| Summary: | Cancer stem cells (CSCs) are closely related to tumor resistance and tumor recurrence in esophageal squamous cell carcinoma (ESCC). The lack of specific biomarkers to identify and isolate CSCs has led to the slow progression of research on CSCs in ESCC. Here, we established a method to identify and isolate CSCs in ESCC using fluorescence-activated cell sorting with combined surface biomarkers including CD71, CD271, and CD338. CD71<sup>−</sup>/CD271<sup>+</sup>/CD338<sup>+</sup> subpopulation cells possessed more stem cell properties in proliferation, self-renewal, differentiation, metastasis, drug resistance, and tumorigenesis. We further explored possible roles that microRNAs played in stem cells. Using microarrays, we identified that has-miR-21-3p was highly expressed in positive sorted cells, and further functional and Luciferase reporter assays verified that has-miR-21-3p promoted proliferation and anti-apoptosis by regulating TRAF4. We further analyzed the relationship between hsa-miR-21-3p and ESCC in 137 patients with ESCC. Statistical analysis showed that up-regulation of hsa-miR-21-3p was associated with a high risk of ESCC. Collectively, we identified surface biomarkers of stem cells in esophageal squamous cell carcinoma, and discovered thathsa-miR-21-3p may be involved in stemness maintenance by regulating TRAF4.
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