一种结肠癌干细胞上皮-间质转化分层模型的建立和验证

马晓莹; 李小嘉; 金文琪; 丁旭枫; 郭修田

doi:10.19401/j.cnki.1007-3639.2019.02.002

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一种结肠癌干细胞上皮-间质转化分层模型的建立和验证

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- 一种结肠癌干细胞上皮-间质转化分层模型的建立和验证
- Establishment and verification of a stratification model of colon cancer stem cell epithelial-mesenchymal transition
- 中国癌症杂志 2019年29卷第2期页码：100-110
- 作者机构：
  
  1. 华东理工大学药学院,上海,200237
  2. 上海中医药大学附属市中医医院肛肠科,上海,200070
- 作者简介：
- 基金信息：
  
  国家自然科学基金（81573977）;上海中医药大学研究生创新培养项目（Y201853）。
- DOI：10.19401/j.cnki.1007-3639.2019.02.002
  中图分类号：
- 网络出版：2019-03-25，
  
  纸质出版：2019-03-25
- 稿件说明：
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李鹏，马晓莹，李小嘉，金文琪，丁旭枫，郭修田. 一种结肠癌干细胞上皮-间质转化分层模型的建立和验证[J]. 中国癌症杂志, 2019, 29(2): 100-110.

LI Peng, MA Xiaoying, LI Xiaojia, et al. Establishment and verification of a stratification model of colon cancer stem cell epithelial-mesenchymal transition[J]. China Oncology, 2019, 29(2): 100-110.
李鹏，马晓莹，李小嘉，金文琪，丁旭枫，郭修田. 一种结肠癌干细胞上皮-间质转化分层模型的建立和验证[J]. 中国癌症杂志, 2019, 29(2): 100-110. DOI： 10.19401/j.cnki.1007-3639.2019.02.002.

LI Peng, MA Xiaoying, LI Xiaojia, et al. Establishment and verification of a stratification model of colon cancer stem cell epithelial-mesenchymal transition[J]. China Oncology, 2019, 29(2): 100-110. DOI： 10.19401/j.cnki.1007-3639.2019.02.002.

摘要

背景与目的：肿瘤干细胞（cancer stem cells，CSC）是肿瘤中具有干细胞特性的一类细胞，同时具有自我更新能力及多向分化潜能。CSC在肿瘤转移过程中起着重要作用。上皮-间质转化（epithelial-mesenchymal transition，EMT）与恶性肿瘤浸润、转移及与肿瘤患者预后紧密联系。本研究以CSC是否经EMT为标准，建立一种新型结肠癌干细胞EMT分层模型，以评价不同活性物质对不同分型的结肠癌干细胞表型的影响。并用当下研究成熟的抑癌基因miR-139-5p及其靶向抑制的转录因子4（transcription factor 4，TCF4）蛋白验证上述模型的可行性，为临床研究提供理论依据。方法：以CD44和细胞上皮黏附分子/上皮特异性抗原（epithelial cell adhesion molecule/epithelial specific antigen，EpCAM/ESA）为结肠癌干细胞标志物，采用流式细胞术分选皮下移植瘤中CD44+ESA+、CD44+ESA-细胞亚群和转移瘤中CD44

ESA

、CD44

ESA

细胞亚群，分别命名为Epi-S、Epi-P、pEMT-S和pEMT-P，亲本细胞株HCT116作为后续实验的阴性对照；验证分选后不同细胞亚群的多药耐药性、克隆形成能力以及干性转录因子SOX2、OCT4和EMT相关蛋白E-cadherin、N-cadherin、vimentin的表达情况；检测miR-139-5p过表达、TCF4敲低对Epi-P和pEMT-P以及TCF4过表达对Epi-S和pEMT-S CSC侵袭能力与EMT标志分子E-cadherin、N-cadherin和vimentin蛋白的影响。结果：相较于HCT116亲本细胞株，Epi-S、Epi-P、pEMT-S和pEMT-P共4种CSC分型均对奥沙利铂（oxaliplatin，LOHP）和氟尿嘧啶（fluorouracil，5-FU）产生了不同程度的抗性；干性相关转录因子OCT4和SOX2在4种CSC分型中表达均升高，与亲本细胞株相比，差异有统计学意义（P0.05）；上皮相关标志物E-cadherin、N-cadherin和vimentin在4种CSC分型中表达均升高，与亲本细胞株相比，差异有统计学意义（P0.05）；过表达miR-139-5p或敲低TCF4均能抑制Epi-P和pEMT-P结肠癌干细胞亚型的侵袭能力（P0.01）；在表型稳定未经历EMT的上皮亚系Epi-S中，过表达TCF4能够显著促进其侵袭能力（P0.01）；Epi-P和pEMT-P中过表达miR-139-5p和敲低TCF4，上皮标志物E-cadherin表达增加，间充质标志物N-cadherin和vimentin表达下降。结论：以CD44和ESA为结肠癌干细胞标志物分选出的4种分型具有不同表型可塑性，该模型可用来评价不同生物活性物质对结肠癌干细胞

上皮-间质转化的影响。

Abstract

Background and purpose: Cancer stem cells (CSC) are a class of cells with stem cell characteristics in tumors

and have self-renewal ability and multi-directional differentiation potential. CSC play an important role in tumor metastasis. Epithelialmesenchymal transition (EMT) is closely associated with malignant tumor invasion

metastasis and prognosis of patients with cancer. We established a novel colon cancer EMT stratification model based on whether CSC undergo EMT as a standard to evaluate the effects of different active substances on various types of colon cancer stem cell phenotypes. The feasibility of the above model was verified by the tumor suppressor gene miR-139-5p and transcription factor 4 (TCF4) protein

providing a theoretical basis for clinical research. Methods: CD44 and epithelial cell adhesion molecule/epithelial specific antigen (EpCAM/ESA) were used as markers of colon cancer stem cells. Flow cytometry was used to sort CD44

ESA

and CD44

ESA

cell subsets in subcutaneous tumors and metastatic tumors

respectively. These four cell subsets were named Epi-S

Epi-P

pEMT-S and pEMT-P respectively

and the parental strain HCT116 was used as a negative control for subsequent experiments. The multi-drug resistance

clonal formation ability and the expression levels of stemness transcription factors (SOX2

OCT4) and EMT-related proteins (E-cadherin

N-cadherin and vimentin) were verified after sorting. We then tested the effects of miR-139-5p overexpression and TCF4 knockdown on the invasive ability of Epi-P and pEMT-P and the expression of EMT marker proteins. At the same time

the effects of TCF4 overexpression on the invasion of Epi-S and pEMT-S and the EMT markers including E-cadherin

N-cadherin and vimentin were detected. Results: Compared with HCT116

these four CSC had different degrees of resistance to oxaliplatin (LOHP) and fluorouracil (5-FU). The express

ion levels of OCT4 and SOX2 were increased in the four CSC compared with the parental strains (P0.05). Compared with parental cell strains

the expression levels of E-cadherin

N-cadherin and vimentin in the four CSC types were also significantly increased (P0.05). Overexpression of miR-139-5p or knockdown of TCF4 inhibited the invasive ability of Epi-P and pEMT-P colon cancer stem cell subtypes (P0.01). Overexpression of TCF4 in Epi-S with phenotypic stability and no EMT significantly promoted its invasive ability (P0.01). When overexpression of miR-139-5p and knockdown of TCF4 were achieved in Epi-P and pEMT-P

the expression of the epithelial cell marker E-cadherin increased

and the expression of the mesenchymal markers including N-cadherin and vimentin decreased. Conclusion: The four subtypes of colon cancer stem cell markers selected by CD44 and ESA have different phenotypic plasticity. The model can be used to evaluate the effects of different active substances on EMT of colon cancer stem cells.

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