金盏花苷E通过自噬途径下调GPX4和SLC7A11抑制肝癌细胞的增殖和迁移

doi:10.12122/j.issn.1673-4254.2024.07.12

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (7): 1327-1335.doi: 10.12122/j.issn.1673-4254.2024.07.12

• • 上一篇

金盏花苷E通过自噬途径下调GPX4和SLC7A11抑制肝癌细胞的增殖和迁移

陈芊伊¹^,²(), 尚书涵¹^,²^,³, 鲁欢¹^,², 李思思¹^,²^,⁴, 孙志勉¹^,²^,³, 范喜瑞¹^,²(), 戚之琳¹^,²()

^1.皖南医学院，基础医学院生物化学与分子生物学教研室，安徽芜湖 241002
^2.皖南医学院，活性生物大分子研究安徽省重点实验室，安徽芜湖 241002
^3.皖南医学院，临床医学院，安徽芜湖 241002
^4.皖南医学院，公共卫生学院，安徽芜湖 241002

收稿日期:2024-05-14 出版日期:2024-07-20 发布日期:2024-07-25
通讯作者: 范喜瑞,戚之琳 E-mail:937441097@qq.com;20210006@wnmc.edu.cn;20010012@wnmc.edu.cn
作者简介:陈芊伊，在读硕士研究生，E-mail: 937441097@qq.com
基金资助:
安徽高校自然科学研究项目(KJ2020ZD54);安徽省学科（专业）拔尖人才学术资助项目(gxbjZD2021060);活性生物大分子研究安徽省重点实验室项目(1306C083008);国家级大学生创新创业训练计划项目(202310368002)

Calenduloside E inhibits hepatocellular carcinoma cell proliferation and migration by down-regulating GPX4 and SLC7A11 expression through the autophagy pathway

Qianyi CHEN¹^,²(), Shuhan SHANG¹^,²^,³, Huan LU¹^,², Sisi LI¹^,²^,⁴, Zhimian SUN¹^,²^,³, Xirui FAN¹^,²(), Zhilin QI¹^,²()

^1.Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences,
^2.Anhui Provincial Key Laboratory of Active Biological Macro-molecules,
^3.School of Clinical Medicine,
^4.School of Public Health, Wannan Medical College, Wuhu 241002, China

Received:2024-05-14 Online:2024-07-20 Published:2024-07-25
Contact: Xirui FAN, Zhilin QI E-mail:937441097@qq.com;20210006@wnmc.edu.cn;20010012@wnmc.edu.cn

摘要/Abstract

摘要：

目的探讨金盏花苷E抑制肝癌细胞增殖和迁移的分子机制。方法金盏花苷E处理肝癌细胞，CCK-8检测细胞活力；Western blotting检测GPX4、SLC7A11、LC3、P62的表达以及Akt/mTOR的磷酸化。自噬抑制剂LY294002和激活剂Rapamycin与金盏花苷E联合处理肝癌细胞，EdU和Transwell实验分别检测肝癌细胞的增殖和迁移能力。TCGA数据库分析GPX4和SLC7A11在肝癌及正常肝组织中的表达水平，以及与肝癌患者存活之间的关系。Western blotting和qPCR分别检测GPX4和SLC7A11在肝癌细胞和正常肝细胞中的表达水平。结果金盏花苷E能够显著抑制肝癌细胞的存活（P<0.05）；GPX4和SLC7A11在肝癌组织和肝癌细胞中均显著高表达；GPX4和SLC7A11的表达与肝癌患者存活呈显著负相关（P<0.001）；金盏花苷E显著抑制GPX4和SLC7A11蛋白的表达，激活Akt-mTOR通路，增强LC3 Ⅱ的表达（P<0.01）；自噬激活剂Rapamycin显著增强金盏花苷E对GPX4和SLC7A11的抑制作用，而自噬抑制剂LY294002则明显逆转了金盏花苷E对GPX4和SLC7A11的抑制作用（P<0.05）；抑制自噬途径能够逆转金盏花苷E对肝癌细胞增殖和迁移的抑制作用，增强自噬途径则发生相反的变化（P<0.01）。结论金盏花苷E经自噬途径下调GPX4和SLC7A11抑制肝癌细胞的增殖和迁移。

关键词: 金盏花苷E, 谷胱甘肽过氧化物酶4, 溶质转运蛋白第7家族11成员, 自噬, 肝细胞癌

Abstract:

Objective To investigate the molecular mechanism through which calenduloside E inhibits hepatocellular carcinoma (HCC) cell proliferation and migration. Methods HCC cell lines HepG2 and Huh7 treated with calenduloside E were examined for changes in cell viability using CCK-8 assay and expressions of GPX4, SLC7A11, LC3, P62 and phosphorylation of Akt/mTOR using Western blotting. The effects LY294002 and Rapamycin (the inhibitor and activator of autophagy, respectively) on proliferation and migration of calenduloside E-treated HCC cells were evaluated using EdU and Transwell assays. The TCGA database was used to explore the expression levels of GPX4 and SLC7A11 in HCC and normal liver tissues and their correlation with the patients' survival outcomes. GPX4 and SLC7A11 expressions were also detected in HCC cells and normal hepatocytes using RT-qPCR and Western blotting. Results Calenduloside E obviously inhibited the viability of HCC cells. GPX4 and SLC7A11 were highly expressed in HCC tissues and cell lines, and their expression levels were negatively correlated with the patients' survival. In HCC cell lines, calenduloside E significantly inhibited the expressions of GPX4 and SLC7A11 proteins, activated the Akt-mTOR pathway, and enhanced the expression of LC3 II. The inhibitory effect of calenduloside E on GPX4 and SLC7A11 expressions was significantly enhanced by rapamycin but attenuated by LY294002. Inhibiting the autophagy pathway obviously diminished the inhibitory effect of calenduloside E on proliferation and migration of HCC cells, while activating this pathway produced the opposite effect. Conclusion Calenduside E inhibits the proliferation and migration of HCC cells by down-regulating GPX4 and SLC7A11 expression via the autophagy pathway.

Key words: calenduside E, glutathione peroxidase 4, solute carrier family 7 member 11, autophagy, hepatocellular carcinoma

陈芊伊, 尚书涵, 鲁欢, 李思思, 孙志勉, 范喜瑞, 戚之琳. 金盏花苷E通过自噬途径下调GPX4和SLC7A11抑制肝癌细胞的增殖和迁移[J]. 南方医科大学学报, 2024, 44(7): 1327-1335.

Qianyi CHEN, Shuhan SHANG, Huan LU, Sisi LI, Zhimian SUN, Xirui FAN, Zhilin QI. Calenduloside E inhibits hepatocellular carcinoma cell proliferation and migration by down-regulating GPX4 and SLC7A11 expression through the autophagy pathway[J]. Journal of Southern Medical University, 2024, 44(7): 1327-1335.

图/表 9

表1 SLC7A11、GPX4以及GAPDH引物序列

Tab.1 Primer sequences for RT-qPCR of SLC7A11, GPX4 and GAPDH

Primers	Sequence of primer
Primers	Forward (form 5′ to 3′)	Reverse (from 5′ to 3′)
SLC7A11	TCTCCAAAGGAGGTTACCTGC	AGACTCCCCTCAGTAAAGTGAC
GPX4	GAGGCAAGACCGAAGTAAACTAC	CCGAACTGGTTACACGGGAA
GAPDH	AAAGCCTGCCGGTGACTAA	AGAGTTAAAAGCAGCCCTGG

图1 金盏花苷E抑制肝癌Huh7细胞的存活

Fig.1 CE inhibits the viability of hepatocellular carcinoma (HCC) Huh7 cells (Mean±SD). *P<0.05, **P<0.01 vs 0 µg/mL group.

图2 GPX4和SLC7A11在肝癌组织和细胞中的表达及其与患者存活的相关性

Fig.2 Expression levels of GPX4 and SLC7A11 in HCC tissues and cells and their correlation with the patients' survival (Mean±SD). A: Expression of GPX4 and SLC7A11 in HCC tissues and normal liver tissues. B: Correlation of GPX4 and SLC7A11 expressions with the patients' survival. The expressions of GPX4 and SLC7A11 mRNA (C) and protein (D) in HCC cells and normal liver cells. **P<0.01 vs HL-7702.

图3 金盏花苷E对GPX4和SLC7A11表达的影响

Fig.3 Effects of CE on GPX4 and SLC7A11 expressions in HepG2 (A, B) and Huh7 cells (C, D). Data are presented as Mean±SD. **P<0.01 vs 0 µg/mL. CE: Calenduloside E.

图4 金盏花苷E对GPX4和SLC7A11降解的影响

Fig.4 Effects of CE on GPX4 and SLC7A11 protein degradation in HepG2 (A, B) and Huh7 cells (C, D). Data are presented as Mean±SD. *P<0.05, **P<0.01 vs CHX group.

图5 金盏花苷E对肝癌细胞自噬途径的影响

Fig.5 Effects of CE on the autophagy pathway in HepG2 (A) and Huh7 cells (B). Data are presented as Mean±SD. *P<0.05, **P<0.01 vs 0 µg/mL.

图6 金盏花苷E对肝癌细胞自噬的影响

Fig.6 Effects of CE on autophagy of HepG2 (A) and Huh7 cells (B)(Original magnification: ×200).

图7 自噬抑制剂或激活剂对金盏花苷E下调GPX4和SLC7A11的影响

Fig.7 Effects of autophagy inhibitor or activator on CE-induced GPX4 and SLC7A11 down-regulation in HepG2 (A) and Huh7 (B) cells (Mean±SD). *P<0.05, **P<0.01 vs Control; &P<0.05, &&P<0.01 vs CE.

图8 自噬抑制剂或激活剂对金盏花苷E抑制肝癌细胞增殖和迁移的影响

Fig.8 Effects of autophagy inhibitor or activator on proliferation and migration of CE-treated HepG2 (A, B) and Huh7 (C, D) cells (×100).

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金盏花苷E通过自噬途径下调GPX4和SLC7A11抑制肝癌细胞的增殖和迁移

Calenduloside E inhibits hepatocellular carcinoma cell proliferation and migration by down-regulating GPX4 and SLC7A11 expression through the autophagy pathway

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