miR-138 suppresses cell proliferation and invasion by inhibiting SOX9 in hepatocellular carcinoma

Abstract

Accumulating evidence suggests that miR-138 expression was frequently downregulated in different cancer types and involves in the progression of tumorigenesis. However, the biological role and molecular mechanism of miR-138 involvement in hepatocellular carcinoma (HCC) still remains largely unknown. Therefore, in the present study, we investigated the role of miR-138 in the progression of HCC. We found that miR-138 expression levels were significantly downregulated in HCC tissues and cell lines compared with the corresponding noncancerous liver tissues and normal hepatic cell line. In addition, we also found that enforced expression of miR-138 inhibited proliferation, colony formation, migration and invasion in HCC cells. Using a luciferase reporter assay, SOX9 was confirmed as a direct target of miR-138. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assay showed that overexpression of miR-138 in HCC cells significantly inhibited SOX9 expression on mRNA level and protein level. Furthermore, SOX9 expression was significantly upregulated in HCC tissues and cell lines, and its mRNA expression is negative correlated with miR-138 expression in clinical HCC tissues (r=-0.689, P<0.01). Of note, downregulation of SOX9 performed similar effects with overexpression of miR-138. These findings suggested that miR-138 functioned as a tumor suppressor in HCC partially via repressing SOX9 expression.

Keywords: Hepatocellular carcinoma; SOX9; invasion; miR-138; proliferation.

Figure 1

miR-138 expression is downregulated in HCC tissues and cell lines. A. qRT-PCR analysis of miR-138 expression in HCC tissues and adjacent no-cancerous tissues from 40 patients with HCC. **P<0.01 versus non-cancerous tissue. B. qRT-PCR analysis of miR-138 expression in four HCC cell lines (SMMC-7721, Hep3B, HepG2, Huh-7) and normal hepatic cell line HL-7702. *P<0.05, **P<0.01 versus HL-7702.

Figure 2

miR-138 inhibits cell proliferation and colony formation in HCC cells. A. HepG2 cells were transfected with miR-138 mimics or miR-NC. The expression level of miR-138 was detected by qRT-PCR. B, C. Cell proliferation and colony formation were determined in HepG2 cells transfected with miR-138 mimic or miR-NC. *P<0.05, **P<0.01 versus miR-NC.

Figure 3

miR-138 inhibits cell migration and invasion in HCC cells. A. Wound healing assay for migration in hepG2 cells transfected with miR-138 mimic or miR-NC. B. Transwell assay for invasion in hepG2 cells transfected with miR-138 mimic or miR-NC. *P<0.05, **P<0.01 versus miR-NC.

Figure 4

miR-138 binds to 3’UTR of SOX9 and decreases expression of SOX9 in HCC cells. A. The potential miR-138 binding sequence of SOX9 3’UTR and the mutant was shown. B. HepG2 cells were co-transfected with miR-138 mimic or miR-NC and Wt or Mut SOX9 3’UTR report plasmid. Luciferase activity was measured. C, D. SOX9 expression on mRNA level and protein level were detected in HepG2 cells transfected with miR-138 mimics or miR-NC. GAPDH was used as a control. *P<0.05, **P<0.01 versus miR-NC.

Figure 5

SOX9 expression was upregulated and inversely correlated with miR-138 expression in HCC tissues. A, B. SOX9 expression on mRNA level and protein level were detected HCC tissues and adjacent no-cancerous tissues. GAPDH was used as a control. *P<0.05, **P<0.01 versus **P<0.01 versus non-cancerous. C. The reverse relationship between SOX9 and miR-138 expression was explored in HCC tissues by pearson product-moment correlation coefficients assay. D. qRT-PCR analysis of SOX9 mRNA expression in four HCC cell lines (SMMC-7721, Hep3B, HepG2, Huh-7) and normal hepatic cell line HL-7702. *P<0.05, **P<0.01 versus HL-7702.

Figure 6

Decreased expression of SOX9 showed similar effect with miR-138 overexpression in HCC cells. A, B. SOX9 expression on mRNA level and protein level was determined in HepG2 cells transfected with si-SOX9 or si-NC. GAPDH was used to an internal control. C-F. Cell proliferation, colony formation, migration and invasion were determined in HepG2 cells transfected with si-SOX9 or si-NC. *P<0.05, **P<0.01 versus miR-NC.