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中华普通外科学文献(电子版)

中华普通外科学文献(电子版) ›› 2024, Vol. 18 ›› Issue (05) : 344 -349. doi: 10.3877/cma.j.issn.1674-0793.2024.05.006

论著

连接蛋白43通过调控细胞周期抑制结直肠癌的增殖机制研究
罗青杉1, 梅海涛2, 郝家领3, 蔡锦锋1, 周润楷1, 温玉刚1,†()   
  1. 1. 200080　上海交通大学医学院附属第一人民医院普外科
    2. 200080　上海交通大学医学院附属第一人民医院普外科；210003　上海长征医院结直肠外科
    3. 100044　北京大学人民医院肝胆外科
  • 收稿日期:2024-06-15 出版日期:2024-10-01
  • 通信作者: 温玉刚
  • 基金资助:
    国家自然科学基金项目(81972215)

Connexin 43 inhibiting the proliferation of colorectal cancer by regulating the cell cycle

Qingshan Luo1, Haitao Mei2, Jialing Hao3, Jinfeng Cai1, Runkai Zhou1, Yugang Wen1,†()   

  1. 1. Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
    2. Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China; Department of Colorectal Surgery, Changzheng Hospital, Shanghai 210003, China
    3. Department of Hepatobiliary Surgery, Peking University People’s Hospital, Beijing 100044, China
  • Received:2024-06-15 Published:2024-10-01
  • Corresponding author: Yugang Wen
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罗青杉, 梅海涛, 郝家领, 蔡锦锋, 周润楷, 温玉刚. 连接蛋白43通过调控细胞周期抑制结直肠癌的增殖机制研究[J]. 中华普通外科学文献(电子版), 2024, 18(05): 344-349.

Qingshan Luo, Haitao Mei, Jialing Hao, Jinfeng Cai, Runkai Zhou, Yugang Wen. Connexin 43 inhibiting the proliferation of colorectal cancer by regulating the cell cycle[J]. Chinese Archives of General Surgery(Electronic Edition), 2024, 18(05): 344-349.

目的

探究连接蛋白（CX43）在结直肠癌中的表达和临床关系及其作用与机制。

方法

在线分析网站检索GJA1在结直肠癌中的表达及其与结直肠癌的临床特征关系，下载TCGA数据库中的结直肠癌转录组数据，通过生物信息学方法分析GJA1在配对结直肠癌中的表达，收集临床结直肠癌样本，通过qPCR、Western blotting及免疫组织化学技术验证CX43在结直肠癌中的表达情况。在过表达和敲低CX43的HCT116和LOVO细胞中，利用克隆形成实验及CCK8实验检验其增殖能力，通过流式细胞仪检测其对细胞周期的影响，进一步裸鼠成瘤实验在体内验证其作用，最后通过qPCR和Western blotting技术验证其作用机制。

结果

TCGA数据库和组织样本中发现CX43在结直肠癌中低表达，且与更差的预后相关。过表达GJA1的HCT116细胞转录组数据分析显示，其可以通过抑制细胞周期来抑制结直肠癌的增殖，过表达和敲低CX43能够通过调控细胞周期蛋白D1（CCND1）的水平来抑制或促进结直肠癌的增殖。

结论

CX43在结直肠癌中低表达，并能够通过抑制细胞周期来抑制结直肠癌的增殖。

关键词: 结直肠肿瘤, 连接蛋白43, 细胞周期, 细胞周期蛋白D1
Objective

To investigate the expression and clinical relevance of Connexin 43 (CX43) in colorectal cancer, as well as its role and mechanisms in colorectal cancer.

Methods

The expression of GJA1 in colorectal cancer and its association with clinical characteristics of colorectal cancer were analyzed on online websites. The transcriptomic data of colorectal cancer from the TCGA database was downloaded, and bioinformatics methods were utilized to analyze the expression of GJA1 in paired colorectal cancer. Clinical colorectal cancer samples were collected for validation of CX43 expression through qPCR, Western blotting, and immunohistochemistry techniques. Transcriptional profiling was performed by overexpressing GJA1 in HCT116 cells to explore its role and mechanisms in colorectal cancer. The proliferative capacity of HCT116 and LOVO cells with overexpressed or knocked-down CX43 was assessed through colony formation and CCK8 assays. Flow cytometry was employed to examine the effects of CX43 on the cell cycle. The in vivo effects of CX43 were validated through xenograft experiments in nude mice. Finally, the mechanisms underlying the observed effects were verified using qPCR and Western blotting techniques.

Results

Low expression of CX43 in colorectal cancer was identified in the TCGA database and tissue samples, and the low expression of GJA1 was associated with poor prognosis in colorectal cancer. Transcriptomic analysis of HCT116 cells overexpressing GJA1 revealed its ability to inhibit colorectal cancer proliferation by suppressing the cell cycle. Overexpression and knockdown of CX43 can inhibit or promote the proliferation of colorectal cancer through the regulation of CCND1 levels.

Conclusion

GJA1 is downregulated in colorectal cancer and can inhibit the proliferation of colorectal cancer by suppressing the cell cycle.

Key words: Colorectal neoplasms, Connexin 43, Cell cycle, CCND1
表1 GJA1敲低质粒的序号及其目标序列
质粒 目标序列
shRNA(GJA1)-1 CAATTCTTCTTGCCGCAATTA
shRNA(GJA1)-2 TGGGTCCTGCAGATCATATTT
shRNA(GJA1)-3 GGGCGTTAAGGATCGGGTTAA
表2 实时荧光定量实验所用引物序列
目标基因 正向引物序列(5’→3’) 反向引物序列(5’→3’)
GAPDH GTCTCCTCTGACTTCAACAGCG ACCACCCTGTTGCTGTAGCCAA
GJA1 GGAGATGAGCAGTCTGCCTTTC TGAGCCAGGTACAAGAGTGTGG
CCNA2 CTCTACACAGTCACGGGACAAAG CTGTGGTGCTTTGAGGTAGGTC
CCNE2 CTTACGTCACTGATGGTGCTTGC CTTGGAGAAAGAGATTTAGCCAGG
CCND1 TCTACACCGACAACTCCATCCG TCTGGCATTTTGGAGAGGAAGTG
图1 CX43在结直肠癌中低表达且与预后相关　A 、B显示GJA1在TCGA数据库结肠癌、直肠癌患者中低表达；C GJA1在TCGA数据库中配对的结直肠癌患者中显著低表达（n=50，*P<0.05）；D为qPCR验证GJA1在结直肠癌患者临床标本中低表达（n=12，*P<0.05）；E、F显示CX43蛋白在结直肠癌组织中低表达（比例尺：200 μm）；G GJA1表达水平与不同分期结直肠癌患者总生存率的关系；H结肠癌患者的GJA1表达水平与治疗前CEA水平呈负相关
图2 构建稳定过表达和敲低CX43的结直肠癌细胞株　A GJA1结直肠癌细胞系中的表达排序；B、C为结直肠癌细胞系中CX43的水平及相对表达水平柱状图；D HCT116细胞中GJA1的相对表达水平（****P<0.000 1）；E、F分别为HCT116细胞、LOVO细胞中CX43的Western blotting结果；G LOVO细胞中CX43水平的柱状图
图3 生物信息学分析表明CX43与细胞周期有关　A过表达GJA1后HCT116细胞的差异基因火山图；B、C分别为差异基因的KEGG、GO富集分析结果；D HCT116细胞的GSEA分析结果；E TCGA数据库中结直肠癌患者的GSEA分析结果
图4 体内和体外实验表明CX43能够抑制结直肠癌的增殖　A、B分别为HCT116、LOVO细胞的CCK8增殖图（**P<0.01，***P<0.001）；C、D分别为HCT116、LOVO细胞的克隆形成图；E、F分别为HCT116、LOVO细胞的流式细胞周期分布及柱状图；G皮下瘤的体积增长曲线（*P<0.05）；H皮下瘤的照片；I皮下瘤的重量图（*P<0.05）；J HCT116细胞中的细胞周期相关基因的表达（***P<0.001）；K、L分别为HCT116和LOVO中相关蛋白的Western blotting结果
[1]
Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2024, 74(3): 229-263.
[2]
Dekker E, Tanis PJ, Vleugels JLA, et al. Colorectal cancer[J]. Lancet, 2019, 394(10207): 1467-1480.
[3]
Varela-Eirin M, Varela-Vazquez A, Rodríguez-Candela Mateos M, et al. Recruitment of RNA molecules by connexin RNA-binding motifs: implication in RNA and DNA transport through microvesicles and exosomes[J]. Biochim Biophys Acta Mol Cell Res, 2017, 1864(4): 728-736.
[4]
Aasen T, Mesnil M, Naus CC, et al. Gap junctions and cancer: communicating for 50 years[J]. Nat Rev Cancer, 2016, 16(12): 775-788.
[5]
Chen R, Chen Y, Yuan Y, et al. Cx43 and AKAP95 regulate G1/S conversion by competitively binding to cyclin E1/E2 in lung cancer cells[J]. Thorac Cancer, 2020, 11(6): 1594-1602.
[6]
Zhou M, Zheng M, Zhou X, et al. The roles of connexins and gap junctions in the progression of cancer[J]. Cell Commun Signal, 2023, 21(1): 8.
[7]
Su YJ, Zhang JX, Li SM, et al. Relationship of vasculogenic mimicry, SphK1 expression, and Cx43 expression to metastasis and prognosis in colorectal cancer[J]. Int J Clin Exp Pathol, 2018, 11(11): 5290-5299.
[8]
Gong K, Hong Q, Wu H, et al. Gap junctions mediate glucose transfer to promote colon cancer growth in three-dimensional spheroid culture[J]. Cancer Lett, 2022, 531: 27-38.
[9]
Behiels E, Elegheert J. High-level production of recombinant eukaryotic proteins from mammalian cells using lentivirus [M]//OWENS R J. Structural proteomics: high-throughput methods. New York, NY; Springer US, 2021: 83-104.
[10]
GBD 2019 Colorectal Cancer Collaborators. Global, regional, and national burden of colorectal cancer and its risk factors, 1990-2019: A systematic analysis for the Global Burden of Disease Study 2019[J]. Lancet Gastroenterol Hepatol, 2022, 7(7): 627-647.
[11]
Tanaka T, Kimura M, Ishiguro H, et al. Connexin 43 expression is associated with poor survival in patients with esophageal squamous cell carcinoma[J]. Mol Clin Oncol, 2016, 4(6): 989-993.
[12]
Sheng J-g, Wang B, Diao Z-p, et al. BRMS1 and Cx43 expression in fine needle aspiration thyroid cancer tissue and their correlation with tumor malignancy[J]. J Hainan Med Univ, 2016, 22(17): 112-115.
[13]
Puzzo L, Caltabiano R, Parenti R, et al. Connexin 43 (Cx43) expression in laryngeal squamous cell carcinomas: preliminary data on its possible prognostic role[J]. Head Neck Pathol, 2016, 10(3): 292-297.
[14]
Han Y, Wang H, Chen H, et al. CX43 down-regulation promotes cell aggressiveness and 5-fluorouracil-resistance by attenuating cell stiffness in colorectal carcinoma[J]. Cancer Biol Ther, 2023, 24(1): 2221879.
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