MicroRNA-26-5p functions as a new inhibitor of hepatoblastoma by repressing lin-28 homolog B and aurora kinase a expression

Abstract

Hepatoblastoma (HB) is the most common liver tumor in children. Despite recent improvements in treatment strategies, the survival of children with hepatoblastoma remains poor. In this study, we identified a novel role of microRNA-26a-5p (miR-26a-5p), lin-28 homolog B (LIN28B), Ras-related nuclear protein (RAN), and aurora kinase A (AURKA) in HB. The expression of LIN28B, RAN, and AURKA was significantly up-regulated in human HB livers and cell lines. Knockdown of LIN28B and RAN by small interfering RNAs inhibited HB tumor cell proliferation and foci formation. We also elucidated miR-26a-5p-mediated translational inhibition of LIN28B and AURKA in HB. Overexpression of miR-26a-5p markedly decreased LIN28B and AURKA 3'-untranslated region activities and protein expression and repressed HB cell proliferation and colony formation. In contrast, re-expression of LIN28B and AURKA rescued miR-26a-5p-mediated suppression of HB cell growth and clonality. Importantly, a decreased miR-26a-5p expression correlated with the poor outcome of patients with HB. Conclusion: miR-26a-5p is a newly identified repressor of HB growth through its inhibition of the oncogenic LIN28B-RAN-AURKA pathway. (Hepatology Communications 2018;2:481-491).

Figure 1

Lin28B, RAN, and AURKA are highly expressed in human HB. (A) Quantitative polymerase chain reaction analysis of LIN28B, RAN, and AURKA expression in nontumor livers and HB. (B) Western blot analysis of LIN28B, RAN, and AURKA protein expression in nontumor livers and HB, with β‐actin as a loading control (n = 4 samples/group). Quantification of the western blot analysis is presented on the right. Data are shown as mean ± SEM (triplicate assays). *P < 0.05 versus nontumor sample.

Figure 2

Lin28B directly regulates AURKA through RAN. (A) qPCR (left) and western blot (right) analysis of Lin28B, RAN, and AURKA expression in HB cell lines HUH6, HepG2, and Hep293TT and normal hepatocyte cell line HC‐04. (B) qPCR analysis of LIN28B, RAN, and AURKA mRNA levels in HUH6, HepG2, and Hep293TT cells transfected with control, siLIN28B, or siRAN after 48 hours. LIN28B, RAN, and AURKA mRNA levels are normalized to β‐actin levels. *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as mean ± SEM (triplicate assays). (C) Western blot analysis of LIN28B, RAN, and AURKA protein expression in HUH6, HepG2, and Hep293TT cells transfected with control, siLIN28B, or siRAN for 2 days, with β‐actin serving as a loading control. Pooled samples (n = 3 per group) were run in a single lane. Numbers indicate density relative to siCon. Abbreviations: qPCR, quantitative polymerase chain reaction; siCon, siControl.

Figure 3

Knockdown of LIN28B and RAN inhibits HB cell growth. (A) Growth curves of control and HUH6 (left), HepG2 (middle), and Hep293TT (right) cells transfected with siLIN28B and siRAN for 5 days. Data are shown as mean ± SEM (triplicate assays). *P < 0.05, **P < 0.01. (B) Colony formation assay of Hep293TT cells transfected with siControl, siLIN28B, or siRNA for 14 days. Quantification of colony numbers is presented on the right. Abbreviation: siCon, siControl.

Figure 4

Lin28B and AURKA are novel target genes of miR‐26a‐5p. (A) Target prediction for miR‐26a‐5p using Targetscan identified miR‐26a‐5p binding sequences within LIN28B 3′‐UTR and AURKA 3′‐UTR. (B) Luciferase reporter assays of LIN28B 3′‐UTR and AURKA 3′‐UTR in HUH6 and HepG2 cells transfected with 100 nmol miR‐26a‐5p. (C) Luciferase reporter assays of LIN28B 3′‐UTR mutant and AURKA 3′‐UTR mutant in HUH6 and HepG2 cells transfected with 100 nmol miR‐26a‐5p. (D) qPCR analysis of miR‐26a‐5p expression in normal hepatocyte cells HC‐04 and HB cell lines HUH6, HepG2, and Hep293TT. (E) Western blot analysis of LIN28B, RAN, and AURKA protein expression in the indicated cells transfected with mimic control and miR‐26a‐5p mimic for 48 hours, with β‐actin serving as a loading control. Numbers indicate density relative to control. (F) qPCR analysis of miR‐26a‐5p and miR‐21‐5p expression in normal liver (n = 16) and FFPE HB tissues (n = 14). All data are shown as mean ± SEM (triplicate assays). *P < 0.05 versus HC‐04. Abbreviations: CON, control; MUT, mutant; qPCR, quantitative polymerase chain reaction.

Figure 5

Re‐expression LIN28B and AURKA promotes HB cell growth. (A) Growth curves of control and HUH6 (left), HepG2 (middle), and Hep293TT (right) cells transfected with mimic control, 100 nmol miR‐26a‐5p mimic, and 100 nmol miR‐29a‐3p mimic. (B) Colony formation assay of Hep293TT cells transfected with mimic control, miR‐26a‐5p mimic, and miR‐29a‐3p mimic. (C) Colony formation assay of Hep293TT cells transfected with mimic control, miR‐26a‐5p mimic, miR‐26a‐5p inhibitor, and miR‐26a‐5p mimic with its inhibitor. (D) Colony formation assay of Hep293TT cells transfected with control, miR‐26a‐5p mimic, miR‐26a‐5p mimic plus LIN28B plasmid, and miR‐26a‐5p mimic plus AURKA plasmid. (E) Hep293TT cells were transfected with mimic control plus 2 μg empty vector control, miR‐26a‐5p mimic plus 2 μg empty vector control, miR‐26a‐5p mimic plus 2 μg LIN28B plasmid, and miR‐26a‐5p mimic plus 2 μg AURKA plasmid. The cell proliferation assay was performed from day 0 to day 5. All data are shown as mean ± SEM (triplicate assays). Abbreviation: CON, control.

Figure 6

Working model of miR‐26a‐5p as a novel HB repressor. The LIN28B–RAN–AURKA signaling pathway plays a crucial role in hepatoblastoma oncogenesis. MiR‐26a‐5p translationally inhibits LIN28B and AURKA expression by targeting their 3′‐UTR, thereby suppressing HB cell growth.