IL-4/IL-4R对肝细胞癌生物学行为的影响及潜在的作用机制

Effect of IL-4/IL-4R on Biological Behavior of Hepatocellular Carcinoma and Its Underlying Mechanism

目的检测白细胞介素-4/白细胞介素-4受体(Interleukin-4/Interleukin-4 receptor,IL-4/IL-4R)对肝细胞癌(hepatocellular carcinoma,HCC)生物学行为的影响及潜在的作用机制。方法体外培养Huh7细胞,并进行转染,沉默细胞中IL-4R基因的表达。CCK8法检测细胞增殖。流式细胞术检测细胞凋亡和细胞周期。Western blot法探索可能的作用机制。结果 IL-4促进Huh7细胞的增殖(P=0.01),沉默IL-4R可抑制Huh7细胞的增殖(P=0.00033),且细胞早期凋亡增加(P=0.014),凋亡蛋白Bax(P=0.016)和Caspase-3(P=0.029)表达增加,抗凋亡蛋白Bcl-2(P=0.003)表达减少。沉默IL-4R并不影响细胞晚期凋亡(P=0.108)及细胞周期(G_0/G_1期:P=0.677、S期:P=0.139、G_2/M期:P=0.855)变化。IL-4促进JAK1(P=0.01)和STAT6(P=0.005)的磷酸化。沉默IL-4R后细胞内JAK1(P=0.016)和STAT6(P=0.019)的磷酸化水平均降低。结论 IL-4/IL-4R可以激活HCC内JAK1/STAT6信号通路、促进HCC的增殖。此外,还通过调节凋亡蛋白和抗凋亡蛋白的表达,最终抑制细胞凋亡。

Objective To investigate the effect of Interleukin-4/Interleukin-4 receptor （IL-4/IL-4R） on the biological behavior of hepatocellular carcinoma（HCC） and its underlying mechanism. Methods Huh7 cells were cultured in vitro and then transfected with small interfering RNAs （siRNAs）. Cell proliferation was assessed by CCK8. Cell cycle and apoptosis were analyzed by flow cytometry. The possible signaling proteins were measured by Western blot. Results The proliferation of Huh7 cells was increased by IL-4 （P=-0.01）, however the proliferation was suppressed by silencing IL-4R（P=0.00033 ） . After knocking down IL-4R, the early apoptosis of Huh7 cells（P=0.014） and the expression of apoptosis-related proteins Bax（P=-0.016）, Caspase-3（P=0.029） were enhanced, and the expression of anti-apoptosis-related protein Bcl-2 was decreased（P=0.003）. However the late apoptosis rate（P=0.108） and the cell cycle（GdGl phase, P=0.677; S phase, P／0.139; G2/M phase, P=0.855） were not changed. Furthermore, IL-4 up-regulated the phosphorylation of JAKI（P=0.01） and STAT6（P=0.005）. The phosphorylation levels of JAKI（P=-0.016） and STAT6（P=0.019） in Huh7 cells were down-regulated by IL-4R depletion. Conclusion IL-4/IL-4R can activate JAK1/STAT6 signaling pathway and enhance the proliferation of HCC. Furthermore, IL-4/IL-4R can inhibit the apoptosis of HCC through regulating the expression of apoptotic proteins and anti-apoptotic proteins.