李凤敏, 韩进美, 黄红茜, 王娜, 张爱华, 韩雪. NaAsO2染毒HaCaT细胞中丝氨酸合成途径关键酶的表达及其作用[J]. 环境与职业医学, 2022, 39(5): 545-549. DOI: 10.11836/JEOM21514
引用本文: 李凤敏, 韩进美, 黄红茜, 王娜, 张爱华, 韩雪. NaAsO2染毒HaCaT细胞中丝氨酸合成途径关键酶的表达及其作用[J]. 环境与职业医学, 2022, 39(5): 545-549. DOI: 10.11836/JEOM21514
LI Fengmin, HAN Jinmei, HUANG Hongqian, WANG Na, ZHANG Aihua, HAN Xue. Expressions and roles of key enzymes in serine synthesis pathway in NaAsO2-treated HaCaT cells[J]. Journal of Environmental and Occupational Medicine, 2022, 39(5): 545-549. DOI: 10.11836/JEOM21514
Citation: LI Fengmin, HAN Jinmei, HUANG Hongqian, WANG Na, ZHANG Aihua, HAN Xue. Expressions and roles of key enzymes in serine synthesis pathway in NaAsO2-treated HaCaT cells[J]. Journal of Environmental and Occupational Medicine, 2022, 39(5): 545-549. DOI: 10.11836/JEOM21514

NaAsO2染毒HaCaT细胞中丝氨酸合成途径关键酶的表达及其作用

Expressions and roles of key enzymes in serine synthesis pathway in NaAsO2-treated HaCaT cells

  • 摘要: 背景 丝氨酸合成途径(SSP)关键酶在肿瘤的生长、增殖、侵袭中发挥重要作用,但其在砷致癌中的作用尚不明确。

    目的 以人永生化皮肤角质形成细胞(HaCaT细胞)和NaAsO2所致恶性转化HaCaT(T-HaCaT)细胞为研究对象,观察NaAsO2染毒对SSP关键酶如磷酸甘油酸脱氢酶(PHGDH)、磷酸丝氨酸转氨酶1(PSAT1)和磷酸丝氨酸磷酸酶(PSPH)表达及对细胞增殖、迁移能力的影响,探讨SSP关键酶在砷致癌中的作用。

    方法 (1)取课题组前期构建的T-HaCaT细胞,实验分组为传代对照(0 μmol·L−1 NaAsO2)组、T-HaCaT(0.5 μmol·L−1 NaAsO2)组、NCT503(PHGDH抑制剂,25 μmol·L−1)组、NCT503(25 μmol·L−1)+T-HaCaT(0.5 μmol·L−1 NaAsO2)组。Western blotting法检测传代对照组和T-HaCaT组细胞的SSP关键酶蛋白表达水平,CCK8法和细胞划痕试验分别检测各组细胞的增殖率和迁移率。(2)取生长良好的对数生长期HaCaT细胞,以0、0.625、1.25和2.5 μmol·L−1 NaAsO2染毒细胞0、24、48和72 h,检测细胞增殖率和SSP关键酶的蛋白表达水平。后续实验给予HaCaT细胞25 μmol·L−1 NCT503预处理6 h后,用2.5 μmol·L−1 NaAsO2继续染毒72 h,实验分组为对照(0 μmol·L−1 NaAsO2)组、染毒(2.5 μmol·L−1 NaAsO2)组、预处理组(25 μmol·L−1 NCT503)、预处理(25 μmol·L−1 NCT503)+染毒(2.5 μmol·L−1 NaAsO2)组,检测各组细胞的增殖率。

    结果 T-HaCaT组细胞中PHGDH的蛋白表达水平是传代对照组的1.60倍(P<0.05),其增殖率(177.51%±14.69%)和迁移率(53.85%±0.94%)高于传代对照组(100.00%±0.00%)、(24.30%±2.26%)(均P<0.05)。NCT503干预后,NCT503+T-HaCaT组细胞的增殖率(144.97%±8.08%)和迁移率(35.80%±0.99%)较T-HaCaT组细胞降低(均P<0.05)。NaAsO2染毒72 h后HaCaT细胞的增殖率随染毒浓度的增加而增加(r=0.862,P<0.05),与此一致,各染毒组HaCaT细胞中SSP关键酶的蛋白水平均较对照组表达增加(均P<0.05)。2.5 μmol·L−1 NaAsO2 染毒后的HaCaT细胞增殖率随染毒时间的延长而增加(r=0.775,P<0.05),与细胞中PHGDH蛋白表达水平的变化一致。NCT503干预后,预处理+染毒组细胞增殖率低于染毒组细胞(P<0.05)。

    结论 SSP关键酶可能在NaAsO2所致的T-HaCaT细胞增殖中扮演重要的角色。

     

    Abstract: Background The key enzymes of serine synthesis pathway (SSP) play an important role in tumor growth, proliferation, and invasion, but their roles in arsenic carcinogenesis are unclear.

    Objective To observe the effects of NaAsO2 treatment on the expressions of key enzymes such as phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) of SSP and on the ability to proliferate and migrate in human immortalized skin keratinocytes (HaCaT) and NaAsO2-induced malignantly transformed HaCaT (T-HaCaT), and to explore the roles of SSP key enzymes in arsenic carcinogenesis.

    Methods (1) The T-HaCaT cells constructed earlier by our research team were divided into a passage control (0 μmol·L−1 NaAsO2) group, a T-HaCaT (0.5 μmol·L−1 NaAsO2) group, a NCT503 (PHGDH inhibitor, 25 μmol·L−1) group, and a NCT503 (25 μmol·L−1) + T-HaCaT (0.5 μmol·L−1 NaAsO2) group. Western blotting was used to detect the protein expression levels of SSP key enzymes in the passage control group and the T-HaCaT group. CCK8 assay and cell scratch test were used to detect the proliferation and migration rates of cells in each group respectively. (2) Well-grown logarithmic-phase HaCaT cells were treated with 0, 0.625, 1.25, and 2.5 μmol·L−1 NaAsO2 for 0, 24, 48, and 72 h to detect cell proliferation rate and protein expression levels of SSP key enzymes. In the subsequent experiment, HaCaT cells were pretreated with 25 μmol·L−1 NCT503 for 6 h, and then treated with 2.5 μmol·L−1 NaAsO2 for 72 h continuously. The experimental groups included a control (0 μmol·L−1 NaAsO2) group, an exposure (2.5 μmol·L−1 NaAsO2) group, a pretreatment (25 μmol·L−1 NCT503) group, and a pretreatment (25 μmol·L−1 NCT503) + exposure (2.5 μmol·L−1 NaAsO2) group, to detect the proliferation rate of cells in each group.

    Results The protein expression level of PHGDH in the T-HaCaT group were 1.60 times higher than that in the passage control group (P<0.05), and its proliferation rate (177.51%±14.69%) and migration rate (53.85%±0.94%) were also higher than the passage control group’s (100.00%±0.00% and 24.30%±2.26%) (bothPs<0.05), respectively. After the NCT503 intervention, the proliferation rate (144.97%±8.08%) and migration rate (35.80%±0.99%) of cells in the NCT503 + T-HaCaT group were lower than those in the T-HaCaT group (bothP<0.05). The proliferation rate of HaCaT cells after NaAsO2 exposure for 72 h increased with the increase of exposure concentration (r=0.862, P<0.05), and consistently, the protein levels of SSP key enzymes in HaCaT cells in each exposure group were higher than those in the control group (allP<0.05). The proliferation rate of HaCaT cells treated with 2.5 μmol·L−1 NaAsO2 increased with the extension of exposure time (r=0.775, P<0.05), which was consistent with the changes of PHGDH levels in cells. After the NCT503 intervention, the proliferation rate of the pretreatment + exposure group was significantly lower than that of the exposure group (P<0.05).

    Conclusion The key enzymes of SSP may play an important role in the proliferation of T-HaCaT cells induced by NaAsO2.

     

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