miR‑185 inhibits non‑small cell lung cancer cell proliferation and invasion through targeting of SOX9 and regulation of Wnt signaling

Abstract

SRY-box 9 (SOX9) is an important transcription factor required for development, which has additionally been reported to be an independent prognostic indicator for the survival of patients with non‑small cell lung cancer (NSCLC). Accumulating evidence has indicated that dysregulation of microRNAs (miRNAs/miRs) may contribute to the initiation and progression of cancer. SOX9 may be regulated by a number of miRNAs in different types of cancer, including in NSCLC. The present study sought to identify novel candidate miRNAs associated with SOX9 in NSCLC using online tools, and investigated the detailed functions of miR‑185, which suppressed SOX9 mRNA expression most strongly out of the candidate miRNAs. It was observed that ectopic miR‑185 expression significantly suppressed NSCLC cell proliferation, invasion and migration. Using luciferase reporter gene and RNA immunoprecipitation assays, SOX9 was confirmed to be a direct target of miR‑185. In addition, the downstream Wnt signaling‑associated factors β‑catenin and c‑Myc proto‑oncogene protein (Myc) were demonstrated to be inhibited by miR‑185 overexpression. SOX9, β‑catenin and c‑Myc mRNA expression was significantly upregulated in NSCLC tissues, and was inversely correlated with miR‑185 expression. The results of the present study demonstrated that rescuing miR‑185 expression in NSCLC, thereby inhibiting SOX9 expression and the downstream Wnt signaling, and leading to the suppression of NSCLC cell proliferation, invasion and migration, may be a promising strategy for the treatment of NSCLC.

Figure 1.

Screening and verification of candidate miRNAs of SOX9. (A) Online tools including miRWalk, miRanda, RNA22, Targetscan and starBase were employed to screen for the candidate miRNAs associated with SOX9. The following miRNAs were screened out: miR-105, miR-138, miR-15a, miR-15b, miR-19a, miR-19b, miR-206, miR-30b, miR-424, miR-497 and miR-185. Overexpression of the indicated miRNAs was achieved through transfection of miRNA mimics, compared with mimics-NC. (B) The miRNA mimics of the indicated miRNAs were transfected into A549 cells to achieve ectopic miRNA expression, as verified using qPCR assays. SOX9 mRNA expression in response to the ectopic miRNA expression of the indicated miRNAs was determined using qPCR assays. The mean value of the expression of controls was adjusted to 1. The data are presented as mean ± standard deviation of three independent experiments. **P<0.01 vs. respective mimics-NC group. NC, negative control; miRNA/miR, microRNA; SOX-9, SRY-box 9; qPCR, quantitative polymerase chain reaction.

Figure 2.

Low miR-185 expression in NSCLC is associated with poorer clinicopathological parameters and shorter overall survival. (A) miR-185 expression in 42 pairs of NSCLC tissues was determined using qPCR assays, compared with the adjacent tissues. (B) The expression of miR-185 in 42 pairs of NSCLC tissues and their corresponding adjacent non-tumorous tissues was analyzed. The expression level of miR-185 was determined by qPCR and normalized to U6. Fold changes were analyzed using the 2^−ΔΔCq method. (C) Kaplan-Meier overall survival curves for 42 patients with NSCLC classified according to relative miR-185 expression level. (D) miR-185 expression in NSCLC cell lines A549, H1299, H1650 and SK-MES-1, and normal cell line BEAS-2B using qPCR assays. The data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. BEAS-2B. miR, microRNA; qPCR, quantitative polymerase chain reaction; NSCLC, non-small cell lung cancer.

Figure 3.

Functions of miR-185 in the regulation of non-small cell lung cancer cell proliferation. (A) A549 and SK-MES-1 cells were transfected with miR-185 mimics or miR-185 inhibitor to achieve ectopic miR-185 expression or miR-185 inhibition, as verified using quantitative polymerase chain reaction assays. The cell viability of (B) A549 and (C) SK-MES-1 cells was determined using MTT assays. The DNA synthesis capability of (D) A549 and (E) SK-MES-1 cells was determined using 5-bromo-2′-deoxyuridine assays. The data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. respective mimics-NC group. NC, negative control; miR, microRNA; OD, optical density.

Figure 4.

Effects of miR-185 on non-small cell lung cancer cell invasion and migration. A549 and SK-MES-1 cells were transfected with miR-185 mimics or miR-185 inhibitor to achieve ectopic miR-185 expression or miR-185 inhibition. The cell invasion capability of A549 and SK-MES-1 cells was determined using Transwell assays. (A) Representative images and (B) statistical analysis are presented. The cell migration capability of A549 and SK-MES-1 cells was determined using a cell scratch test. (C) Representative images and (D) statistical analysis are presented. The data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. respective mimics-NC group. NC, negative control; miR, microRNA.

Figure 5.

miR-185 inversely regulates SOX9 expression through direct binding to the 3′UTR of SOX9. (A) Wt-SOX9 3′UTR and mut-SOX9 3′UTR, containing a 6-bp mutation in the predicted binding site of miR-185, luciferase reporter gene vectors were constructed. A549 cells were co-transfected with the indicated vectors and miR-185 mimics and miR-185 inhibitor, respectively. (B) Luciferase assays were performed to determine the luciferase activity 48 h following transfection using a dual luciferase reporter assay system. **P<0.01 vs. mimics-NC. The association between miR-185/SOX9 and AGO2 was determined. A549 cellular lysates were used for RNA immunoprecipitation with an AGO2 antibody. Detection of AGO2 and IgG using (C) western blotting, and detection of miR-185 or SOX9 using (D) reverse transcription-quantitative polymerase chain reaction analysis. All data of SOX9 expression were normalized to GAPDH mRNA expression levels. miR-185 expression data was normalized to U6 small RNA expression. The data are presented as the mean ± standard deviation of three independent experiments. **P<0.01 vs. respective IgG group. NC, negative control; SOX9, SRY-box 9; UTR, untranslated region; mut, mutated; wt, wild type; miR, microRNA; IgG, immunoglobulin G; AGO2, protein argonaute-2.

Figure 6.

Downstream Wnt signaling of SOX9 is involved in the miR-185 regulation of NSCLC cell proliferation, invasion and migration. A549 and SK-MES-1 cells were transfected with miR-185 mimics or miR-185 inhibitor; the protein levels of SOX9, β-catenin and c-Myc in A549 and SK-MES-1 cells were determined using (A) western blotting and (B) densitometric analysis. The data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 vs. respective mimics-NC group. The mRNA expression of (C) SOX9, (D) β-catenin and (E) c-Myc in NSCLC tissues and adjacent normal tissues was determined using quantitative polymerase chain reaction analysis. The correlation between miR-185 and (F) SOX9, (G) β-catenin and (H) c-Myc, respectively, was analyzed by Spearman's rank correlation analysis. SOX9, SRY-box 9; miR, microRNA; c-Myc, c-Myc proto-oncogene protein; NC, negative control; NSCLC, non-small cell lung cancer.