Conclusion
miR-148a regulates the stem cell-like side populations distribution by inhibiting the expression of ACVR1 in ESCC. miR-148a may be a promising targeted therapy for ESCC.
Methods
A total of 20 human ESCC samples were collected from the Affiliated Tumor Hospital of Xinjiang Medical University. Eca109 and Kyse510 cells, which are ESCC cell lines, were subjected to FACS analysis to get side population (SP) cells and non-SP cells. Cell cycle and cell proliferation were analyzed by flow cytometry. Cell migration and invasion were detected using a transwell assay. Quantitative PCR and Western blot were performed to analyze the expression levels of ABCG2, KLF4, OCT4, and ACVR1, which are related to the stemness of stem cells. The target genes of hsa-miR-148a were predicted using TargetScan (version 7.2) and verified by a dual luciferase reporter assay. A chromatin immunoprecipitation (ChIP) assay was carried out to demonstrate direct interaction between miR-148a and ACVR1.
Results
The expression of miR-148a was significantly down-regulated in ESCC cells and significantly decreased in SP esophageal squamous cells when compared to the tumor cells. By analyzing the stem cell stemness of ESCC, overexpression of miR-148a decreased the expression of ABCG2, KLF4, SOX2, OCT4, and Nanog, indicating that miR-148a may regulate stem cell function. Target gene prediction and functional annotation of miR-148a suggested that miR-148a is involved in stem cell stemness of ESCC via ACVR1. Expression of the dual luciferase-labeled gene indicates that overexpression of miR-148a inhibits the expression of ACVR1, thereby affecting stem cell stemness.