| Objective:From the perspective of calcium signal and calcium channels,to explore the mechanism of TGF-βon regulating the expression of stem cell markers,Nanog and Oct4,in gastric cancer cells and their ability of tumor ball formation,with the purpose of providing new insight of the regulatory mechanism of gastric cancer stem cells.Methods:（1）The ability of tumor ball test was measured before and after TGF-βstimulation on MKN45 cells,and the effects of BAPTA-AM on these functions were checked.（2）The dynamic high-speed calcium imaging was used to detect the changes of intracellular calcium after adding TGF-β（20ng/mL）and other different calcium channel blockers（BAPTA-AM,2-APB,SKF96365）.（3）qPCR was used to screen the genes that had distinct changes in expression in gastric cancer cell line MKN45 after treated with TGF-β（10ng/mL）.（4）qPCR and Western-Blot were used to detect the expression of tumor stem cell factor Nanog and Oct4 after adding TGF-β,BAPTA-AM and TRPC6channel inhibitor respectively.（5）The expression of phosphorylatedβ-catenin（ser675）in TGF-β-stimulated gastric cancer cells were investigated with 1h,45min,30min,15min and 5min by Western-Blot,and the expression of phosphorylatedβ-catenin in MKN45cells,which treated with TGF-βfor 1h and then added 2-APB,was detected.Results:（1）After TGF-β（10ng/mL）stimulation,the ability of tumor ball forming of MKN45 gastric cancer cells increased significantly（P<0.05）;and after adding BAPTA-AM（10μmol/L）,this ability reduced significantly（P<0.05）.（2）TGF-β（10ng/mL）could induce the increase of intracellular calcium in MKN45 cells,While BAPTA-AM（10μmol/L）,2-APB（40μmol/L）,and SKF96365（5μmol/L）inhibited the increase of intracellular calcium concentration which was induced by TGF-β（P<0.05）.（3）The expression of Nanog and Oct4 were up-regulated after treated with TGF-β（10ng/m L）at mRNA and protein levels,and this effect could be inhibited by BAPTA-AM（10μmol/L）and 2-APB（40μmol/L）（P<0.05）.（4）.The expression of TRPC1,TRPC3,TRPC4 and TRPC6 were up-regulated in MKN45 cells after TGF-β（10ng/m L）treating（P<0.05）,but the expression of TRPM7,TRPV6,TRPC2,TRPC5,TRPC7did not shown significant changes.（5）Expression of phosphorylatedβ-catenin in MKN45cells was up-regulated and it reached the peak after treated with TGF-β（10ng/mL）for 1h.Meanwhile,expression of phosphorylatedβ-catenin was significant decreased When2-APB（40μmol/L）was added（P<0.05）.Conclusion:1.The change of intracellular Ca2+signal is a key link of TGF-βto promote the expression of stem genes and the ability for ball forming in gastric cancer cells;2.TRPC,especially TRPC6,may be the most important calcium channels in the progress of increasing intracellular calcium induced by TGF-β;3.Wnt/β-catenin may be an important relative pathway for calcium signal downstream regulation of stem markers by gastric cancer. |
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