GLDC通过PI3K/Akt/mTOR通路调控卵巢癌细胞的增殖与凋亡

doi:10.3760/cma.j.cn371439-20210514-00142

摘要/Abstract

摘要： 目的探究甘氨酸脱氢酶(GLDC)基因通过PI3K/Akt/mTOR调控卵巢癌细胞增殖与凋亡的机制。方法采用RNA干扰技术沉默卵巢癌HEY、SK-OV-3细胞中GLDC的表达,分别将细胞分为si-control组、si-GLDC#1组和si-GLDC#2组,si-control组为转染siRNA-control的细胞,si-GLDC#1组为转染siRNA-GLDC#1的细胞,si-GLDC#2组为转染siRNA-GLDC#2的细胞。采用蛋白质印迹法检测GLDC蛋白表达水平以及PI3K/Akt/mTOR蛋白磷酸化水平,分别采用CCK-8法、Transwell小室迁移实验、细胞划痕实验和流式细胞仪检测细胞增殖、迁移和凋亡能力。结果 GLDC在卵巢癌HEY细胞si-control组、si-GLDC#1组和si-GLDC#2组中的相对表达量分别为1.00±0.01、0.68±0.10、0.80±0.08,差异有统计学意义(F=13.80,P=0.006);GLDC在卵巢癌SK-OV-3细胞si-control组、si-GLDC#1组、si-GLDC#2组中的相对表达量分别为1.02±0.01、0.58±0.17、0.60±0.25,差异具有统计学意义(F=6.08,P=0.036)。si-control组、si-GLDC#1组和si-GLDC#2组3组HEY细胞培养24 h后吸光度(A)值分别为1.04±0.03、0.91±0.02、0.82±0.01,48 h后分别为1.53±0.13、1.30±0.03、1.29±0.07,72 h后分别为1.44±0.08、1.25±0.01、1.15±0.03,差异均具有统计学意义(F=83.14,P<0.001;F=8.96,P=0.007;F=29.55,P<0.001),进一步两两比较,24、48、72 h si-GLDC#1组、si-GLDC#2组细胞增殖活力均低于si-control组,差异均有统计学意义(均P<0.05)。在HEY细胞中,si-control组、si-GLDC#1组、si-GLDC#2组迁移细胞数目分别为57.33±6.43、27.67±5.13、30.67±2.31,差异具有统计学意义(F=32.88,P=0.001);si-GLDC#1组、si-GLDC#2组迁移细胞数较si-control组显著下降,差异均有统计学意义(P<0.001;P=0.001);在SK-OV-3细胞中也观察到类似结果。在SK-OV-3细胞中,si-control组、si-GLDC#1组、si-GLDC#2组的划痕愈合率分别为(51.27±1.59)%、(26.35±2.94)%、(26.34±7.69)%,差异具有统计学意义(F=26.54,P=0.001);si-GLDC#1组、si-GLDC#2组细胞划痕愈合率较si-control组显著下降,差异均有统计学意义(均P=0.001)。在HEY细胞中,si-control组、si-GLDC#1组、si-GLDC#2组细胞凋亡率分别为(7.11±0.82)%、(10.44±1.50)%、(17.39±1.55)%,差异具有统计学意义(F=46.52,P<0.001);si-GLDC#1组、si-GLDC#2组细胞凋亡率较si-control组均显著上升,差异均有统计学意义(P=0.022;P<0.001);在SK-OV-3细胞中也观察到类似结果。在HEY细胞中,si-control组、si-GLDC#1组、si-GLDC#2组中PI3K总蛋白差异无统计学意义(F=0.54,P=0.631),但pAkt/Akt、pmTOR/mTOR水平差异具有统计学意义(F=22.14,P=0.016;F=10.57,P=0.044);其中si-GLDC#1组、si-GLDC#2组较si-control组pAkt/Akt、pmTOR/mTOR均显著降低(P=0.015,P=0.008;P=0.039,P=0.023);在SK-OV-3细胞中也观察到类似结果。结论在卵巢癌细胞中,沉默GLDC可通过抑制PI3K/Akt/mTOR通路抑制细胞增殖并促进细胞凋亡。

关键词: 卵巢肿瘤, 细胞增殖, 细胞运动, 细胞凋亡, 甘氨酸脱氢酶

Abstract: Objective To explore the mechanism of glycine dehydrogenase (GLDC) regulating the proliferation and apoptosis of ovarian cancer cells through PI3K/Akt/mTOR pathway. Methods RNA interference method was used to silence the expression of GLDC in ovarian cancer cell lines HEY and SK-OV-3. The HEY and SK-OV-3 cells were divided into si-control group (transfected with siRNA-control), si-GLDC#1 group (trans-fected with siRNA-GLDC#1) and si-GLDC#2 group (transfected with siRNA-GLDC#2). The expression level of GLDC and the protein phosphorylation level of PI3K/Akt/mTOR were detected by Western blotting. Cell proli-feration, migration and apoptosis were detected by CCK-8 method, Transwell chamber test, cell scratch test and flow cytometry. Results The relative expression levels of GLDC in the si-control group, si-GLDC#1 group amd si-GLDC#2 group of HEY cells were 1.00±0.01, 0.68±0.10, 0.80±0.08, and there was a statistically significant difference (F=13.80, P=0.006). The relative expression levels of GLDC in the si-control group, si-GLDC#1 group and si-GLDC#2 group of SK-OV-3 cells were 1.02±0.01, 0.58±0.17, 0.60±0.25, and there was a statistically significant difference (F=6.08, P=0.036). The absorbance (A) values in the si-control group, si-GLDC#1 group and si-GLDC#2 group of HEY cells were 1.04±0.03, 0.91±0.02, 0.82±0.01 at 24 h after transfection, 1.53±0.13, 1.30±0.03, 1.29±0.07 at 48 h after transfection, 1.44±0.08, 1.25±0.01, 1.15±0.03 at 72 h after transfection, and there were statistically significant differences (F=83.14, P<0.001; F=8.96, P=0.007; F=29.55, P<0.001). Further pairwise comparison showed that the proliferation abilities of the si-GLDC#1 and si-GLDC#2 group at 24, 48 and 72 h were significantly lower than those of the si-control group (all P<0.05). In HEY cells, the migration numbers of cells in the si-control group, si-GLDC#1 group and si-GLDC#2 group were 57.33±6.43, 27.67±5.13 and 30.67±2.31, and there was a statistically significantly difference (F=32.88, P=0.001). The migration numbers of cells in the si-GLDC#1 group and si-GLDC#2 group were significantly lower than that in the si-control group (P<0.001; P=0.001). Similar results were also observed in SK-OV-3 cells. In SK-OV-3 cells, the scratch healing rates in the si-control group, si-GLDC#1 group and si-GLDC#2 group were (51.27±1.59)%, (26.35±2.94)% and (26.34±7.69)%, and there was a statistically significant difference (F=26.54, P=0.001). The scratch healing rates in the si-GLDC#1 group and si-GLDC#2 group were significantly lower than that in the si-control group (both P=0.001). In HEY cells, the apoptosis rates in the si-control group, si-GLDC#1 group and si-GLDC#2 group were (7.11±0.82)%, (10.44±1.50)%, (17.39±1.55)%, and there was a statistically significantly difference (F=46.52, P<0.001). The apoptosis rates in the si-GLDC#1 group and si-GLDC#2 group were significantly higher than that in the si-control group (P=0.022; P<0.001). Similar results were also observed in SK-OV-3 cells. In HEY cells, there was no significant difference in total PI3K protein in the si-control group, si-GLDC#1 group and si-GLDC#2 group (F=0.54, P=0.631), but there were significant differences in pAkt/Akt and pmTOR/mTOR levels (F=22.14, P=0.016; F=10.57, P=0.044). The pAkt/Akt and pmTOR/mTOR levels in the si-GLDC#1 group and si-GLDC#2 group were significantly lower than those in the si-control group (P=0.015, P=0.008; P=0.039, P=0.023). Similar results were also observed in SK-OV-3 cells. Conclusion In ovarian cancer cells, GLDC silencing can inhibit cell proliferation and promote apoptosis by inhibiting the PI3K/Akt/mTOR pathway.

Key words: Ovarian neoplasms, Cell proliferation, Cell movement, Cell apoptosis, Glycine dehydrogenase

李哲丰, 李洁, 赵晓婷, 岳文涛. GLDC通过PI3K/Akt/mTOR通路调控卵巢癌细胞的增殖与凋亡[J]. 国际肿瘤学杂志, 2021, 48(12): 716-722.

Li Zhefeng, Li Jie, Zhao Xiaoting, Yue Wentao. GLDC regulates proliferation and apoptosis of ovarian cancer cells through PI3K/Akt/mTOR pathway[J]. Journal of International Oncology, 2021, 48(12): 716-722.

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组别	HEY				SK-OV-3
组别	PI3K	pAkt/Akt	pmTOR/mTOR		PI3K	pAkt/Akt	pmTOR/mTOR
si-control组	1.00±0.01	1.19±0.16	1.00±0.06	0.71±0.09		0.94±0.09	1.01±0.09
si-GLDC#1组	1.30±0.39	0.71±0.01^a	0.52±0.16^a	0.93±0.01		0.50±0.17^a	0.62±0.01^a
si-GLDC#2组	1.14±0.31	0.59±0.05^a	0.41±0.16^a	0.71±0.29		0.50±0.06^a	0.50±0.11^a
F值	0.54	22.14	10.57	0.97		9.67	21.14
P值	0.631	0.016	0.044	0.475		0.049	0.017

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