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

1	Shafa A, Kumar A, Torres D, et al. Minimally invasive hysterectomy and bariatric surgery to improve endometrial cancer survivorship[J]. Obstet Gynecol, 2019, 134(3):570-572.
2	Lai Y, Sun C. Association of abnormal glucose metabolism and insulin resistance in patients with atypical and typical endometrial cancer[J]. Oncol Lett, 2018, 15(2):2173-2178.
3	Erkan G, Tayyar AT, Açmaz G, et al. Role of osteocalcin, tumor necrosis factor-alpha and adiponectin in polycystic ovary syndrome patients with insulin resistance[J]. Turk J Obstet Gynecol, 2017, 14(2):89-93.
4	蔡志福, 邓露, 王毛毛, 等. 脂联素对子宫内膜癌细胞AMPK/mTOR/S6K1信号通路及胰岛素增敏的影响[J]. 中华妇产科杂志, 2018, 53(8):554-560.
5	Song J, Kim OY. Melatonin modulates neuronal cell death induced by endoplasmic reticulum stress under insulin resistance condition[J]. Nutrients, 2017, 9(6):593.
6	Ramos-Lopez O, Riezu-Boj JI, Milagro FI, et al. DNA methylation signatures at endoplasmic reticulum stress genes are associated with adiposity and insulin resistance[J]. Mol Genet Metab, 2018, 123(1):50-58.
7	Guo Q, Jin S, Hu H, et al. Hypoxia in 3T3-L1 adipocytes suppresses adiponectin expression via the PERK and IRE1 unfolded protein response[J]. Biochem Biophys Res Commun, 2017, 493(1):346-351.
8	刘思邈, 沈影, 李佳, 等. 基于抗炎机制的小檗碱防治多囊卵巢综合征相关子宫内膜癌的研究进展[J]. 中国药房, 2019, 30(16):2294-2297.
9	Hanawa S, Mitsuhashi A, Shozu M. Antitumor effects of metformin via indirect inhibition of protein phosphatase 2A in patients with endometrial cancer[J]. PLoS ONE, 2018, 13(2):e0192759.
10	Bizzarri M, Dinicola S, Cucina A. Modulation of both insulin resistance and cancer growth by inositol[J]. Curr Pharm Des, 2017, 23(34):5200-5210.
11	Shang CG, Liu ZH, Wang XH, et al. Effect of high-fat diet-induced disorders on rat with endometrial hyperplasia and adiponectin system in circulation and uterus[J]. Chin Med J (Engl), 2017, 130(15):1831-1837.
12	邓露, 梁慧瑾, 蔡志福, 等. 血清脂联素水平与Ⅰ型子宫内膜癌的相关性研究[J]. 中国癌症防治杂志, 2018, 10(3):225-228.
13	Li X, Yu Z, Fang L, et al. Expression of adiponectin receptor-1 and prognosis of epithelial ovarian cancer patients[J]. Med Sci Monit, 2017, 23(1):1514-1521.
14	郭会敏, 范毛川, 华方方, 等. 脂联素对子宫内膜癌Ishikawa细胞增殖及侵袭和转移能力的影响[J]. 新乡医学院学报, 2018, 35(7):563-567.
15	Song J, Kim OY. Melatonin modulates neuronal cell death induced by endoplasmic reticulum stress under insulin resistance condition[J]. Nutrients, 2017, 9(6):593
16	Mostafa DG, Khaleel EF, Badi RM, et al. Rutin hydrate inhibits apoptosis in the brains of cadmium chloride-treated rats via preserving the mitochondrial integrity and inhibiting endoplasmic reticulum stress[J]. Neurol Res, 2019, 41(7):594-608.
17	Chen J, Zheng G, Guo H, et al. The effect of metformin treatment on endoplasmic reticulum (ER) stress induced by status epilepticus (SE) via the PERK-eIF2α-CHOP pathway[J]. Bosn J Basic Med Sci, 2018, 18(1):49-54.
18	徐亚杰, 鲍红光, 王晓亮, 等. 外源性脂联素对术后认知功能障碍老龄小鼠海马AGEs-ROS-ERS通路的影响[J]. 中华麻醉学杂志, 2015, 35(7):801-804.
19	Zhao YL, Li F, Liu YW, et al. Adiponectin attenuates endoplasmic reticulum stress and alveolar epithelial apoptosis in COPD rats[J]. Eur Rev Med Pharmacol Sci, 2017, 21(21):4999-5007.
20	Karadeniz Z, Aynacıoğlu AŞ, Bilir A, et al. Inhibition of midkine by metformin can contribute to its anticancer effects in malignancies: A proposal mechanism of action of metformin in context of endometrial cancer prevention and therapy[J]. Med Hypotheses, 2020, 134(1):109420.
21	Mu N, Xu T, Gao M, et al. Therapeutic effect of metformin in the treatment of endometrial cancer[J]. Oncol Lett, 2020, 20(5):156.

[1]	梁志清. 子宫内膜癌微创与个性化治疗的进化与发展[J/OL]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 621-621.
[2]	孙鸿坤, 艾虹, 陈正. 内质网应激介导的牙周炎骨改建失衡的研究进展[J/OL]. 中华口腔医学研究杂志(电子版), 2024, 18(04): 211-218.
[3]	许娟, 张党锋. 尼达尼布对肺纤维化小鼠肺功能及内质网应激反应的影响[J/OL]. 中华肺部疾病杂志(电子版), 2023, 16(05): 673-675.
[4]	刘洪云, 李翠, 张海堂, 张玉英, 黄成香. 经脐单孔腹腔镜技术在子宫内膜癌手术中的应用[J/OL]. 中华腔镜外科杂志(电子版), 2024, 17(03): 182-184.
[5]	王酉, 严斌, 狄文, 楼微华. 经脐单孔腹腔镜前哨淋巴结活检术在早期子宫内膜癌手术中的探讨[J/OL]. 中华腔镜外科杂志(电子版), 2024, 17(03): 173-176.
[6]	周福兴, 贺艳丽, 张潍. 单孔腹腔镜技术在子宫内膜癌全面分期手术中的应用[J/OL]. 中华腔镜外科杂志(电子版), 2024, 17(01): 55-57.
[7]	陈珂瑶, 柳祎, 孙力. vNOTES全子宫＋前哨淋巴结切除治疗Ⅰ期子宫内膜癌[J/OL]. 中华腔镜外科杂志(电子版), 2024, 17(01): 21-25.
[8]	王细文, 刘恒炜, 黄桔园, 易跃雄, 张蔚. 单孔腹腔镜UGF两翼法腹主动脉旁淋巴结切除术[J/OL]. 中华腔镜外科杂志(电子版), 2023, 16(04): 246-248.
[9]	桑田, 赵磊, 佟琰, 欧阳清, 陈香美. 急性肾损伤的内质网应激相关基因和通路的生物信息学分析[J/OL]. 中华肾病研究电子杂志, 2024, 13(01): 26-33.
[10]	张萌, 喻中华. 阿尔茨海默病患者血清脂联素、Lp-PLA2、IL-17的表达及与认知功能的相关性分析[J/OL]. 中华脑科疾病与康复杂志(电子版), 2023, 13(06): 358-363.
[11]	吴迪, 闫志风, 李明霞, 孟元光. 晚期子宫内膜癌免疫治疗的探索[J/OL]. 中华临床医师杂志(电子版), 2024, 18(03): 231-237.
[12]	袁媛, 赵良平, 刘智慧, 张丽萍, 谭丽梅, 閤梦琴. 子宫内膜癌组织中miR-25-3p、PTEN的表达及与病理参数的关系[J/OL]. 中华临床医师杂志(电子版), 2023, 17(09): 1016-1020.
[13]	郭如烨, 孟黎明, 陈楠, 宋玉莹, 尹海燕, 郭岩. Apelin/APJ系统对帕金森病模型的神经保护作用及机制研究进展[J/OL]. 中华诊断学电子杂志, 2023, 11(04): 276-282.
[14]	吴晓明, 翟仰魁, 王娟, 张硕, 许杰, 潘从清. 男性2 型糖尿病患者空腹C 肽和定量胰岛素敏感性检测指数与血浆致动脉粥样硬化指数的相关性[J/OL]. 中华肥胖与代谢病电子杂志, 2024, 10(04): 288-294.
[15]	殷雨来, 李雪, 何晓阳, 张晓宇. 体质量指数和4种女性特征性癌症的因果关系：一项两样本孟德尔随机化研究[J/OL]. 中华肥胖与代谢病电子杂志, 2023, 09(04): 253-260.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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