TRPC3在胰腺癌细胞系中的表达及功能

Expression and function of TRPC3 in pancreatic cancer cell lines

目的:研究TRPC3在胰腺癌(pancreatic cancer,PC)细胞系中的表达及对细胞增殖和凋亡的影响。方法:采用qRT-PCR和Western blot检测正常和PC细胞系中TRPC3表达;采用Western blot检测GdCl3和TRPC3 siRNA干预PC细胞系后TRPC3、PI3K、p-PI3K、AKT、p-AKT和PTEN蛋白表达;CCK-8法检测细胞增殖;DAPI染色法检测细胞形态学;Annexin V/PI双标法检测细胞凋亡;钙成像法检测细胞内[Ca2+]i。结果:PC细胞系中TRPC3 mRNA和蛋白表达明显高于正常胰腺上皮细胞HDPE6-C7;与溶剂和转染阴性对照细胞比较,GdCl3和转染TRPC3 siRNA的Panc1、A8184和T3M4细胞核固缩数量明显增加,Annexin V/PI双阳性凋亡细胞比例明显增多,T3M4细胞的静息[Ca2+]i、达峰[Ca2+]i和恢复相[Ca2+]i浓度明显降低;A8184和T3M4细胞的p-PI3K、p-AKT和PTEN蛋白表达明显降低。结论:TRPC3在PC细胞系中表达上调,且TRPC3通过调节细胞Ca2+介导的PI3K/AKT信号通路从而影响PC细胞增殖和凋亡。

Objective:To investigate the expression of TRPC3 in pancreatic cancer(PC)cell lines and its effects on proliferation and apoptosis.Methods:The expression of TRPC3 in normal and PC cell lines was detected by qRT-PCR and Western blot.Western blot was used to detect the expression of TRPC3,PI3K,p-PI3K,AKT,p-AKT and PTEN protein in PC cell line treated with GdCl3 and TRPC3 siRNA.Cell proliferation was detected by CCK-8 method.Cell morphology was detected by DAPI staining.Apoptosis was detected by Annexin V/PI double labeling method.[Ca2+]i was detected by calcium imaging method.Results:The expression of TRPC3 mRNA and protein in PC cell lines was significantly higher than that in normal pancreatic epithelial cells HDPE6-C7.Compared with solvent and transfection negative control group,the nuclear pyknosis of Panc1,A8184 and T3M4 cells and Annexin V+PI+were significantly increased,the concentration of resting[Ca2+]i,peak[Ca2+]i and recovery phase[Ca2+]i in T3M4 cells were significantly decreased,the expression of p-PI3K,p-AKT and PTEN protein in A8184 and T3M4 cells were significantly decreased after GdCl3 treatment and TRPC3 siRNA transfection.Conclusion:TRPC3 is up-regulated in PC cell lines,and it affects PC cell proliferation and apoptosis by regulating PI3K/AKT signaling pathway mediated by cellular Ca2+.