miR-200a-3p- and miR-181-5p-Mediated HOXB5 Upregulation Promotes HCC Progression by Transcriptional Activation of EGFR

Abstract

Background: Hepatocellular carcinoma (HCC) remains a worldwide burden. However, the mechanisms behind the malignant biological behavior of HCC remain unclear. The homeobox (HOX) family could act as either promoters or suppressors in different kinds of malignancies. Our study discovered the role of HOXB5 in regulating HCC progression.

Methods: The HOXB5 expression was assessed by RT-PCR analysis in human HCC samples and cell lines. HOXB5 transcriptional regulation of the EGFR was verified by the luciferase reporter assay and chromatin immunoprecipitation experiment. The oncogenic role of HOXB5 in HCC progression was analyzed by CCK8, colony-forming, and transwell assays.

Results: Upregulation of HOXB5 was found in human HCC, and was strongly correlated with HCC tumor size, tumor-nodule metastasis, TNM stage, and relatively unfavorable OS and DFS. Ectopic expression of HOXB5 promoted the capacity of cell growth and clonogenicity, while the inhibition of HOXB5 decreased the proliferation and clonogenicity potential in vitro by CCK8 and colony-forming assays. In addition, HOXB5 also promoted cell migration by transwell experiment. Mechanism studies elucidated that HOXB5 triggers HCC progression via direct transcriptional activation of EGFR. The upregulation of HOXB5 is regulated by miR-200a-3p and miR-181-5p. Transfection of miR-200a-3p and miR-181-5p mimics blocked the cell proliferation and migration regulated by HOXB5, while overexpression of the 3'-UTR mutant HOXB5 abolished the suppressive effect of miR-200a-3p and miR-181-5p, but not the wild-type HOXB5.

Conclusion: HOXB5 is a promising prognostic factor in human HCC. Targeting miR-200a-3p and the miR-181-5p/HOXB5/EGFR signaling pathway may provide new options for the treatment strategies of HCC.

Keywords: EGFR; HCC; HOXB5; miR-181-5p; miR-200a-3p.

Figure 1

Elevated expression of HOXB5 is frequently found in HCC patients and positively correlates with poor prognosis. (A) mRNA expression level of HOXB5 in 100 pairs of HCC tissues and adjacent non-tumor tissues was evaluated by qRT-PCR. Data are mean ± SD, n = 100. (B) mRNA levels of HOXB5 were significantly higher in recurrence patients than in patients free of recurrence. (C) The HOXB5 expression level was much higher in metastasis patients than in patients free of metastasis. (D, E) Kaplan–Meier curve of HOXB5 depicting the OS and DFS of HCC patients. (F) mRNA expression of HOXB5 in human HCC was evaluated in a public database.

Figure 2

HOXB5 promotes cell proliferation and migration of HCC cells. (A) Expression of HOXB5 in HepG2, Huh-7 and Hep3B HCC cell lines as tested by qRT-PCR and Western bolt. Data are mean ± SD. **p < 0.01, ***p < 0.001; (B, C) Expression of HOXB5 in HepG2-HOXB5 and Hep3B-shHOXB5, and their corresponding control cell lines as tested by qRT-PCR and Western bolt. Data are mean ± SD. **p < 0.01, ***p < 0.001; (D) CCK8 and clonogenicity assays of proliferation capability of HCC cells. Data are mean ± SD. *p < 0.05, ***p < 0.001; (E) Invasion and migration assays of HepG2-HOXB5 and Hep3B-shHOXB5, and their corresponding control cell lines as tested by transwell experiment. Data are mean ± SD. *p <0.05. Scale bars, 100µm.

Figure 3

EGFR is a direct transcriptional target of HOXB5. (A, B) Expression of EGFR in HepG2-HOXB5 and Hep3B-shHOXB5, and their corresponding control cell lines as tested by qRT-PCR and Western blot. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. (C) The luciferase reporter assay demonstrated that ectopic expression of HOXB5 promoted the luciferase activity of the EGFR promoter. (D) EGFR promoter. Serially truncated and mutated EGFR promoter constructs were co-transfected with pCMV-HOXB5, and relative luciferase activities were determined. (E, F) ChIP assays demonstrated the direct binding of HOXB5 to the EGFR promoter in HepG2-HOXB5 cells and the enriched binding of endogenous HOXB5 to the EGFR promoter in primary HCC tissues.

Figure 4

HOXB5 promotes HCC progression by upregulating EGFR expression, and positively correlated with EGFR expression in human HCC tissues. (A) Expression of EGFR in HepG2-HOXB5 and Hep3B-shHOXB5, and their corresponding control cell lines as tested by Western bolt. Data are mean ± SD. **p < 0.01; (B) CCK8 and clonogenicity assays of proliferation capability of HCC cells. Data are mean ± SD. *p < 0.05; (C) Migration assay of HepG2-HOXB5 and Hep3B-shHOXB5, and their corresponding control cell lines as tested by transwell experiment. Data are mean ± SD. *p < 0.05, **p < 0.01. Scale bars, 100µm; (D) mRNA expression level of EGFR in 100 pairs of HCC tissues and adjacent non-tumor tissues was evaluated by qRT-PCR. Data are mean ± SD, n=100; (E) EGFR expression is positively corelated with HOXB5 expression; (F) Kaplan-Meier analysis showed that HCC patients with positive co-expression of EGFR and HOXB5 showed lowest OS and DFS times.

Figure 5

miR-200a-3p and miR-181-5p upregulates HOXB5 expression to contributes HCC progression. (A) Luciferase reporter assay confirmed that miR-200a-3p mimic could successfully made a dramatic reduction in the relative luciferase activity when HOXB5 3′ UTR sequences containing WT miR-200a-3p binding sites compared with HOXB5 3′ UTR sequences containing mutant miR-200a-3p binding sites; (B) Expression of 200a-3p in 100 pairs of HCC tissues and adjacent non-tumor tissues was evaluated by qRT-PCR. Data are mean ± SD, n=100; (C) Luciferase reporter assay confirmed that miR-181-5p mimic could successfully made a dramatic reduction in the relative luciferase activity when HOXB5 3′ UTR sequences containing WT miR-181-5p binding sites compared with HOXB5 3′ UTR sequences containing mutant miR-181-5p binding sites; (D) Expression of miR-181-5p in 100 pairs of HCC tissues and adjacent non-tumor tissues was evaluated by qRT-PCR. Data are mean ± SD, n=100. ***p < 0.001.