WAS-like 蛋白在肠癌组织中低表达并抑制人结肠癌细胞的干性

doi:10.16098/j.issn.0529-1356.2022.01.007

摘要/Abstract

摘要：

目的探讨WAS-like 蛋白(WASL)在肠癌的表达情况，以及对肠癌细胞干性的影响。方法运用UALCAN数据库预测WASL蛋白的表达量与肠癌组织中的表达及其与肠癌患者的性别、临床分期、淋巴结转移的关系；组织免疫荧光和细胞免疫荧光染色验证WASL在肠癌组织和细胞中的表达。Western blotting检测人正常结直肠黏膜细胞（FHC细胞）与肠癌细胞HCT-116和SW-480中WASL蛋白的表达情况及WASL的转染效率；应用干细胞球（sphere）形成实验和集落形成实验检测转染后，WASL对肠癌细胞中癌干细胞（CSCs）的影响；Real-time PCR检测CSCs相关基因八聚体结合转录因子4（OCT4），性别决定区Y框蛋白2（SOX2），Lin28，Krüppel样因子（KLF）的表达；采用缺氧，血清剥夺和对5-氟尿嘧啶（5-FU）耐药实验检测WASL对肠癌细胞干性的影响。结果 WASL在肠癌组织中mRNA和蛋白均低表达（P<0.05）；WASL在肠癌组织中低表达，在HCT-116和SW-480中，WASL的表达明显弱于FHC细胞；成功构建稳定WASL表达的肠癌细胞系；Real-time PCR显示，WASL抑制了CDCs相关基因OCT4，SOX2，Lin28，Krüppel样因子（KLF）的表达；干细胞球形成和集落形成实验发现，WASL抑制肠癌细胞形成干细胞球形成和集落形成；缺氧实验和血清剥夺实验发现WASL上调后，能够抑制肠癌对缺氧和缺血清耐受，5-FU耐药实验显示，WASL也能抑制肠癌细胞对5-FU的耐受。结论 WASL蛋白在肠癌组织和肠癌细胞中低表达，并与肠癌的不良预后有关，WASL蛋白的过表达能够抑制肠癌细胞的干性。

关键词: WASL-like蛋白, 肠癌, 干性, 缺氧, 耐药, 免疫印迹法, 人

Abstract:

Objective To explore the expression of Wiskot-Aldrich syndrome-like（WASL）in colon cancer tissues and its function on colon cancer stemness. Methods The UALCAN database was used to analyze the expression of WASL in colon cancer tissues and its correlation among the gender, lymphnode stage and metastasis in colon cancer patients. Immunohistofluorescence and immunocyte fluorescence were used to verify the date from UALCAN. Western blotting was used to detect the expression and transfection efficiency of WASL in human normal colorectal mucosal cells (FHC cells) and colon cancer cells HCT-116, SW-480. Sphere formation assay and colony formation were used to detect the effect of WASL on the stemness of colon cancer. Hypoxia assay, serum deprivation, drug resistance assay were used to detect the stemness of colon cancer cells. Results WASL was lower expressed in colon cancer tissues (P<0.05). WASL was lower expressed in colon cancer tissues than peri-cancer tissues. WASL was obviously lower expressed in HCT-116 and SW-480 than FHC cells. WASL overexpression stable cells were constructed suscessfully in HCT-116 and SW-480.Real-time PCR showed WASL obviously down-regulated the expression of cancer stemcells(CSCs) related markers ［octamer binding transcription factor 4(OCT4), sex determining region Y-box 2(SOX2), Lin28, krüppel-like factor(KLF)］. Sphere formation assay and colony formation assay showed WASL could inhibited the sphere formation and colony formation. Hypoxia assay and serum deprivation showed WASL inhibited the resistance of cancer cells to hypoxia and serum deprivation. Drug resistance assay showed WASL inhibit the resistance of cancer cells to 5-fluorouracil(5-FU). Conclusion WASL is lower expressed in colon cancer tissues and cells, and is related with poor prognosis of colon cancer. Overexpression of WASL can inhibit the stemness of colon cancer cells.

Key words: Wiskot-Aldrich syndrome-like, Colon cancer, Stemness, Hypoxia, Drug resistance, Western blotting, Human

中图分类号:

R735.3

李宏丹杨成 . WAS-like 蛋白在肠癌组织中低表达并抑制人结肠癌细胞的干性[J]. 解剖学报, 2022, 53(1): 50-59.

LI Hong-dan YANG Cheng. Wiskot-Aldrich syndrome-like lower expressed in colon cancer tissues and inhibited the stemness of human colon cancer[J]. Acta Anatomica Sinica, 2022, 53(1): 50-59.

参考文献

［1］ Ferlay J, Colombet M, Soerjomataram I, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods ［J］. Int J Cancer, 2019, 144(8):1941-1953.

［2］ Goldberg RM, Fleming TR, Tangen CM,et al. Surgery for recurrent colon cancer: strategies for identifying resectable recurrence and success rates after resection. Eastern Cooperative Oncology Group,the North Central Cancer Treatment Group,and the Southwest Oncology Group ［J］.Ann Intern Med, 1998, 129(1):27-35.

［3］ Kuo CT, Chiang CL, Chang CH, et al.Modeling of cancer metastasis and drug resistance via biomimetic nano-cilia and microfluidics ［J］.Biomaterials, 2014, 35(5):1562-1571.

［4］ Jung P, Sommer C, Barriga FM, et al. Isolation of human colon stem cells using surface expression of PTK7 ［J］. Stem Cell Reports, 2015, 5(6):979-987.

［5］ Yan PP, Liu J, Zhou R, et al. LASP1 interacts with N-WASP to activate the Arp23 complex and facilitate colorectal cancer metastasis by increasing tumour budding and worsening the pattern of invasion ［J］.Oncogene, 2020, 39(35):5743-5755.

［6］ Hidalgo-Sastre TA, Yan P, Liu J, et al. Loss of Wasl improves pancreatic cancer outcome［J］.JCI Insight, 2020, 5(10):e127275-e127292.

［7］ Martin TA, Pereira G, Watkins G, et al. N-WASP is a putative tumour suppressor in breast cancer cells,in vitro and in vivo,and is associated with clinical outcome in patients with breast cancer ［J］. Clin Exp Metastasis, 2008, 25(2):97-108.

［8］ Chandrashekar DS, Bashel B, Balasubramanya SAH, et al. UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses ［J］. Neoplasia, 2017, 19(8):649-658.

［9］ Nishimura T, Nakata A, Chen XX, et al. Cancer stem-like properties and gefitinib resistance are dependent on purine synthetic metabolism mediated by the mitochondrial enzyme MTHFD2 ［J］.Oncogene, 2019, 38(14):2464-2481.

［10］ Siegel RL, Miller KD, Jemal A. Cancer statistics,2019 ［J］.CA Cancer J Clin, 2019, 69(1):7-34.

［11］ Stein A, Folprecht G. Immunotherapy of colon cancer ［J］.Oncol Res Treat, 2018, 41(5):282-285.

［12］ Huang JZ, Chen M, Chen D, et al. A peptide encoded by a putative lncRNA HOXB-AS3 suppresses colon cancer growth［J］.Mol Cell, 2017, 68(1):171-184.

［13］ Yuan YN, Du MN, Yan XL, et al. Expression of miR-145-5p in colorectal cancer and its relationship with multidrug resistance ［J］. Acta Anatomica Sinica，2020,51(4):553-556. (in Chinese)

袁亚男，杜梦楠，严晓丽，等. MiR-145-5p 在大肠癌中的表达及与多药耐药的关系［J］. 解剖学报，2020，51（4）:553-556.

［14］ Zhang TJ, Han S, Zhang W, et al. Triptolide promoting colon cancer CT26 cell death through induction of autophagy ［J］. Acta Anatomica Sinica, 2016, 47(6): 774-778. (in Chinese)

张天娇，韩森，张玮，等. 雷公藤甲素通过诱导细胞自噬促进结肠癌CT26细胞死亡［J］. 解剖学报，2016，47（6）: 774-778.

［15］ Li H, Courtois ET, Sengupta D, et al. Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors ［J］. Nat Genet, 2017, 49(5): 708-718.

［16］ Morris HT, Fort L, Spence HJ, et al. Loss of N-WASP drives early progression in an Apc model of intestinal tumourigenesis ［J］. J Pathol, 2018, 245(3):337-348.

［17］ Sanchez AM, Flamini MI, Baldacci C, et al. Estrogen receptor-alpha promotes breast cancer cell motility and invasion via focal adhesion kinase and N-WASP ［J］. Mol Endocrinol, 2010, 24(11):2114-2125.

［18］ Jin KM, Lu D, Liu FF, et al. N-WASP is highly expressed in hepatocellular carcinoma and associated with poor prognosis ［J］.Surgery, 2013, 153(4):518-525.

［19］ Liu GH, Chen J, Ji ZG, et al.Expression of neural Wiskott-Aldrich syndrome protein in clear cell renal cell carcinoma and its correlation with clinicopathological features ［J］.Urol Int, 2015, 95(1):79-85.

[1]	洪晓敏李三强崔钦奕郑润月杨孟利罗仁利李前辉 . 酒精性肝纤维化核因子κB信号通路与性别的差异 [J]. 解剖学报, 2024, 55(1): 55-61.
[2]	魏茜茜李超红赵晨露唐家萍刘玉珍. 大鼠颈上神经节中Ⅰ组代谢型谷氨酸受体表达及慢性间歇性低氧对其的影响 [J]. 解剖学报, 2024, 55(1): 3-9.
[3]	袁蕾杨智伟杨惠刘政海何洁万炜. 三七总皂苷抑制小鼠星形胶质细胞活化缓解完全弗氏佐剂所致炎性痛 [J]. 解剖学报, 2024, 55(1): 25-31.
[4]	马婷婷陈建红刘爱翠李海宁. 抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用 [J]. 解剖学报, 2024, 55(1): 32-42.
[5]	邹成功冯浩陈兵唐辉邵川孙谋杨荣何家全. 创伤性颅脑损伤中神经功能障碍与认知功能损伤的动态变化 [J]. 解剖学报, 2024, 55(1): 43-48.
[6]	吕海燕沈西雅赵富生朱梅. 松茸多糖对 1-甲基-4-苯基-吡啶离子诱导 PC12 细胞损伤的影响 [J]. 解剖学报, 2024, 55(1): 49-54.
[7]	郑丽平刘霞杜晓华吴亚娟刘珊珊 . 脑源性神经营养因子在牦牛与黄牛端脑不同区域的表达与分布 [J]. 解剖学报, 2024, 55(1): 10-16.
[8]	郝永明郭磊周程辉裴汉军李莉赵哲 . 改良腹主动脉缩窄法建立心肌肥厚模型[J]. 解剖学报, 2024, 55(1): 120-124.
[9]	许亚平余悦欣陈金福王柯柯郭志坤 . 高龄大鼠心室肌间质弹性纤维和胶原纤维的结构分布特征及其机制 [J]. 解剖学报, 2023, 54(6): 716-721.
[10]	王文娟丁雨桐苏昊任捷孙艳荣王涵菲陆嘉莉张林倩白钰秦丽华. 低雌激素状态下大鼠海马及纹状体Nogo-A的表达变化[J]. 解剖学报, 2023, 54(6): 620-627.
[11]	张琴杨俊霞蒋青松. 福辛普利对糖尿病视网膜病变小鼠血管紧张素转化酶2和血管内皮生长因子的影响[J]. 解剖学报, 2023, 54(6): 628-634.
[12]	梁盼盼禹爱梅杜静寇文辉王欢欢宋爱霞. 基于c-Jun氨基末端激酶/p38丝裂原活化蛋白激酶信号通路探讨阿魏酸钠对偏头痛大鼠炎症反应的抑制作用[J]. 解剖学报, 2023, 54(6): 652-659.
[13]	徐颖刘斌郑兴何宗钊. 补体C3a受体在盲肠结扎穿孔致脓毒症大鼠认知障碍中的作用及机制 [J]. 解剖学报, 2023, 54(6): 668-675.
[14]	刘哲川李坤马帅男孟家琪王艳芹. 利拉鲁肽对百草枯诱导的小鼠帕金森病模型炎症及线粒体融合/分裂的影响 [J]. 解剖学报, 2023, 54(6): 676-681.
[15]	吴俊波杨杰肖锋李金亮 . 微小RNA-138调控Wnt通路对体外人脑胶质瘤细胞生物学行为的影响 [J]. 解剖学报, 2023, 54(6): 682-688.