Epigenetic silenced miR-125a-5p could be self-activated through targeting Suv39H1 in gastric cancer

Abstract

Emerging evidence suggests that microRNAs (miRNAs) serve an important role in tumorigenesis and development. Although the low expression of miR-125a-5p in gastric cancer has been reported, the underlying mechanism remains unknown. In the current study, the low expression of miR-125a-5p in gastric cancer was verified in paired cancer tissues and adjacent non-tumour tissues. Furthermore, the GC islands in the miR-125a-5p region were hypermethylated in the tumour tissues. And the hypermethylation was negatively correlated with the 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 the Suv39H1 expression and decrease the H3K9me3 levels. On the other hand, the 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 gastric cancer cells, which suppressed cell migration, invasion and proliferation in vitro and inhibited cancer progression in vivo. Thus, we uncovered here that the epigenetic silenced miR-125a-5p could be self-activated through targeting Suv39H1 in gastric cancer, suggesting that miR-125a-5p might be not only the potential prognostic value as a tumour biomarker but also potential therapeutic targets in gastric cancer.

Keywords: Suv39H1; epigenetic silence; gastric cancer; miR-125a-5p.

Figure 1

Reduced miR‐125a‐5p is correlated with the poor prognosis of gastric cancer. A, miR‐125a‐5p levels in cancer tissues and adjacent normal tissues were determined by qPCR (n = 286). Result is depicted as box plots; middle line indicates median; bottom of box, 25th percentile; and top of box, 75th percentile. *P < .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 = 286). Result is depicted as box plots; middle line indicates median; bottom of box, 25th percentile; and top of box, 75th percentile. *P < .05. D, Correlation between the mRNA level and the methylation level of miR‐125a‐5p. Inset corresponds to Pearson's R correlation and corresponding P value

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 overexpressing 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 < .05. C, Suv39H1 levels in cancer tissues and adjacent normal tissues were determined by qPCR (n = 286). Result is depicted as box plots; middle line indicates median; bottom of box, 25th percentile; and top of box, 75th percentile. *P < .05. D, Overexpressing miR‐125a‐5p could suppress the Suv39H1 expression in two primary gastric cancer cell line (GC‐114 and GC‐026), determined by qPCR. n = 3. *P < .05. E, Overexpressing miR‐125a‐5p could suppress the Suv39H1 expression in two primary gastric cancer cell line (GC‐114 and GC‐026), determined Western blot. NC: empty vector. F, Cell morphology of the primary gastric cancer cell line GC‐114 and GC‐026

Figure 3

Demethylation and activation of endogenous miR‐125a‐5p through exogenous overexpressing miR‐125a‐5p. A, Overexpressing miR‐125a‐5p could suppress the H3K9me3 level in two primary gastric cancer cell line (GC‐114 and GC‐026), determined by Western blot. B, Overexpressing miR‐125a‐5p could suppress the miR‐125a‐5p methylation levels in two primary gastric cancer cell line (GC‐114 and GC‐026). n = 3. *P < .05. C, Overexpressing miR‐125a‐5p could up‐regulate the endogenous miR‐125a‐5p precursor expression in two primary gastric cancer cell line (GC‐114 and GC‐026), determined by qPCR. n = 3. *P < .05. D, Successful overexpression of Suv39H1 in two primary gastric cancer cell line (GC‐114 and GC‐026), determined by Western blot. E, Up‐regulation of endogenous miR‐125a‐5p precursor by exogenous miR‐125a‐5p overexpression could be abolished by Suv39H1 overexpression in two primary gastric cancer cell line (GC‐114 and GC‐026), determined by qPCR. n = 3. *P < .05. F, The demethylation of miR‐125a‐5p by exogenous miR‐125a‐5p overexpression could be abolished by Suv39H1 overexpression in two primary gastric cancer cell line (GC‐114 and GC‐026). n = 3. *P < .05. NC: empty vector

Figure 4

Increasing miR‐125a‐5p levels suppressed gastric cancer cell activities. A, Wound‐healing assay with overexpressing miR‐125a‐5p or empty vector (NC) in two primary gastric cancer cell line (GC‐114 and GC‐026). n = 3. *P < .05. B, Cell invasion assay with overexpressing miR‐125a‐5p or empty vector (NC) in two primary gastric cancer cell line (GC‐114 and GC‐026). n = 3. *P < .05. C, The survival rate analysis with overexpressing miR‐125a‐5p or empty vector (NC) in two primary gastric cancer cell line (GC‐114 and GC‐026). n = 3. *P < .05. D, The cell apoptosis analysis with overexpressing miR‐125a‐5p or empty vector (NC) in two primary gastric cancer cell line (GC‐114 and GC‐026). n = 3. *P < .05

Figure 5

Overexpressing miR‐125a‐5p suppressed gastric cancer development in vivo. A, Overexpressing miR‐125a‐5p decreased the tumour size in vivo. n = 12. *P < .05. B, Overexpressing miR‐125a‐5p decreased the methylation level of miR‐125a‐5p in vivo. n = 12. *P < .05. C, Overexpressing miR‐125a‐5p increased the expression of endogenous miR‐125a‐5p precursor in vivo. n = 12. *P < .05. D, Overexpressing miR‐125a‐5p suppressed the expression of Suv39H1 in vivo. n = 12. *P < .05. NC: empty vector