Reck-Notch1 Signaling Mediates miR-221/222 Regulation of Lung Cancer Stem Cells in NSCLC

Abstract

Cancer stem cells (CSCs) contribute to the cancer initiation, metastasis and drug resistance in non-small cell lung cancer (NSCLC). Herein, we identified a miR-221/222 cluster as a novel regulator of CSCs in NSCLC. Targeted overexpression or knockdown of miR-221/222 in NSCLC cells revealed the essential roles of miR-221/222 in regulation of lung cancer cell proliferation, mammosphere formation, subpopulation of CD133⁺ CSCs and the expression of stemness genes including OCT4, NANOG and h-TERT. The in vivo animal study showed that overexpression of miR-221/222 significantly enhanced the capacity of lung cancer cells to develop tumor and grow faster, indicating the importance of miR-221/222 in tumorigenesis and tumor growth. Mechanistically, Reck was found to be a key direct target gene of miR-221/222 in NSCLC. Overexpression of miR-221/222 significantly suppressed Reck expression, activated Notch1 signaling and increased the level of NICD. As an activated form of Notch1, NICD leads to enhanced stemness in NSCLC cells. In addition, knockdown of Reck by siRNA not only mimicked miR-221/222 effects, but also demonstrated involvement of Reck in the miR-221/222-induced activation of Notch1 signaling, verifying the essential roles of the miR-221/222-Reck-Notch1 axis in regulating stemness of NSCLC cells. These findings uncover a novel mechanism by which lung CSCs are significantly manipulated by miR-221/222, and provide a potential therapeutic target for the treatment of NSCLC.

Keywords: Notch1 signaling; Reck; cancer stem cell; miR-221/222; non-small cell lung cancer.

FIGURE 1

Upregulation of miR-221/222 in the CD133⁺ lung cancer stem cells. (A) CD133⁺ CSC isolation from A549 and H1299 cells using FACS analysis. (B) miRNA screening analysis on the CD133⁺ CSC subpopulation, compared with matched CD133^– cells. (C) Chromosomal structure of the miR-221/222 cluster, in which miR-221 and miR-222 share same “seed” sequence. (D) Upregulation of miR-221 and miR-222 in the NSCLC cell lines A549 and H1299, compared with non-tumorigenic lung epithelial cell line BEAS-2B. Data are presented as mean SEM (N = 3), *p < 0.05, **p < 0.01. (E) Higher levels of miR-221 and miR-222 in the NSCLC tumors at stages II/III, compared with the tumors at stage I. **p < 0.01.

FIGURE 2

miR-221/222 promoted cell proliferation in NSCLC. (A,B) CCK8 assays indicating increased cell proliferation by overexpression of miR-221 and miR-222 in A549 (A) and H1299 (B) cells. (C) Colony formation assays indicating the positive regulation of cell proliferation by miR-221/222 in A549 cells. Both overexpression and knockdown of miR-221 or miR-222 were applied. (D) Quantitative analysis of panel (C). (E) Colony formation assays indicating the positive regulation of cell proliferation by miR-221/222 in H1299 cells. Both overexpression and knockdown of miR-221 or miR-222 were applied. (F) Quantitative analysis of panel (E). Data are presented as mean ± SEM (N = 3), *p < 0.05, **p < 0.01.

FIGURE 3

miR-221/222 promoted the cancer cell stemness in NSCLC. (A) Increased percentage of CD133⁺ CSCs in A549 cells after treatment with miR-221 or miR-222. Conversely, CD133⁺ CSC percentage decreased after treatment with anti-miR-221 or anti-miR-222. (B) Quantitative analysis of panel (A). (C) H1299 cells were applied with same assays with panel (A). (D) Quantitative analysis of panel (C). (E) Sphere formation assays using A549 cells treated with miR-221 or miR-222 mimics. (F) Sphere formation assays using A549 cells treated with anti-miR-221 or anti-miR-222. (G) Quantitative analysis of the number and size of the spheres in panel (E). (H) Quantitative analysis of the number and size of the spheres in panel (F). (I,J) Quantitative real-time PCR (I) and western blot (J) analyses indicating the increased expression of Oct4, Nanog and h-Tert in A549 cells after treatment with miR-221 or miR-222 mimics. Consistent results were obtained after treatment with anti-miR-221 or anti-miR-222. (K,L) Doxycycline-induced miR-221/222 overexpression promoted CD133⁺ CSC percentage in A549 cells. (M) Doxycycline-induced miR-221/222 overexpression increased the expression of Oct4, Nanog, and h-Tert in A549 cells. Data are presented as mean ± SEM (N = 3), *p < 0.05, **p < 0.01.

FIGURE 4

miR-221/222 promoted tumor growth in the xenograft model with NSCLC. (A) Schematic representation of the procedure for in vivo assays. (B) Tumor growth curves of the two groups of mice (miR-221/222-overexpressing group and control group). (C) Tumor images isolated from the mice. (D) Weight of the tumors from both groups of mice. Data are presented as mean ± SEM (N = 8), *p < 0.05, **p < 0.01.

FIGURE 5

Reck gene mediated the miR-221/222-induced Notch1 signaling activation in NSCLC. (A) 2,313 predicted target genes of miR-221/222, 5,346 stem cell-related genes and 87 NSCLC-regulating genes overlapped 3 genes including Ect2, Braf and Reck. (B) Quantitative real-time PCR analysis validated downregulation of Reck by overexpression of miR-221/222 in A549 cells. (C) Upregulation of Reck was shown after knockdown of miR-221/222 in A549 cells. (D) Wild type (WT) or point mutated (MU) Reck 3′UTR were cloned into pGL-3 Luciferase reporter vector. The miR-221/222 binding site was mutated in MU. (E) Luciferase reporter assays indicating the direct inhibition of Reck WT 3′UTR by miR-221/222, which was attenuated by the binding-site mutation. (F) Western blot analyses indicating the activation of Notch1 signaling by miR-221/222 overexpression in A549 cells, including increased level of Notch1 intracellular domain (NICD) and decreased level of uncleaved Notch1, which were associated with downregulation of Reck. Consistent results were obtained in A549 cells after knockdown of miR-221/222. (G) Western blot analyses indicating the increase of NICD by knockdown of Reck. But miR-221/222 did not show regulation on the level of NICD in the si-Reck-treated A549 cells. (H) Schematic representation of the mechanism through which miR-221/222 promoted CSCs properties in NSCLC by suppressing Reck and activating Notch1 signaling. Data are presented as mean ± SEM (N = 3), *p < 0.05, **p < 0.01, ns means non significant.