吞噬和细胞运动蛋白2对胰腺癌细胞趋化性和转移能力的影响

Effects of engulfment and cell motility 2 protein on chemotaxis and metastasis of pancreatic cancer cells

目的观察吞噬和细胞运动蛋白2(ELMO2)对胰腺癌细胞株人源胰腺癌细胞系-1(PANC-1)的趋化运动能力、细胞黏附能力和侵袭能力的影响,探讨其作用机制。方法2018年9月至2019年7月应用小干扰RNA技术对PANC-1中ELMO2蛋白进行敲减。采用配对设计方法,分为ELMO2蛋白敲减组、阴性敲减链对照组和空白对照组。建立不同浓度的CXC趋化因子配体12(CXCL12)浓度梯度,应用趋化运动实验、细胞黏附实验和细胞侵袭实验来研究ELMO2蛋白对细胞迁移运动能力、趋化运动能力和转移侵袭能力的影响。应用免疫共沉淀技术对ELMO2蛋白与G蛋白阿尔法亚基i2蛋白(Gαi2)的相互作用机制进行研究。组间比较采用双因素方差分析方法。结果将RNAi干扰链转染进入PANC-1细胞株,细胞中ELMO2蛋白表达量明显降低。在趋化因子浓度为10μg/L时,实验结果显示,敲减组定向跨膜的移动细胞数量明显低于阴性对照组[(36.67±1.21)个比(75.67±1.76)个,t=18.270,P<0.01],差异有统计学意义。在趋化因子浓度为10μg/L的条件下,实验结果显示,敲减组细胞黏附率明显低于阴性对照组[(0.47±0.02)%比(0.79±0.01)%,t=19.850,P<0.01],差异有统计学意义。趋化因子CXCL12浓度为10μg/L时,实验结果显示,敲减组穿越基质胶的细胞数量明显低于阴性对照组,侵袭能力明显减弱[(84.33±2.03)个比(132.00±0.58)个,t=22.610,P<0.01],差异有统计学意义。构建吞噬与细胞运动蛋白2的过表达质粒(代号质粒362)载体,在Flag标签蛋白抗体的免疫沉淀物中发现Gαi2蛋白的条带。结论ELMO2蛋白的敲减可以减弱胰腺癌细胞的趋化运动能力、黏附能力和侵袭能力。外源性免疫共沉淀试验结果表明,ELMO2蛋白与Gαi2蛋白存在直接相互作用关系。

Objective To observe the effects of engulfment and cell motility protein 2(ELMO2)on the chemotactic motility,migration motility,cell adhesion ability and invasion ability of pancreatic cancer cell line pancreatic cancer cell line-1(PANC-1)[American type culture collection(ATCC)],and investigate the underlying mechanism.Methods The small interfering RNA technique was used to knock down the ELMO2 protein in the pancreatic cancer cell line PANC-1.The experimental group was designed and PANC-1 cells were transfected with small interfering RNA knockdown strands and negative control strands,respectively,and composed of ELMO2 protein knockdown group,negative control group and normal group.We established concentration gradients of CXC chemokine ligand 12(CXCL12)with different concentrations,and the chemotaxis assay,adhesion assay and cell invasion assay were used to detect the role of ELMO2 on chemotaxis and metastasis of the pancreatic cancer cells.Exogenous co-immunoprecipitation was performed to show the ELMO2 interaction with the Gαi2 subunit.Two-way ANOVAwas used for statistical analysis.Results RNAi interfering strands were transfected into PANC-1 cell line,and the expression of ELMO2 protein in the cells was significantly reduced.After the expression of ELMO2 protein was reduced in PANC-1 cells,when the chemokine concentration was 10μg/L,the number of mobile cells capable of directing transmembrane in the knockdown group was significantly reduced as compared with that in the negative control group[(36.67±1.21)vs.(75.67±1.76),t=18.270,P<0.01].Under the condition of chemokine concentration of 10μg/L,the adhesion rate of PANC-1 cells in the ELMO2 knockdown group was significantly lower than that in the negative control group[(0.47±0.02)vs(0.79±0.01),t=19.850,P<0.01].When the concentration of chemokine CXCL12 was 10μg/L,the invasion ability of PANC-1 cells in ELMO2 knockdown group was significantly weakened as compared with that in the negative control group[(84.33±2.03)vs.(132.00±0.58),t=22.610,P<0.01].A GV362-ELMO2 overexpression plasmid vector was constructed.The band of Gαi2 protein was found in the immunoprecipitation of anti-flag antibody.Conclusion ELMO2 knockdown inhibited chemotaxis,adhesion and invasion of pancreatic cancer cells.Our results confirmed the physical association between ELMO2 and Gαi2 in pancreatic cancer cells through exogenous co-immunoprecipitation.