through transwell filter chambers. Real-time PCR was adopted to detect the expression of EMT-related factors E-cadherin, Vimentin, N-cadherin, and MMPs (MMP2 and MMP9), critical subunits of mitochondrial complex I, and TGF-β1. The expression of EMT-related factors and content of TGF-β1 was detected after carbonylcyanide-m-chlorophenylhydrazone(CCCP) treatment. Meanwhile, the migration potential of pancreatic cells was detected after small interfering RNA (siRNA) knockdown of the expression of TGF-β1.Results:After irradiation, the migration capacities of the cancer cells increased ( t=21.90, 35.64, P<0.05). The expression of N-cadherin ( t=4.42, 4.77, P<0.05), Vimentin ( t=4.57, 3.02, P<0.05), MMP2 ( t=7.27, 26.08, P<0.05), and MMP9 ( t=13.26, 7.29, P<0.05) all increased, while the expression of E-cadherin deceased ( t=8.37, 6.77, P<0.05). The expression of TGF-β1 ( t=90.49, 35.17, P<0.05) increased. The expression of TGF-β1 decreased with small interfering RNA, which paralleled the inhibition of the epithelial-mesenchymal transition and migration ( t=38.66, 11.54, P<0.05). Mitochondrial dysfunction was reflected by the decline in the membrane potential ( t=6.94, 29.71, P<0.05) and complex-related subunits. The expression of TGF-β1 ( t=47.93, P<0.05) and EMT-related factors further increased after mitochondrial function was destroyed ( t=16.51, P<0.05). Conclusions:Radiation-induced mitochondrial dysfunction can increase the expression of TGF-β1, which promotes epithelial-mesenchymal transition, and result in the migration of pancreatic cancer cell line.
ABSTRACT
Objective To investigate the regulatory effects of cyclic diguanylate (c-di-GMP) signaling on CheB and CheR, which were chemotaxis regulatory proteins relating to the motility of Leptospira interrogans.Methods Real-time PCR was used to determine the expression of cheB1, cheB2, cheB3, cheR1 and cheR2 genes at mRNA level during Leptospira interrogans infection.Fragments of these genes were amplified and cloned into the expression vector pET-28a, respectively, to construct the prokaryotic expression system for them.Colony morphologies of Escherichia coli (E.coli) strains that overexpressed the target genes were observed to determine the regulatory effects of c-di-GMP on CheB and CheR.Results The expression of cheB1 gene at mRNA level increased 60 min after infection and reached the peak at 90 min.Compared with the control group, the expression of cheB3 gene at mRNA level were up-regulated, while no significant difference in the expression of cheB2 and cheR genes was observed 60 min after infection.The prokaryotic expression system for the five genes was successfully constructed and the purified proteins were obtained.CheB1, CheB3 and CheR2 improved the motility of E.coli, but that was inhibited by the inhibitor of diguanylate cyclase (DGC) or phosphodiesterase (PDE).Conclusion CheB and CheR regulate the swarming motility of E.coli and are affected by intracellular c-di-GMP.