Reduced miR-125a-5p level in non-small-cell lung cancer is associated with tumour progression

Abstract

Emerging evidence suggests that microRNAs (miRNAs) serve an important role in tumourigenesis and development. Although the low expression of miR-125a-5p in non-small-cell lung cancer (NSCLC) has been reported, the underlying mechanism remains unknown. In the current study, the low expression of miR-125a-5p in NSCLC was verified in paired cancer tissues and adjacent non-tumour tissues. Furthermore, the CpG island in the miR-125a-5p region was hypermethylated in the tumour tissues, and the hypermethylation was negatively correlated with miR-125a-5p expression. Target gene screening showed that the histone methyltransferase Suv39H1 was one of the potential target genes. In vitro studies showed that miR-125a-5p could directly suppress Suv39H1 expression and decrease the H3K9me3 levels. On the other hand, Suv39H1 could induce demethylation of miR-125a-5p, resulting in re-activation of miR-125a-5p. What is more, overexpessing miR-125a-5p could also self-activate the silenced miR-125a-5p in NSCLC cells, which suppressed cell migration, invasion and proliferation in vitro and inhibited cancer progression in vivo Thus, we found that the epigenetic silenced miR-125a-5p could be self-activated through targeting Suv39H1 in NSCLC, suggesting that miR-125a-5p might not only have the potential prognostic value as a tumour biomarker but also be a potential therapeutic target in NSCLC.

Keywords: Suv39H1; miR-125a-5p; non-small-cell lung cancer.

Figure 1.

Reduced miR-125a-5p is correlated with the poor prognosis of NSCLC. (a) miR-125a-5p levels in cancer tissues and adjacent normal tissues were determined by qPCR (n = 384). The middle line indicates median; bottom of box, 25th percentile; top of box, 75th percentile. *p < 0.05. (b) Kaplan–Meier survival curve of patients with high or low level of miR-125a-5p. (c) miR-125a-5p methylation levels in cancer tissues and adjacent normal tissues were determined (n = 384). The middle line indicates median; bottom of box, 25th percentile; top of box, 75th percentile. *p < 0.05. (d) Correlation between the mRNA level and the methylation level of miR-125a-5p.

Figure 2.

Suv39H1 is the miR-125a-5p target gene. (a) The potential binding site of miR-125a-5p in the 3′UTR of Suv39H1. (b) Luciferase assay performed by over-expressing miR-125a-5p and the wild-type of Suv39H1 3′UTR (WT-3′UTR) or the 3′UTR without potential miR-125a-5p binding site (Mut-3′UTR). n = 3. *p < 0.05. (c) Suv39H1 levels in cancer tissues and adjacent normal tissues were determined by qPCR (n = 384). The middle line indicates median; bottom of box, 25th percentile; top of box, 75th percentile. *p < 0.05. (d) Over-expressing miR-125a-5p could suppress the Suv39H1 expression in NSCLC cell lines (A549 and SK-MES-1), determined by qPCR. n = 3. *p < 0.05. (e) Over-expressing miR-125a-5p could suppress the Suv39H1 expression in NSCLC cell lines (A549 and SK-MES-1), determined by western blot. NC: empty vector.

Figure 3.

Demethylation and activation of endogenous miR-125a-5p through exogenous over-expressing miR-125a-5p. (a) Over-expressing miR-125a-5p could suppress the H3K9me3 level in NSCLC cell lines (A549 and SK-MES-1), determined by western blot. (b) Over-expressing miR-125a-5p could suppress the miR-125a-5p methylation levels in NSCLC cell lines (A549 and SK-MES-1). n = 3. *p < 0.05. (c) Over-expressing miR-125a-5p could upregulate the endogenous miR-125a-5p precursor expression in NSCLC cell lines (A549 and SK-MES-1), determined by qPCR. n = 3. *p < 0.05. (d) Successful over-expression of Suv39H1, determined by western blot in NSCLC cell lines (A549 and SK-MES-1). (e) Upregulation of endogenous miR-125a-5p precursor by exogenous miR-125a-5p over-expression could be abolished by Suv39H1 over-expression in NSCLC cell lines (A549 and SK-MES-1), determined by qPCR. n = 3. *p < 0.05. (f) The demethylation of miR-125a-5p by exogenous miR-125a-5p over-expression could be abolished by Suv39H1 over-expression in NSCLC cell lines (A549 and SK-MES-1). n = 3. *p < 0.05. NC: empty vector.

Figure 4.

Increasing miR-125a-5p level suppressed NSCLC cell activities. (a) Wound healing assay with over-expressing miR-125a-5p or empty vector (NC) in NSCLC cell lines (A549 and SK-MES-1). n = 3. *p < 0.05. (b) Cell invasion assay with over-expressing miR-125a-5p or empty vector (NC) in NSCLC cell lines (A549 and SK-MES-1). n = 3. *p < 0.05. (c) The survival rate analysis with over-expressing miR-125a-5p or empty vector (NC) in NSCLC cell lines (A549 and SK-MES-1). n = 3. *p < 0.05. (d) The cell apoptosis analysis with over-expressing miR-125a-5p or empty vector (NC) in NSCLC cell lines (A549 and SK-MES-1). n = 3. *p < 0.05.

Figure 5.

Over-expressing miR-125a-5p suppressed NSCLC development in vivo. (a) Over-expressing miR-125a-5p decreased the tumour size in vivo. n = 12. *p < 0.05. (i) Statistical analysis; (ii) representative figures of the tumour formation. (b) Over-expressing miR-125a-5p decreased the methylation level of miR-125a-5p in vivo. n = 12. *p < 0.05. (c) Over-expressing miR-125a-5p increased the expression of endogenous miR-125a-5p precursor in vivo. n = 12. *p < 0.05. (d) Over-expressing miR-125a-5p suppressed the expression of Suv39H1 in vivo. n = 12. *p < 0.05. NC: empty vector.