CD74/MIF信号通路对Capan-2细胞神经侵袭力及HIF-1α表达的影响

Effect of CD74/MIF signaling pathway on perineural invasion and HIF-1α expression of Capan-2 cells

目的:探讨应用小干扰RNA(siRNA)沉默CD74和巨噬细胞移动抑制因子(MIF)基因对Capan-2细胞神经侵袭力及缺氧诱导因子1α(HIF-1α)表达的影响及其机制。方法:空白载体处理Capan-2细胞设为对照组,CD74-siRNA慢病毒稳定感染Capan-2细胞设为实验组。Transwell实验检测细胞侵袭能力,体外嗜神经侵袭(PNI)实验检测细胞神经侵袭力。靶向MIF的siRNA进一步处理两组细胞,免疫印迹法检测CD74、MIF、HIF-1α、CX3CR1和ERK1/2蛋白表达。结果:Transwell实验显示,对照组和实验组穿膜细胞数分别为(139.1±9.9)个/孔、(102.5±9.0)个/孔;体外PNI实验显示,对照组和实验组的穿膜细胞数分别为(144.9±13.1)个/孔、(104.6±10.4)个/孔,组间比较差异均有统计学意义(均P<0.01),显示沉默CD74基因可显著抑制Capan-2细胞的侵袭能力和PNI能力。免疫印迹显示沉默CD74基因可明显抑制Capan-2细胞CD74蛋白表达,HIF-1α和CX3CR1等蛋白表达量也同时下降(均P<0.01)。MIFsiRNA不影响Capan-2细胞CD74蛋白表达(P>0.05); CD74-siRNA或MIF-siRNA均明显降低ERK1/2蛋白表达,二者联合转染抑制ERK1/2蛋白表达的效能最大(均P<0.01)。结论:沉默CD74可能通过MIFERK1/2信号通路,降低HIF-1α和CX3CR1蛋白表达而抑制Capan-2细胞侵袭和PNI能力。

Objective:To investigate the effect of small interfering RNA(siRNA)-induced silencing of CD74 gene and macrophage migration inhibitory factor(MIF)on the perineural invasion(PNI)and hypoxiainducible factor 1α(HIF-1α)expression of Capan-2 cells and its mechanism.Methods:The control group was treated with empty vectors,and the study group was stably infected with CD74-siRNA lentivirus.Transwell assay was used to determine cell invasion,and in vitro PNI assay was used to determine PNI.The MIF-targeting siRNA further processed two groups of cells.The protein expression of CD74,MIF,HIF-1α,CX3CR1 and ERK1/2 were determined by Western blot.Results:Transwell assay showed that the number of transmembrane cells in the control group and study group were(139.1±9.9)/well and(102.5±9.0)/well,respectively.In vitro PNI assay showed that the number of transmembrane cells in the control group and study group were(144.9±13.1)/well and(104.6±10.4)/well,respectively,with statistically significant difference between the two groups(both P<0.01).It suggested that silencing CD74 could significantly inhibit the invasion and PNI of Capan-2 cells.Western blot showed that silencing CD74 could significantly inhibit the expression of CD74 protein in Capan-2 cells,and the protein expression levels of HIF-1αand CX3CR1 decreased at the same time(both P<0.01).MIF-siRNA did not affect the expression of CD74 protein in Capan-2 cells(P>0.05).CD74-siRNA or MIF-siRNA significantly decreased the expression of ERK1/2 protein,and the combined transfection showed the greatest inhibitory effect(both P<0.01).Conclusion:Silencing CD74 may reduce the expression of HIF-1αand CX3CR1 proteins via MIF-ERK1/2 signaling pathway,and thereby inhibit the invasion and PNI of Capan-2 cells.