生长抑素联合人转录因子叉头框蛋白D1通过磷脂酰肌醇3激酶/蛋白激酶B通路调控胰腺癌细胞增殖、迁移和侵袭的机制研究

Study on the mechanism of somatostatin combined with human transcription factor forkhead box protein D1 in regulating the proliferation,migration and invasion of pancreatic cancer cells through the phosphatidylinositol 3-kinase/protein kinase B pathway

目的探讨生长抑素联合人转录因子叉头框蛋白D1(FOXD1)通过磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)通路调控胰腺癌细胞增殖、迁移和侵袭的机制研究。方法研究于2018年12月至2019年12月进行,通过体外培养PANC-1细胞,经过不同浓度(0 mg/L、100 mg/L、200 mg/L、400 mg/L)的生长抑素(SST)处理细胞,记为生长抑素各剂量组,其中0 mg/L和400 mg/L的生长抑素作为对照组(Control)和生长抑素组(SST);将过表达载体(pcDNA)和过表达FOXD1(FOXD1)转染至PANC-1细胞,经过400 mg/L的生长抑素及20 mol/L的PI3K抑制剂LY294002处理细胞,记为SST+pcDNA组、SST+FOXD1组和SST+FOXD1+LY294002组。四甲基偶氮唑盐微量酶反应比色法(MTT)检测细胞增殖;Transwell检测细胞迁移和侵袭;蛋白质印迹法(Western blotting)检测增殖细胞核抗原(PCNA)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、FOXD1和PI3K/AKT相关蛋白的表达;实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测FOXD1 mRNA的表达。结果与Control组相比,SST组可以抑制FOXD1 mRNA(0.47±0.03)及蛋白表达(0.42±0.04)、增殖(0.52±0.05)、迁移(66.8±7.2)和侵袭(63.7±6.5)能力,下调p-PI3K(0.43±0.04)、p-AKT(0.39±0.03)蛋白表达;与SST+pcDNA组相比,SST+FOXD1组可以促进以抑制FOXD1 mRNA(0.67±0.05)及蛋白表达(0.64±0.06)、增殖(0.71±0.07)、迁移(86.9±8.5)和侵袭(81.5±7.3)能力,上调p-PI3K(0.62±0.05)、p-AKT(0.65±0.06)蛋白表达。与SST+FOXD1组相比,SST+FOXD1+LY294002组可以抑制FOXD1 mRNA(0.37±0.04)及蛋白表达(0.35±0.03)、增殖(0.53±0.05)、迁移(67.2±6.5)和侵袭(62.5±5.2)。结论生长抑素通过调控FOXD1的表达抑制胰腺癌细胞增殖、迁移和侵袭,其作用机制可能与抑制PI3K/AKT通路有关。

Objective To investigate the mechanism of somatostatin combined with human transcription factor forkhead box protein D1(FOXD1)regulating the proliferation,migration and invasion of pancreatic cancer cells through the phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)pathway.Methods The experiment was conducted from December 2018 to December 2019,PANC-1 cells were cultured in vitro,and treated with somatostatin(SST)at different concentrations(0 mg/L,100 mg/L,200 mg/L,400 mg/L),and the cells were recorded as somatostatin dose groups,among them,0 mg/L and 400 mg/L somatostatin were used as the control group(Control)and somatostatin group(SST);the transfection of overexpression vector(pcDNA)and overexpression FOXD1(FOXD1)into PANC-1 cells,and the cells were treated with 400 mg/L somatostatin and 20 mol/L PI3K inhibitor LY294002,denoted as SST+pcDNA group,SST+FOXD1 group,and SST+FOXD1+LY294002 group.Colorimetric method of microenzyme reaction of tetramethylazazole salt(MTT)detected cell proliferation;Transwell detected cell migration and invasion;Western blot detected the expression of proliferating cell nuclear antigen(PCNA),matrix metalloproteinase-2(MMP-2),matrix metalloproteinase(MMP-9),FOXD1 and PI3K/AKT related protein;real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR)detected the expression of FOXD1 mRNA.Results Compared with the Control group,the SST group could inhibit FOXD1 mRNA(0.47±0.03)and protein(0.42±0.04)expression,proliferation(0.52±0.05),migration(66.8±7.2)and invasion(63.7±6.5),and down-regulate the expression of p-PI3K(0.43±0.04)and p-AKT(0.39±0.03)protein;compared with the SST+pcDNA group,the SST+FOXD1 group could promote and suppress FOXD1 mRNA(0.67±0.05)and protein(0.64±0.06)expression,proliferation(0.71±0.07),migration(86.9±8.5)and invasion(81.5±7.3),up-regulation of p-PI3K(0.62±0.05),p-AKT(0.65±0.06)protein expression.Compared with the SST+FOXD1 group,the SST+FOXD1+LY294002 group could inhibit FOXD1 mRNA(0.37±0.04)and protein(0.35±0.03)expression,proliferation(0.53±0.05),migration(67.2±6.5)and invasion(62.5±5.2).Conclusion Somatostatin inhibits the proliferation,migration and invasion of pancreatic cancer cells by regulating the expression of FOXD1,and its mechanism may be related to the inhibition of the PI3K/AKT pathway.