癌-睾丸抗原CT63在慢性髓系白血病中的作用及其机制

doi:10.3969/j.issn.1674-8115.2024.11.002

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (11): 1347-1358.doi: 10.3969/j.issn.1674-8115.2024.11.002

• 创新团队成果专栏 • 上一篇

癌-睾丸抗原CT63在慢性髓系白血病中的作用及其机制

孔汝心(), 周亚群, 魏婷宜(), 雷鸣()

上海交通大学医学院附属第九人民医院精准医学研究院，上海 200125

收稿日期:2024-03-21 接受日期:2024-05-14 出版日期:2024-11-28 发布日期:2024-11-28
通讯作者: 魏婷宜,雷鸣 E-mail:wskrx008@sjtu.edu.cn;leim@shsmu.edu.cn;weitingyi@sjtu.edu.cn
作者简介:孔汝心（1998—），男，硕士生；电子信箱：wskrx008@sjtu.edu.cn。
基金资助:
国家重点研发计划(2018YFA0107004);国家自然科学基金(32300609)

Function and mechanism of cancer-testis antigen CT63 in chronic myeloid leukemia

KONG Ruxin(), ZHOU Yaqun, WEI Tingyi(), LEI Ming()

Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China

Received:2024-03-21 Accepted:2024-05-14 Online:2024-11-28 Published:2024-11-28
Contact: WEI Tingyi,LEI Ming E-mail:wskrx008@sjtu.edu.cn;leim@shsmu.edu.cn;weitingyi@sjtu.edu.cn
Supported by:
National Key Research and Development Program of China(2018YFA0107004);National Natural Science Foundation of China(32300609)

摘要/Abstract

摘要：

目的·研究癌-睾丸抗原（cancer-testis antigen，CTA）家族成员CT63对慢性髓系白血病细胞增殖、分化和成瘤方面的影响，阐述其分子机制。方法·采用生物信息学方法分析癌症基因组图谱（TCGA）数据库中髓系白血病患者的CT63表达差异及其对疾病预后的影响；构建CT63敲降的人慢性髓系白血病K562细胞系，通过实时荧光定量PCR和蛋白质印迹法确定CT63的敲降效果；通过细胞实时成像和CCK-8实验评估CT63对慢性髓系白血病细胞增殖能力的影响；进行小鼠皮下成瘤实验，探究CT63在体内环境下对慢性髓系白血病肿瘤发生、生长和细胞分化的影响；通过佛波酯诱导的K562细胞向单核/巨噬细胞的分化实验，探究CT63对慢性髓系白血病细胞分化能力的影响；检测敲降CT63后细胞内线粒体功能的变化。结果·Kaplan-Meier生存曲线分析显示，CT63差异表达与髓系白血病患者的预后显著相关；敲降CT63抑制K562细胞的增殖和成瘤；敲降CT63促进K562细胞在体内和体外的分化；敲降CT63抑制K562细胞中线粒体呼吸链复合物Ⅳ的活性，并导致包括细胞色素C氧化酶Ⅳ（COX Ⅳ）、丙酮酸脱氢酶、琥珀酸脱氢酶A（SDHA）和电压依赖性阴离子通道（VDAC）的线粒体相关标志物表达下降，影响K562细胞的线粒体代谢活性。结论·CT63的表达水平与髓系白血病患者的预后相关；CT63通过维持线粒体的代谢活性，促进K562细胞的增殖和体内成瘤；CT63是维持慢性髓系白血病细胞自我更新和分化的重要分子开关。

关键词: 癌-睾丸抗原63, 慢性髓系白血病, 细胞增殖, 细胞分化, 线粒体

Abstract:

Objective ·To explore the effects of the cancer-testis antigen (CTA) family member CT63 on proliferation, differentiation, and tumorigenicity of chronic myeloid leukemia (CML) cells, and uncover the underlying molecular mechanisms. Methods ·The link between CT63 expression and the prognosis of myeloid leukemia patients was analyzed using bioinformatics methods (TCGA database). A K562 cell line with CT63 knockdown was established. The knockdown efficiency of CT63 was confirmed by qRT-PCR and Western blotting. Live-cell imaging and CCK-8 methods were adopted to evaluate the inhibitory effect of CT63 knockdown in CML cells. A subcutaneous tumorigenesis assay in nude mice was conducted to examine the effects of CT63 on tumorigenesis, tumor growth, and differentiation of K562 cells invivo. Phorbol 12-myristate 13-acetate (PMA)-induced monocyte/macrophage differentiation experiment was carried out to investigate the role of CT63 in the differentiation of K562 cells in vitro. Mitochondrial function was assessed to determine the impact of CT63 on CML cells both in vivo and in vitro. Results ·The Kaplan-Meier survival curve indicated that low expression levels of CT63 were correlated with longer survival in patients with myeloid leukemia. Down-regulation of CT63 in K562 cells inhibited proliferation and promoted differentiation. Live-cell imaging and CCK-8 assays displayed that knockdown of CT63 inhibited cell proliferation and extended cell doubling time in K562 cells. In the subcutaneous xenotransplantation model, down-regulation of CT63 inhibited tumor growth in nude mice. K562 cells expressing lower levels of CT63 were more prone to differentiate into monocyte/macrophage both in vivo and in vitro under PMA exposure condition. Knockdown of CT63 suppressed the activity of mitochondrial respiratory chain complex Ⅳ. This led to decreased expression of mitochondrial markers, including cytochrome C oxidase Ⅳ (COX Ⅳ), pyruvate dehydrogenase, succinate dehydrogenase A (SDHA), and voltage-dependent anion channel (VDAC), thus affecting the mitochondrial metabolic activity of K562 cells. Conclusion ·CT63 is related to the prognosis of myeloid leukemia patients. CT63 plays an important role in promoting proliferation and inhibiting differentiation of K562 cells in vivo and in vitro. CT63 serves as a switch to regulate the balance between proliferation and differentiation of CML cells via the modulation of mitochondrial activity.

Key words: cancer-testis antigen 63, chronic myeloid leukemia, cell proliferation, cell differentiation, mitochondria

中图分类号:

R733.72

孔汝心, 周亚群, 魏婷宜, 雷鸣. 癌-睾丸抗原CT63在慢性髓系白血病中的作用及其机制[J]. 上海交通大学学报（医学版）, 2024, 44(11): 1347-1358.

KONG Ruxin, ZHOU Yaqun, WEI Tingyi, LEI Ming. Function and mechanism of cancer-testis antigen CT63 in chronic myeloid leukemia[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(11): 1347-1358.

图/表 7

表1 shRNA片段的目的序列

Tab 1 Target sequences of shRNA

shRNA	Target sequence (5′→3′)
PG-shCT63-1	GCTTTGCATATCCATGTTCTA
PG-shCT63-2	GCCATTGATAGATACCTCAAA
PG-shCT63-3	CCAAACACATTACGCCACGTT
PG-shCT63-4	GCTACAGTTATTAGATGGCT
PG-shCT63-5	GCCTGAGGTGAATCCATTGTA
PG-shCtrl	CCTAAGGTTAAGTCGCCCTCG

表2 qRT-PCR的引物序列

Tab 2 Sequence of primers used for qRT-PCR

Gene	Forward primer (5′→3′)	Reverse primer (5′→3′)
CT63	CATCGTGGGGAATGAGAGGG	GATTGCAGAGGGGCACAGAT
mtDNA	CGAAAGGACAAGAGAAATAAGG	CTGTAAAGTTTTAAGTTTTATGCG
Nuclear	CAACTTCATCCACGTTCACC	GAAGAGCCAAGGACAGGTAC
HSP60	GTGTAGACCTTTTAGCCGATGC	GTGCCAGTACAGTAGCAGTGG

图1 CT63 敲降后K562细胞增殖能力变化Note： A. Kaplan-Meier survival curves of high and low expression of CT63 in myeloid leukemia patients. B. qRT-PCR analysis of CT63 knockdown efficiency in K562 cells. C. Western blotting analysis of CT63 levels in stable CT63 knockdown cell lines. D. Proliferation of stable shCtrl and CT63 knockdown cell lines assessed by live cell image assay. E. Live cell image analysis of proliferation rate of K562 after down-regulation of CT63 protein. F. Cell doubling time of K562 after down-regulation of CT63 protein. G. CCK-8 proliferation assay of indicated K562 cells. ①P=0.004, ②P=0.001, ③P=0.004, ④P=0.004, ⑤P=0.001, ⑥P=0.003, ⑦P=0.017, ⑧P=0.095, ⑨P=0.463, ⑩P=0.145, compared with the shCtrl group.

Fig 1 Proliferation of K562 cells after down-regulation of CT63

图2 CT63 敲降后K562细胞在裸鼠皮下成瘤的能力变化Note： A. Subcutaneous tumor of nude mice. B. Average radiant efficiency of region of interest. C. Tumor size. D. Tumor weight. E. H-E staining of tumor. F/H. Immunofluorescence staining of CT63 and Ki67 in tumor tissue. G/I. Relative fluorescence density of Immunofluorescence staining in F and H. ①P=0.000, ②P=0.029, ③P=0.000, ④P=0.000, compared with the shCtrl group.

Fig 2 Tumorigenic ability of K562 cells in nude mice after down-regulation of CT63

图3 CT63下调后K562细胞分化能力变化Note： A/B. CT63 knockdown efficiency in K562 in RNA and protein level. C. Heat maps and hierarchical clustering of differently expressed mRNA in shCtrl and shCT63 K562 cells. D. GO function analysis of differently expressed mRNA in shCtrl and shCT63 K562 cells. E. qRT-PCR analysis of the expression of myeloid cell differentiation genes. ①P=0.000, ②P=0.000, ③P=0.063, ④P=0.001, ⑤P=0.000, ⑥P=0.000, ⑦P=0.001, ⑧P=0.077, ⑨P=0.001, ⑩P=0.003, compared with the shCtrl group.

Fig 3 Differentiation of K562 cells after CT63 downregulation

图4 敲降 CT63 对K562细胞向单核/巨噬细胞分化的影响Note： A, B, E. Immunofluorescence staining of CT63, CD11b, and CD14 after 48 h of PMA exposure in K562 cells. C, D, F. Relative fluorescence density of immunofluorescence staining. G. Western blotting analysis of CT63, CD11b and CD14 in K562 cells after 48 h of PMA exposure. H. CD11b positive cells of CT63-knockdown K562 cells and control after 48 h of PMA exposure. I. The percentage of CD11b positive cells. J. Immunofluorescence staining for CD11b and CD14 in tumor tissues. ①P=0.007, ②P=0.001, ③P=0.024, ④P=0.021, compared with the shCtrl group.

Fig 4 Effect of knockdown of CT63 on the differentiation of K562 cells into monocytes/macrophages

图5 CT63对细胞代谢的调控作用Note： A. Activity of mitochondria complex Ⅳ in shCtrl and shCT63 K562 cells. B. Immunofluorescence staining of mitochondrial markers in tumors. PD—pyruvate dehydrogenase C. Relative fluorescence density of immunofluorescence staining in B. D. Western blotting analysis of mitochondrial markers in shCtrl and shCT63 K562 cells with PMA treatment. E. Relative average fluorescence density of Western blotting analysis. F. Immunofluorescence staining and subcellular localization of COX Ⅳ, G. Relative mtDNA level in shCtrl and shCT63 K562 cells. ①P=0.006, ②P=0.000, ③P=0.021, ④P=0.007, ⑤P=0.001, ⑥P=0.000, ⑦P=0.000, ⑧P=0.001, ⑨P=0.000, ⑩P=0.035, compared with the shCtrl group.

Fig 5 Regulation of CT63 on cell metabolism

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癌-睾丸抗原CT63在慢性髓系白血病中的作用及其机制

Function and mechanism of cancer-testis antigen CT63 in chronic myeloid leukemia

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