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中华临床医师杂志(电子版) ›› 2024, Vol. 18 ›› Issue (11) : 1019 -1029. doi: 10.3877/cma.j.issn.1674-0785.2024.11.008

基础研究

ST6GAL2 表达下调促进结肠癌细胞生长和转移的多维度研究
程玉1, 顾晋2,()   
  1. 1.100142 北京,北京大学肿瘤医院胃肠肿瘤中心
    2.100144 北京,北京大学首钢医院胃肠外科
  • 收稿日期:2024-10-08 出版日期:2024-11-15
  • 通信作者: 顾晋
  • 基金资助:
    国家自然科学基金(资助编号:82073223)

Downregulation of ST6GAL2 expression promotes colon cancer cell growth and metastasis: a multidimensional study

Yu Cheng1, Jin Gu2,()   

  1. 1.Gastrointestinal Oncology Center, Peking University Cancer Hospital, Beijing 100142, China
    2.Department of Gastrointestinal Surgery, Peking University Shougang Hospital, Beijing 100144, China
  • Received:2024-10-08 Published:2024-11-15
  • Corresponding author: Jin Gu
引用本文:

程玉, 顾晋. ST6GAL2 表达下调促进结肠癌细胞生长和转移的多维度研究[J/OL]. 中华临床医师杂志(电子版), 2024, 18(11): 1019-1029.

Yu Cheng, Jin Gu. Downregulation of ST6GAL2 expression promotes colon cancer cell growth and metastasis: a multidimensional study[J/OL]. Chinese Journal of Clinicians(Electronic Edition), 2024, 18(11): 1019-1029.

目的

探讨ST6β-半乳糖苷α-2,6-唾液酸转移酶2(ST6GAL2)对结肠癌细胞生长和转移的影响。

方法

从生物信息学-组织学-细胞学层面研究ST6GAL2 对结肠癌细胞生长和转移的影响。分析TCGA 和GEO 数据库中ST6GAL2 在结直肠癌中的表达,Kaplan-Meier 方法评估ST6GAL2 的表达高低与总生存期的关系。免疫组化技术验证ST6GAL2 在结直肠癌组织样本中的表达;RT-qpCR 和Western blot 检测ST6GAL2 在结直肠癌细胞系中的表达水平。利用慢病毒感染SW480 和HT29 细胞,构建ST6GAL2 敲降和过表达的稳定细胞系。通过CCK-8 增殖实验、克隆形成实验、细胞划痕实验和Transwell 侵袭实验分别检测ST6GAL2 对细胞增殖、迁移及侵袭能力的影响。

结果

在TCGA 和GEO 数据库观察到ST6GAL2 在结直肠癌组织中表达低于正常组织(P=0.032),且ST6GAL2 在右半结肠癌中较左半结肠癌表达下调(P<0.0001)。预后分析显示在MSS 型结肠癌中,ST6GAL2的表达量与总生存期成正相关(P=0.0209)。免疫组化结果显示ST6GAL2在正常组织高表达,而肿瘤组织中相对低表达(P<0.0001);ST6GAL2 在右半结肠癌中的表达明显低于左半(P=0.018)。CCK-8 与克隆形成实验结果显示敲低ST6GAL2 后结肠癌细胞增殖能力增强(P<0.0001)、克隆形成能力增强(P<0.05)。Transwell 实验表明ST6GAL2 表达下调能促进结肠癌细胞的迁移(P<0.01)和侵袭(P<0.05)。同样的实验方法结果表明过表达ST6GAL2 可抑制细胞的增殖和迁移。

结论

ST6GAL2 在结直肠癌组织中表达降低且在右半结肠癌中较左半低表达。MSS 型结肠癌中,ST6GAL2的表达与预后正相关。ST6GAL2 表达下调会促进CRC 细胞的生长、迁移和侵袭;过表达ST6GAL2可抑制细胞的生长和转移。

Objective

To investigate the effect of ST6GAL2 on the growth and metastasis of colon cancer cells.

Methods

The effect of ST6GAL2 on the growth and metastasis of colon cancer cells was studied from the bioinformatics, histology, and cytology levels. The expression of ST6GAL2 in colorectal cancer was analyzed based on the TCGA and GEO databases, and the Kaplan-Meier method was used to evaluate the relationship between ST6GAL2 expression and overall survival. Immunohistochemistry was used to verify the expression of ST6GAL2 in colorectal cancer tissue samples. RT-qpCR and Western blot were used to detect the expression level of ST6GAL2 in colorectal cancer cell lines. SW480 and HT29 cells were infected with lentivirus to construct stable cell lines with ST6GAL2 knockdown and overexpression.The effects of ST6GAL2 on cell proliferation, migration, and invasion were detected by CCK-8 assay,colony formation assay, cell scratch assay, and Transwell invasion assay.

Results

Based on the TCGA and GEO databases, the expression of ST6GAL2 in colorectal cancer tissues was lower than that in normal tissues (P=0.032), and the expression of ST6GAL2 in right-sided colon cancer was lower than that in leftsided colon cancer (P<0.0001). Prognostic analysis showed that the expression of ST6GAL2 was positively correlated with overall survival in MSS-type colon cancer (P=0.0209). Immunohistochemistry results showed that ST6GAL2 was highly expressed in normal tissues but relatively low in tumor tissues (P<0.0001); the expression of ST6GAL2 in right-sided colon cancer was significantly lower than that in left-sided colon cancer (P=0.018). CCK-8 and colony formation assays showed that the proliferation ability (P<0.0001) and colony formation ability (P<0.05) of colon cancer cells were enhanced after ST6GAL2 knockdown. Transwell assay showed that down-regulation of ST6GAL2 expression promoted the migration (P<0.01) and invasion(P<0.05) of colon cancer cells. The results of the same experimental methods showed that overexpression of ST6GAL2 inhibited cell proliferation and migration.

Conclusion

The expression of ST6GAL2 decreases in colorectal cancer tissues and is lower in right-sided colon cancer than in left-sided colon cancer. In MSStype colon cancer, the expression of ST6GAL2 is positively correlated with prognosis. Downregulation of ST6GAL2 expression promotes CRC cell growth, migration, and invasion, while overexpression of ST6GAL2 inhibits cell growth and metastasis.

图1 公共数据库筛选差异基因。图a 为CRCvs Normal 的差异基因;图b 为RCC vs LCC 的差异基因;图c 为CRC vs Normal 的差异基因与RCC vs LCC 差异基因交集;图d、e、f 为TCGA 训练数据集、验证数据集、GSE39582 数据中,ST6GAL2 在左/右半结肠癌间差异表达 注:CRC 为结直肠癌;RCC 为右半结肠癌;LCC 为左半结肠癌
图2 ST6GAL2 在CRC 中的生存分析。图a 为MSI 型CRC 组,STA6GAL2 对总生存期无显著影响,P=0.0871;图b 为MSS 型CRC 组,STA6GAL2 与总生存期正相关,P=0.0209 注:CRC 为结直肠癌;TCGA 为癌症基因组图谱;MSI 为微卫星不稳定性;MSS 为微卫星稳定性;OS 为总生存期
图3 ST6GAL2 在CRC 组织中的差异表达。图a 为免疫组化染色并扫描后的组织中ST6GAL2 的表达情况;可见ST6GAL2 的表达:Normal>LCC>RCC;图b 为20 例LCC 与20 例RCC 的ST6GAL2 表达有显著差异(Mann-Whitney U 检验,P=0.018);图c 为40 例CRC 组织与配对正常组织的ST6GAL2 表达有显著差异(配对t 检验,P<0.0001) 注:CRC 为结直肠癌;Normal 为正常组织;LCC 为左半结肠癌组织;RCC 为右半结肠癌组织
图4 敲降ST6GAL2 促进结肠癌细胞系的增殖。图a 为ST6GAL2 在CRC 细胞系中的表达;图b 为显微镜下慢病毒载体感染后细胞GFP 绿色荧光,western blot 验证SW480 细胞中的ST6GAL2 在蛋白水平明显降低;图c 为RT-qPCR 验证SW480 细胞中的ST6GAL2 在mRNA 水平明显降低(P<0.0001);图d 为敲低ST6GAL2 促进SW480 细胞的克隆形成;图e 为Image J 软件统计2 组克隆数,P<0.05;图f 为CCK-8 实验结果:敲低ST6GAL2 促进SW480 细胞的增殖能力,P<0.0001;*P<0.05,**P<0.01,***P<0.001,****P<0.0001
图5 敲降ST6GAL2 促进结肠癌细胞系的迁移、侵袭。图a 为划痕实验结果;图b 为Image J 软件分析划痕愈合面积,Wound closure(%)=(1-Area 48 h/ Area 0 h)×100,独立样本t 检验,P<0.001;图c 为Transwell 迁移实验结果;图d 为Image J 软件计数迁移的细胞数目,P<0.01;图e 为Transwell 侵袭实验结果;图f 为2 组细胞发生侵袭的细胞数目,P<0.05
图6 过表达ST6GAL2 降低结肠癌细胞系的增殖能力。图a 为显微镜下慢病毒载体感染后细胞GFp 绿色荧光;图b 和c 为HT29 以及SW480 细胞中的ST6GAL2 在mRNA 水平明显升高(P<0.0001;P<0.01);图d 和e 为Western Blot 验证HT29 及SW480 细胞中的ST6GAL2 在蛋白水平明显升高;图f 为过表达ST6GAL2 降低HT29细胞的克隆形成;图g 为Image J 软件统计克隆数,2 组数据使用SPSS 统计(独立样本t 检验),P<0.01;图h为CCK-8 实验显示过表达ST6GAL2 降低HT29 细胞的增殖能力,Two-way ANOVA 分析数据,P<0.0001
图7 过表达ST6GAL2 降低结肠癌细胞系的迁移能力。图a 为划痕实验结果;图b 为划痕愈合面积,Wound closure(%)=(1-Area 48 h/Area 0 h)×100,数据使用SPSS 统计(独立样本t 检验),P<0.001;图c 是Transwell 迁移实验结果;图d 为Image J 计数迁移的细胞数目,SPSS 进行数据分析(独立样本t 检验),P<0.01
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