癌-睾丸抗原CT57促进肝癌细胞增殖、侵袭、迁移和上皮间质转化

doi:10.3969/j.issn.1674-8115.2024.11.001

摘要/Abstract

摘要：

目的·研究癌-睾丸抗原（cancer-testis antigen，CTA）家族成员CT57对人肝癌细胞增殖、侵袭、迁移和裸鼠皮下成瘤的影响及可能的作用机制。方法·通过生物信息学方法分析CT57在多种肿瘤组织和正常组织中的表达差异及其对肝癌患者预后的影响；用慢病毒载体分别构建稳定敲低和过表达CT57的肝癌细胞系模型，并借助Western blotting验证CT57蛋白水平的变化；通过CCK-8细胞增殖实验、软琼脂克隆形成实验和细胞周期实验，检测CT57对肝癌细胞增殖与集落形成能力的影响；通过流式细胞术检测CT57对细胞周期的影响；通过划痕实验和Transwell实验检测CT57对肝癌细胞侵袭和迁移能力的影响；通过实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测上皮间质转化（epithelial-mesenchymal transition，EMT）标志物的表达变化；通过在裸鼠皮下注射敲低CT57的肝癌细胞株（实验组）和对照细胞株（对照组）构建皮下成瘤模型，探究CT57在体内环境下的生物学功能。结果·对肿瘤基因组图谱计划（The Cancer Genome Atlas Program，TCGA）数据库中的多种肿瘤和相对应正常组织的生物信息学分析表明，CT57在包括肝癌在内的大部分肿瘤组织中高表达，并且其表达水平与肝癌患者预后相关。细胞增殖实验、软琼脂克隆形成实验和细胞周期实验证明，敲低CT57可抑制肝癌细胞增殖和集落形成，并导致细胞周期阻滞；划痕实验和Transwell实验证明，敲低CT57可抑制肝癌细胞侵袭和迁移，而过表达CT57则促进这些过程；qRT-PCR结果表明，过表达CT57可导致上皮细胞标志物E-钙黏蛋白（E-cadherin，ECAD）和闭合蛋白（occludin，OCLN）表达下调，间质细胞标志物波形蛋白（vimentin，VIM）、Twist相关蛋白1（twist family bHLH transcription factor 1，TWIST1）和基质金属肽酶2（matrix metallopeptidase 2，MMP2）表达上调；体内实验表明，敲低CT57可降低肝癌细胞在裸鼠中的成瘤率及肿瘤的体积和质量。结论·敲低肝癌细胞CT57导致细胞周期阻滞，从而抑制肝癌细胞增殖和裸鼠皮下成瘤；CT57可以促进肝癌细胞上皮间质转化，增强其侵袭和迁移能力。

关键词: 癌-睾丸抗原, 肝癌, 细胞增殖, 侵袭, 迁移, 上皮间质转化

Abstract:

Objective ·To investigate the effect of cancer-testis antigen family member CT57 on proliferation, migration and invasion of the human liver cancer cells and tumorigenesis in nude mice, and the possible mechanism. Methods ·Bioinformatics methods were used to analyze the differential expression of CT57 in several cancer tissues and normal tissues, and its effect on the prognosis of liver cancer patients. Lentiviral vectors were used to establish liver cancer cell lines with stable knockdown and overexpression of CT57, which were confirmed by Western blotting. CCK-8 cell proliferation assay, soft agar colony formation assay and cell cycle experiment were used to detect the effect of CT57 on the proliferation and colony formation ability of liver cancer cells. Wound healing and Transwell assays were used to detect the effect of CT57 on the migration and invasion of liver cancer cells, and the expression of epithelial-mesenchymal transition (EMT) markers was detected by quantitative real-time PCR (qRT-PCR). To explore the effect of CT57 on liver cancer cells in vivo, CT57 knockdown liver cancer cells (experimental group) and control liver cancer cells (control group) were used to conduct subcutaneous tumor formation experiments in nude mice. Results ·Bioinformatics analysis of multiple tumors and corresponding normal tissues in The Cancer Genome Atlas Program (TCGA) database showed that CT57 was highly expressed in most tumor tissues, including liver cancer, and the expression level of CT57 was significantly correlated with the prognosis of liver cancer patients. Cell proliferation assay, soft agar colony formation assay, and cell cycle experiment showed that knockdown of CT57 inhibited the proliferation and colony formation of liver cancer cells and led to cell cycle arrest. In wound healing and Transwell assays, knockdown of CT57 inhibited the invasion and migration of liver cancer cells, while overexpression of CT57 promoted it. The results of qRT-PCR indicated that overexpression of CT57 resulted in downregulation of epithelial cell markers ECAD (E-cadherin) and OCLN (occludin), and upregulation of mesenchymal cell markers VIM (vimentin), TWIST1 (twist family bHLH transcription factor 1), and MMP2 (matrix metallopeptidase 2). In the in vivo experiments, knockdown of CT57 significantly reduced the tumor formation rate of liver cancer cells, tumor volume, and tumor weight in nude mice. Conclusion ·Knockdown of CT57 leads to cell cycle arrest, thereby inhibiting the proliferation of liver cancer cell and subcutaneous tumorigenesis in nude mice; CT57 promotes the invasion, migration and EMT of liver cancer cells.

Key words: cancer-testis antigen, liver cancer, cell proliferation, invasion, migration, epithelial-mesenchymal transition (EMT)

中图分类号:

R735.7

罗蓝鸽, 郑超, 雷鸣. 癌-睾丸抗原CT57促进肝癌细胞增殖、侵袭、迁移和上皮间质转化[J]. 上海交通大学学报（医学版）, 2024, 44(11): 1335-1346.

LUO Lange, ZHENG Chao, LEI Ming. Promotive effect of cancer-testis antigen CT57 on proliferation, invasion, migration and epithelial-mesenchymal transition of liver cancer cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(11): 1335-1346.

图/表 8

表1 qRT-PCR引物序列

Tab 1 Primer sequences for qRT-PCR

Primer	Forward (5′→3′)	Reverse (5′→3′)
GAPDH	AATGGGCAGCCGTTAGGAAA	GCCCAATACGACCAAATCAGAG
CT57	GAACATCGTGAACTACCTACCG	CAAGGGTGTCTCCGTGATGAT
ECAD	TCTTCAATCCCACCACGTACA	CTGGGGTATTGGGGGCATC
VIM	GACGCCATCAACACCGAGTT	CTTTGTCGTTGGTTAGCTGGT
OCLN	GGCACCTGCATACTCACCC	CTGGGAGAGCAACTCATCCTC
TWIST1	GTCCGCAGTCTTACGAGGAG	GCTTGAGGGTCTGAATCTTGCT
MMP2	TACAGGATCATTGGCTACACACC	GGTCACATCGCTCCAGACT
FOXM1	CGTCGGCCACTGATTCTCAAA	GGCAGGGGATCTCTTAGGTTC
SNA	TCGGAAGCCTAACTACAGCGA	AGATGAGCATTGGCAGCGAG

图1 CT57 在肝癌组织及细胞株中的表达情况以及与患者预后的关系Note： A. CT57 expression levels of different tumor types in the TCGA database. BLCA—bladder urothelial carcinoma; BRCA—breast invasive carcinoma; CHOL—cholangiocarcinoma; COAD—colon adenocarcinoma; ESCA—esophageal carcinoma; HNSC—head and neck squamous cell carcinoma; KICH—kidney chromophobe; KIRC—kidney renal clear cell carcinoma; KIRP—kidney renal papillary cell carcinoma; LIHC—liver hepatocellular carcinoma; LUAD—lung adenocarcinoma; LUSC—lung squamous cell carcinoma; PRAD—prostate adenocarcinoma; READ—rectum adenocarcinoma; STAD—stomach adenocarcinoma; THCA—thyroid carcinoma; UCEC—uterine corpus endometrial carcinoma. The expression of CT57 in the liver cancer is marked by the red box. B. Kaplan-Meier survival curves comparison between the liver cancer patients with high expression and low expression of CT57 in livers. C. The mRNA expression levels of CT57 in cell lines. D. Western blotting analysis of CT57 expression level in four human liver cancer cells.

Fig 1 Expression of CT57 in liver cancer tissues and cell lines and its relationship with patients′ prognosis

图2 敲低 CT57 对肝癌细胞增殖能力及细胞周期的影响Note： A. CT57 knockdown (sh-1 and sh-2) efficiency detected by Western blotting analysis. B. CCK?8 proliferation assay. C. Soft agar colony formation assay. D. Flow cytometry for analyzing cell cycle. ①P=0.016, ②P=0.039, ③P=0.015, ④P=0.014, ⑤P=0.026, ⑥P=0.012, ⑦P=0.003, compared with the corresponding control (Ctrl) group.

Fig 2 Effect of CT57 knockdown on proliferation and cell cycle in liver cancer cells

图3 敲低 CT57 后肝癌细胞Ki67阳性细胞比例的变化Note：Ki67 immunostaining of indicated cells (×200 in big boxes; ×800 in small boxes). ①P=0.020, ②P=0.002, ③P=0.015, ④P=0.000, ⑤P=0.027, ⑥P=0.003, compared with the corresponding control (Ctrl) group.

Fig 3 Changes in the proportion of Ki67-positive cells in liver cancer cells after knocking down CT57

图4 敲低 CT57 对肝癌细胞侵袭和迁移能力的影响Note： A. Wound healing assay (×200) of indicated cells. B. RTCA coupled Transwell migration/invasion assay of indicated cells. C. 3D culture of indicated cells (×200).

Fig 4 Effect of CT57 knockdown on invasion and migration ability of liver cancer cells

图5 CT57过表达对肝癌细胞侵袭和迁移能力的影响Note： A. Western blotting analysis of CT57 overexpression in Hep3B cells. B. RTCA coupled Transwell migration/invasion assay of Hep3B cells. C. Wound healing assay (×200) of Hep3B cells.

Fig 5 Effect of CT57 overexpression on invasion and migration ability of liver cancer cells

图6 CT57过表达对肝癌细胞EMT的影响Note： A. The mRNA expression of EMT markers after CT57 overexpression in Hep3B cells detected by qRT-PCR. B. The cell morphology after overexpression of CT57 in Hep3B cells. C. FOXM1 and SNA expression after CT57 overexpression in Hep3B cells detected by qRT-PCR. ①P=0.031, ②P=0.002, ③P=0.006, ④P=0.021, ⑤P=0.035, ⑥P=0.012, compared with the corresponding control (Ctrl) group.

Fig 6 Effect of CT57 overexpression on EMT in liver cancer cells

图7 敲低 CT57 对肝癌细胞在裸鼠皮下成瘤的影响Note： A. The tumorigenic ability of Hep3B cells after knockdown of CT57 in nude mice. B. Tumor and liver size in nude mice. C. Western blotting analysis of CT57 knockdown efficiency in the tumors. D. H-E staining of tumor tissues.

Fig 7 Effect of CT57 knockdown on subcutaneous tumor formation of liver cancer cells in nude mice

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