microRNA-506 regulates proliferation, migration and invasion in hepatocellular carcinoma by targeting F-spondin 1 (SPON1)

Abstract

Our previous study indicates microRNA-506 (miR-506) is downregulated in hepatocellular carcinoma (HCC). In the current study, we investigate the effects of miR-506 on proliferation, migration and invasion in HCC. We report that enforced expression of miR-506 inhibits proliferation, migration and invasion in vitro, and suppresses tumor growth in vivo. Conversely, suppression of miR-506 exhibits promoting effects on proliferation, migration and invasion in vitro, and on tumor growth in vivo. In addition, miR-506 binds to the 3'UTR of F-spondin 1(SPON1), and enforced expression of miR-506 decreases accumulation of SPON1. Moreover, enforced expression of SPON1 and suppression of SPON1 alleviates effects of miR-506 mimics and inhibitors on proliferation, migration and invasion in vitro, respectively. In conclusion, microRNA-506 regulates proliferation, migration and invasion in HCC by targeting SPON1.

Keywords: SPON1; hepatocellular carcinoma; microRNA-506; migration and invasion; proliferation.

Figure 1

Expression of miR506 was assessed by qRT-PCR in HCC tumor specimens and adjacent non-tumor tissues (A). U6 served as internal control. Data represented as 2^-ΔΔCT. Clinical association of miR506 with gender (B), age (C), tumor size (D), lymph node migration (E), drinking history (F) and clinical stages (G) in HCC specimens was analyzed using the two-tailed Student’s t-test. A P value of < 0.05 was considered statistically significant.

Figure 2

miR506 inhibits proliferation, migration and invasion in huh7 cells. A. Expression of miR-506 in huh7 and hepG2 cells. U6 served as internal control. Data represent as means ± SD of three independent experiments. ***P < 0.001 vs NC. B. MTT assay of huh7 and hepG2 cells. Cells were transfected with mimic or inhibitor of miR506 and MTT assay was performed as previously described. Data represents means ± SD of at least three independent experiments. *P < 0.05 vs NC. **P < 0.01 vs NC. C. Transwell assay for migration in huh7 and hepG2 cells. magnification, 40×. Data represents means ± SD of at least three independent experiments. *P < 0.05 vs NC. D. Transwell assay for invasion in huh7 and hepG2 cells. magnification, 40×. Data represents means ± SD of at least three independent experiments. *P < 0.05 vs NC. **P < 0.01 vs. NC.

Figure 3

miR-506 inhibits tumor growth in nude mice. A. Representative pictures of xenograft tumors isolated from nude mice injected subcutaneously with huh7 cells which were transfected with mimic or inhibitor of miR506. B. HE staining of tumor tissues. magnification, 200×. C. Growth curve of tumors. *P < 0.05. **P < 0.01. D. Expression of miR-506 in tumor tissues. U6 served as internal control. Data represent as means ± SD of three independent experiments. ***P < 0.001.

Figure 4

miR-506 negatively regulates SPON1 by binding to the SPON1 3’UTR. A. The putative miR-506 binding site in 3’UTR of SPON1 is indicated with red characters. B. Luciferase reporter assay of huh7 cells transfected with luciferase reporter construct containing wild type (psiCheck2-SPON1-WT) or mutated 3’UTR of SPON1 (psiCheck2-SPON1-mutant). The luciferase activity was normalized to Renilla luciferase activity. Data represents means ± SD of at least three independent experiments. **P < 0.01. C. Protein expression of SPON1 in huh7 cells. D. Protein expression of SPON1 in tumor tissues isolated from nude mice. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 5

Enforced expression of SPON1 alleviates effects of miR-506 mimics on proliferation, migration and invasion. A. Expression of SPON1 in huh7 cells. U6 served as internal control. Data represent as means ± SD of three independent experiments. **P < 0.01 vs miR-506 mimics. B. MTT assay of huh7 cells. Data represents means ± SD of at least three independent experiments. *P < 0.05 vs miR-506 mimics. C. Transwell assay for migration in huh7 cells. original magnification, 40×. Data represents means ± SD of at least three independent experiments. **P < 0.01 vs miR-506 mimics. D. Transwell assay for invasion in huh7 cells. original magnification, 40×. Data represents means ± SD of at least three independent experiments. **P < 0.01 vs miR-506 mimics.

Figure 6

Silencing SPON1 alleviates effects of miR-506 inhibitors on proliferation, migration and invasion. A. Expression of SPON1 in huh7 cells. U6 served as internal control. Data represent as means ± SD of three independent experiments. **P < 0.01 vs miR-506 inhibitors. B. MTT assay of huh7 cells. Data represents means ± SD of at least three independent experiments. **P < 0.01 vs miR-506 inhibitors. C. Transwell assay for migration in huh7 cells. original magnification, 40×. Data represents means ± SD of at least three independent experiments. *P < 0.05 vs miR-506 inhibitors. D. Transwell assay for invasion in huh7 cells. original magnification, 40×. Data represents means ± SD of at least three independent experiments. *P < 0.05 vs miR-506 inhibitors.