miR-125a is upregulated in cancer stem-like cells derived from TW01 and is responsible for maintaining stemness by inhibiting p53

Abstract

microRNA (miR)-125a and miR-125b were demonstrated to translationally and transcriptionally inhibit the mRNA level of p53 following the induction of chemo-reagents in our previous report. As a small subpopulation of nasopharyngeal carcinoma (NPC), cancer stem-like cells (CSCs) function critically in multi-malignant behaviors, including tumorigenesis and metastasis; however, the expression pattern and regulatory role of miR-125a, miR-125b and p53 in CSCs derived from NPC remain unclear. In order to investigate the potential regulatory role of miR-125 on p53, firstly CSCs was isolated from TW01 by culturing in serum-free medium. The stemness of isolated CSCs was examined via self-renewal capacity and side population assays. Following this, the miR-125a, miR-125b and p53 mRNA levels were evaluated via reverse-transcription quantitative polymerase chain reaction. Following the transfections of wild-type p53 or p53 without DNA binding activity (p53-mutR^248Q) into TW01 or CSCs, Chromatin Immunoprecipitation (ChIP), and cell cycle analyses using flow cytometry or Cell Counting Kit-8 assays were performed. Notably, it was determined that miR-125a was significantly upregulated in CSCs derived from TW01, but not miR-125b, and the mRNA and protein levels of p53 were downregulated. The transfection of p53 significantly decreased the cell viability and stopped cell cycle at the G₀/G₁ phases in TW01 and CSCs. The ChIP assay confirmed that the ectopic expression of wild-type p53 transcriptionally regulates its downstream gene, p21, but not B-cell lymphoma 2 nor Sco2. Taken together, the results of the present study indicated that p53 regulates CSCs via its DNA binding activity and potentially, in CSCs, miR-125a regulates the expression of p53, maintaining stemness.

Keywords: cancer stem-like cells; cell cycle arrest; miR-125a; nasopharyngeal carcinoma; p53.

Figure 1.

Identification of isolated CSCs from TW01 and detection of the changes in miR-125a and p53 mRNA levels. (A) CSCs isolated from TW01 imaged at days 4, 8 and 15 (×40). (B) Side population analysis of isolated CSCs, compared with parental TW01 cells. (C) Expression level of miR-125a in CSCs, compared with parental TW01 cells by reverse transcription-quantitative polymerase chain reaction relative to U6 snRNA. *P<0.05, vs. TW01 group. (D) The mRNA and protein levels of p53 in CSCs and TW01 using western blot analysis relative to β-actin. *P<0.05, vs. TW01 group. (E) Regulation of the p53 3′-UTR by miR-125a. *P<0.05, vs. p53 3′UTR+empty vector group. (F) The mRNA (left panel) and protein (right panel) levels were detected following transfection of scrambled mimics or miR-125a mimics. (G) The mRNA (left panel) and protein (right panel) levels were detected following transfection of antago-scrambled or antago-miR-125a.

Figure 2.

p53's DNA binding activity is necessary for its cell cycle regulation in TW01 and CSCs. (A) Confirmation of transfection in TW01 and CSCs. (B) Cell Counting Kit-8 assay to detect the proliferation in TW01 and CSCs. (C) The cell cycle analyses in TW01 and CSCs transfected. *P<0.05 vs. p53 group; CSCs, cancer stem-like cells.

Figure 3.

p53 transcriptionally upregulates p21, but not Sco2 or Bcl2, in CSCs. (A) Chromatin Immunoprecipitation using anti-Flag antibodies demonstrated the binding activity of Flag-p53 or Flag-p53-mut^R248Q to the p21 promoter. *P<0.05 vs. vector group. The detection of (B) mRNA and (C) protein levels of p21, Sco2 and BCL2 in transfected CSCs. *P<0.05 vs. vector group. Bcl2, B-cell lymphoma-2; CSCs, cancer stem-like cells; UTR, untranslated region; DHFR, dihydrofolate reductase.

Figure 4.

miR-125a regulates the proliferation and self-renewal capacity in CSCs. (A) western blot analysis was conducted to detect the expression levels of p53 and p21 following antago-miR-125a transfection. (B) Cell-Counting Kit-8 assay analysis of proliferation for antago-miR-125a and antago-scrambled. *P<0.05 vs. antago-scrambled group. (C) Serial replating assay analysis of the self-renewal capacity of CSCs following transfection of antago-miR-125a, compared with antago-scrambled. *P<0.05 vs. antago-miR-125a group. miR, microRNA; OD, optical density; CSCs, cancer stem-like cells; miR, miRNA.