MicroRNA-337 regulates the PI3K/AKT and Wnt/β-catenin signaling pathways to inhibit hepatocellular carcinoma progression by targeting high-mobility group AT-hook 2

Abstract

MicroRNAs (miRNAs) serve as major regulators during the tumorigenesis and tumor development of hepatocellular carcinoma (HCC). In addition, miRNAs may serve as new promising biomarkers for the diagnosis and prognosis and as effective therapeutic targets for patients with this malignancy. Therefore, understanding the association between miRNAs and HCC may be beneficial to discover novel therapeutic approaches towards diagnosis and treatments. Results of this study showed that miRNA-337 (miR-337) was markedly downregulated in HCC tissues and cell lines. Decreased miR-337 expression was significantly associated with the TNM stage and lymph node metastasis of HCC. Ectopic expression of miR-337 prohibited the proliferation, colony formation, migration, and invasion of HCC cells. It also promoted the apoptosis in vitro and reduced the tumor growth in vivo of these cells. High-mobility group AT-hook 2 (HMGA2) was identified as a direct target gene of miR-337 in HCC through a series of experiments. HMGA2 was significantly overexpressed in HCC tissues and negatively correlated with miR-337 expression. Moreover, the functions of HMGA2 inhibition were similar to those induced by miR-337 in HCC. Restored HMGA2 expression rescued the tumor-suppressive roles of miR-337 overexpression in HCC. Furthermore, miR-337 overexpression inhibited the activation of PI3K/AKT and Wnt/β-catenin signaling pathways in HCC both in vitro and in vivo. This study demonstrated that miR-337 may play tumor-suppressing roles in HCC, at least partly, via directly targeting HMGA2 and inhibiting the PI3K/AKT and Wnt/β-catenin signaling pathways. Therefore, miR-337 may be a novel and effective target for the therapeutic treatment of patients with HCC.

Keywords: Hepatocellular carcinoma; high-mobility group AT-hook 2; microRNA-337; tumor suppressor.

Figure 1

MiR-337 is downregulated in HCC tissues and cell lines. A. The expression level of miR-337 in 47 paired HCC tissues and adjacent non-cancerous tissues was detected using RT-qPCR. *P<0.05 compared with non-cancerous tissues. B. MiR-337 expression was examined in human HCC cell lines HepG2, Hep3B, and Huh-7, and in the normal human liver cell line LO2 (control). *P<0.05 compared with LO2.

Figure 2

Ectopic expression of miR-337 inhibits cell proliferation and induces apoptosis in HCC. A. HepG2 and Hep3B cells transfected with miR-337 mimics or miR-NC were subjected to RT-qPCR for the detection of miR-337 expression. *P<0.05 compared with miR-NC. B. Proliferative activity of HepG2 and Hep3B cells was determined by CCK-8 assay following transfection of miR-337 mimics or miR-NC. *P<0.05 compared with miR-NC. C. The effect of miR-337 overexpression on the colony formation of HepG2 and Hep3B cells was evaluated using colony formation assay. *P<0.05 compared with miR-NC. D. Cell apoptosis rate was detected in HepG2 and Hep3B cells transfected with miR-337 mimics or miR-NC with an Annexin V-FITC apoptosis detection kit. *P<0.05 compared with miR-NC.

Figure 3

MiR-337 overexpression decreases HCC cell migration and invasion. A, B. Migration and invasion of HepG2 and Hep3B cells were investigated using Transwell migration and invasion assays following transfection of miR-337 mimics or miR-NC. *P<0.05 compared with miR-NC.

Figure 4

HMGA2 is a direct target of miR-337 in HCC. A. MiR-337 and its putative binding sequences in the 3’-UTR of HMGA2. Mutation was generated on the 3’-UTR of HMGA2 in the complementary sites for the seed region of miR-337. B. HepG2 and Hep3B cells were cotransfected with miR-337 mimics or miR-NC and wild-type or mutant HMGA2 3’-UTR. The relative firefly luciferase activity was determined at 48 h posttransfection. *P<0.05 compared with miR-NC. C, D. HepG2 and Hep3B cells that were transfected with miR-337 mimics or miR-NC were subjected to RT-qPCR and Western blot analysis for detection of HMGA2 mRNA and protein expression, respectively. *P<0.05 compared with miR-NC.

Figure 5

HMGA2 is upregulated in HCC tissues and negatively correlated with miR-337 expression. A, B. RT-qPCR and Western blot analysis were performed to measure HMGA2 mRNA and protein expression in HCC tissues and adjacent non-cancerous tissues. *P<0.05 compared with non-cancerous tissues. C. Western blot analysis was utilized to determine HMGA2 protein expression in three HCC cell lines and the normal human liver cell line LO2 (control). *P<0.05 compared with LO2. D. The association between miR-337 and the mRNA expression of HMGA2 was determined using Spearman’s correlation analysis. r = -0.5731, P<0.0001.

Figure 6

HMGA2 knockdown decreases the proliferation, migration, and invasion while increases the apoptosis of HCC cells. A. HMGA2 siRNA or NC siRNA was introduced into HepG2 and Hep3B cells. At 72 h after transfection, the transfection efficiency was evaluated using Western blot analysis. *P<0.05 compared with NC siRNA. B-F. CCK-8 assay, colony formation assay, Transwell migration and invasion assays, and cell apoptosis assay were conducted to investigate the effect of HMGA2 knockdown on HepG2 and Hep3B cell proliferation, colony formation, migration, invasion, and apoptosis, respectively. *P<0.05 compared with NC siRNA.

Figure 7

Re-expression of HMGA2 reverses the tumor-suppressing effects of miR-337 on HCC cells. HepG2 and Hep3B cells were transfected with miR-337 mimics along with pcDNA3.1 or pcDNA3.1-HMGA2 lacking the respective 3’-UTR. A. Expression level of HMGA2 protein was analyzed by Western blot analysis in differently treated cells. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2. B. CCK-8 assay was carried out to analyze cell proliferation in indicated cells. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2. C. Representative images and quantification of colony formation assay in indicated cells. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2. D, E. Transwell migration and invasion assays were employed to assess cell migration and invasion in indicated cells, respectively. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2. F. Cell apoptosis in differently treated cells was quantified by flow cytometry analysis. *P<0.05 compared with miR-NC. #P<0.05 compared with miR-337 mimics + pcDNA3.1-HMGA2.

Figure 8

Upregulation of miR-337 suppresses the PI3K/AKT and Wnt/β-catenin signaling pathways in HCC. MiR-337 mimics was transfected into HepG2 and Hep3B cells together with pcDNA3.1 or pcDNA3.1-HMGA2. At 72 h after transfection, Western blot analysis was conducted to measure AKT, p-AKT, β-catenin, and p-β-catenin expression.

Figure 9

MiR-337 reduces HCC tumor growth in vivo. A. MiR-337 expression was determined in tumor xenografts using RT-qPCR. *P<0.05 compared with miR-NC. B. Mice were sacrificed, and representative images of the miR-337 mimics and miR-NC xenograft tumors were obtained. C. Tumor volume was calculated every 2 days after inoculation for 2 weeks. *P<0.05 compared with miR-NC. D. Tumor weight in the different groups. *P<0.05 compared with miR-NC. E. Western blot analysis was performed to detect HMGA2, AKT, p-AKT, β-catenin, and p-β-catenin expression in xenograft tumor tissues of the miR-337 and miR-NC groups.