脂联素通过PERK/eIF2a介导的内质网应激通路调节子宫内膜癌细胞增殖、凋亡和胰岛素敏感性

Adiponectin regulates proliferation,apoptosis,and insulin sensitivity through PERK/eIF2α-mediated endoplasmic reticulum stress in endometrial cancer cells

目的探讨脂联素(ADPN)通过PERK/eIF2α调节子宫内膜癌细胞内质网应激以及增殖、凋亡和胰岛素敏感性的机制。方法将HEC-1A细胞分为对照组(细胞不进行任何处理)、ADPN组和ADPN+Salubrinal(eIF2α去磷酸化抑制剂)组。ADPN组和ADPN+Salubrinal组加入20μg/mL ADPN,ADPN+Salubrinal组培养基中加入10μmol/L Salubrinal以促进eIF2α的磷酸化水平。通过CCK-8法、克隆形成实验、流式细胞术检测细胞活力、增殖和凋亡情况。通过葡萄糖吸收转运荧光探针(2-NBDG)检测葡萄糖摄取情况评估胰岛素敏感性。通过Western blot检测PERK、eIF2α和p-eIF2α蛋白表达水平。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果与对照组比较,ADPN组和ADPN+Salubrinal组的克隆形成数目[(127.63±5.28)个比(42.62±4.35)个、(73.05±5.86)个]减少;与ADPN组比较,ADPN+Salubrinal组的细胞克隆形成数目[(42.62±4.35)个比(73.05±5.86)个]增多,差异有统计学意义(P均<0.001)。与对照组比较,ADPN组和ADPN+Salubrinal组的凋亡率[48 h:(3.48±0.11)﹪比(7.38±0.20)﹪、(5.57±0.18)﹪,72 h:(7.25±0.24)﹪比(21.47±0.52)﹪、(12.06±0.48)﹪]均升高;与ADPN组比较,ADPN+Salubrinal组凋亡率均降低,差异有统计学意义(P均<0.001)。与对照组比较,ADPN组细胞摄取2-NBDG水平(1.00±0.06比1.85±0.13)升高;与ADPN组比较,ADPN+Salubrinal组摄取2-NBDG水平降低,差异具有统计学意义(P均<0.001)。与对照组比较,ADPN组和ADPN+Salubrinal组的PERK和p-eIF2α/eIF2α水平(3.24±0.31比1.72±0.14、1.74±0.15,1.08±0.09比0.42±0.03、0.94±0.09)均降低;与ADPN组比较,ADPN+Salubrinal组的p-eIF2α/eIF2α水平升高,差异具有统计学意义(P均<0.001)。结论在子宫内膜癌细胞中,PERK/eIF2α通路与ADPN调节胰岛素的敏感性、细胞增殖和凋亡的机制有关。

Objective To explore whether adiponectin(ADPN)can regulate endoplasmic reticulum(ER)stress and cells proliferation,apoptosis or insulin sensitivity in endometrial cancer cells through PERK/eIF2α.Methods endometrial cancer cells(HEC-1A)were divided into control group(no treatment),ADPN group and ADPN+Salubrinal(eIF2αdephosphorylation inhibitor)group.And the latter of two groups were treated with 20μg/mL ADPN.And 10μmol/L Salubrinal was added in the ADPN+Salubrinal group.The cell viability,proliferation and apoptosis were detected by CCK-8 method,clone formation experiment,and flow cytometry.PERK/eIF2αfactors were detected by Western blot.Results The cell viability and cell proliferation ability(42.62±4.35 points)in the ADPN group were significantly lower than those of the control group(P<0.001).The cell viability and cell proliferation ability(127.63±5.28 points)in the ADPN+Salubrinal group were significantly higher than those of the ADPN group(P<0.001).The apoptotic rate of ADPN group at 48 h and 72 h[48 h:(7.38±0.20)﹪,(5.57±0.18)﹪,72 h:(21.47±0.52)﹪,(12.06±0.48)﹪]were significantly higher than those of control group[48 h:(3.48±0.11)﹪,72 h:(7.25±0.24)﹪](P<0.001).Compared with the ADPN group,the apoptotic rate in the ADPN+Salubrinal group decreased,and the difference was statistically significant(P<0.001).The expression level of 2-NBDG uptake in ADPN group(1.85±0.13)was significantly higher than that in control group(1.00±0.06)(P<0.001).The expression level of 2-NBDG in ADPN+Salubrinal group(1.13±0.09,1.08±0.09)was significantly lower than that in ADPN group(P<0.001).The protein levels of PERK and p-eIF2α/eIF2αin the ADPN group(1.72±0.14,0.42±0.03)were significantly lower than those in the control group(P<0.001).The protein level of p-eIF2α/eIF2αin ADPN+Salubrinal group(0.94±0.09)was significantly higher than that in ADPN group(P<0.001).Conclusion ADPN may regulate cell proliferation,apoptosis and insulin sensitivity through PERK/eIF2αpathway in endometrial cancer cells.