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

doi:10.3877/cma.j.issn.2095-1221.2021.04.007

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20 μg/mL ADPN，ADPN+Salubrinal组培养基中加入10 μmol/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。

关键词: 子宫内膜癌, 脂联素, 胰岛素敏感性, 内质网应激, PERK, eIF2α

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 (eIF2α dephosphorylation inhibitor) group. And the latter of two groups were treated with 20 μg/mL ADPN. And 10 μmol/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/eIF2α factors 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/eIF2α in 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/eIF2α in 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/eIF2α pathway in endometrial cancer cells.

Key words: Endometrial cancer, Adiponectin, Insulin sensitivity, Endoplasmic reticulum stress, PERK, eIF2α

表1 ADPN和Salubrinal对子宫内膜癌细胞活力的影响（ ± s）

分组	实验次数	24 h	48 h	72 h
对照	3	0.38±0.03	0.65±0.06	1.12±0.09
ADPN	3	0.33±0.04	0.46±0.06^a	0.68±0.07^a
ADPN+Salubrinal	3	0.35±0.01	0.55±0.08^ab	0.92±0.08^ab
F值		2.192	5.978	22.515
P值		0.193	0.037	0.002

表1 ADPN和Salubrinal对子宫内膜癌细胞活力的影响（ ± s）

分组	实验次数	24 h	48 h	72 h
对照	3	0.38±0.03	0.65±0.06	1.12±0.09
ADPN	3	0.33±0.04	0.46±0.06^a	0.68±0.07^a
ADPN+Salubrinal	3	0.35±0.01	0.55±0.08^ab	0.92±0.08^ab
F值		2.192	5.978	22.515
P值		0.193	0.037	0.002

图1 克隆形成实验检测ADPN和Salubrinal对子宫内膜癌细胞系HEC-1A增殖能力的影响

表2 克隆形成实验检测ADPN和Salubrinal对子宫内膜癌细胞系HEC-1A增殖能力的影响（ ± s）

分组	实验次数	克隆形成数目（个）
对照	3	127.63±5.28
ADPN	3	42.62±4.35^a
ADPN+Salubrinal	3	73.05±5.86^ab
F值		205.785
P值		< 0.001

表2 克隆形成实验检测ADPN和Salubrinal对子宫内膜癌细胞系HEC-1A增殖能力的影响（ ± s）

分组	实验次数	克隆形成数目（个）
对照	3	127.63±5.28
ADPN	3	42.62±4.35^a
ADPN+Salubrinal	3	73.05±5.86^ab
F值		205.785
P值		< 0.001

图2 ADPN和Salubrinal对子宫内膜癌细胞凋亡的影响（72 h）

表3 ADPN和Salubrinal对子宫内膜癌细胞凋亡影响（﹪， ± s）

分组	实验次数	24 h	48 h	72 h
对照	3	2.14±0.01	3.48±0.11	7.25±0.24
ADPN	3	3.01±0.09	7.38±0.20^a	21.47±0.52^a
ADPN+Salubrinal	3	2.56±0.08	5.57±0.18^ab	12.06±0.48^ab
F值		116.692	405.696	843.193
P值		< 0.001	< 0.001	< 0.001

表3 ADPN和Salubrinal对子宫内膜癌细胞凋亡影响（﹪， ± s）

分组	实验次数	24 h	48 h	72 h
对照	3	2.14±0.01	3.48±0.11	7.25±0.24
ADPN	3	3.01±0.09	7.38±0.20^a	21.47±0.52^a
ADPN+Salubrinal	3	2.56±0.08	5.57±0.18^ab	12.06±0.48^ab
F值		116.692	405.696	843.193
P值		< 0.001	< 0.001	< 0.001

表4 ADPN和Salubrinal对子宫内膜癌细胞胰岛素敏感性的影响( ± s)

分组	实验次数	2-NBDG水平
对照	3	1.00±0.06
ADPN	3	1.85±0.13^a
ADPN+Salubrinal	3	1.13±0.09^b
F值		65.969
P值		<0.001

表4 ADPN和Salubrinal对子宫内膜癌细胞胰岛素敏感性的影响( ± s)

分组	实验次数	2-NBDG水平
对照	3	1.00±0.06
ADPN	3	1.85±0.13^a
ADPN+Salubrinal	3	1.13±0.09^b
F值		65.969
P值		<0.001

图3 Western blot检测ADPN和Salubrinal对子宫内膜癌细胞系HEC-1A中PERK/eIF2α通路的影响

表5 ADPN和Salubrinal对子宫内膜癌细胞中PERK、p-eIF2α/eIF2α蛋白水平的影响（ ± s）

分组	实验次数	PERK	p-eIF2α/ eIF2α
对照	3	3.24±0.31	1.08±0.09
ADPN	3	1.72±0.14^a	0.42±0.03^a
ADPN+Salubrinal	3	1.74±0.15^a	0.94±0.09^ab
F值		49.502	63.649
P值		< 0.001	< 0.001

表5 ADPN和Salubrinal对子宫内膜癌细胞中PERK、p-eIF2α/eIF2α蛋白水平的影响（ ± s）

分组	实验次数	PERK	p-eIF2α/ eIF2α
对照	3	3.24±0.31	1.08±0.09
ADPN	3	1.72±0.14^a	0.42±0.03^a
ADPN+Salubrinal	3	1.74±0.15^a	0.94±0.09^ab
F值		49.502	63.649
P值		< 0.001	< 0.001

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

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