关键词: 结肠癌干细胞, CD44, 上皮细胞黏附分子, 上皮-间质转化

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 expression levels of OCT4 and SOX2 were increased in the four CSC compared with the parental strains (P<0.05). Compared with parental cell strains, the expression levels of E-cadherin, N-cadherin and vimentin in the four CSC types were also significantly increased (P<0.05). Overexpression of miR-139-5p or knockdown of TCF4 inhibited the invasive ability of Epi-P and pEMT-P colon cancer stem cell subtypes (P<0.01). Overexpression of TCF4 in Epi-S with phenotypic stability and no EMT significantly promoted its invasive ability (P<0.01). 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.

Key words: Colon cancer stem cells, CD44, Epithelial cell adhesion molecule, Epithelial-mesenchymal transition