The role of AURKA/miR-199b-3p in hepatocellular carcinoma cells

Abstract

Background: Previous studies proved that AURKA functions as an oncogene in several cancers. This article aimed to probe the miRNA-induced regulatory mechanism of AURKA in hepatocellular carcinoma (HCC).

Methods: Differentially expressed genes in TCGA-LIHC dataset were screened by bioinformatics methods. Levels of miR-199b-3p and AURKA mRNA were examined by qRT-PCR. Western blot was utilized to evaluate protein levels of AURKA, p-AKT, and AKT. Dual-luciferase assay was introduced to explore their interaction. MTT, colony formation, scratch healing, transwell, and flow cytometry assays were introduced into cell proliferation, migration, invasion, and apoptosis assessment. The impact of miR-199b-3p/AURKA axis on HCC tumor growth was determined in a tumor xenograft model.

Results: We found that AURKA was highly expressed in HCC and was coupled to poor prognosis of HCC. As manifested by cellular assays, compared to the normal cells HL-7702, AURKA presented notably high expression in HCC cell lines. Overexpressed AURKA evidently impelled the proliferation, colony formation, migration, and invasion of HCC cells while suppressing apoptosis. The regulatory gene upstream of AURKA was predicted to be miR-199b-3p by bioinformatics method, and there was a markedly negative correlation between the two. Overexpressed miR-199b-3p constrained HCC cell proliferation, migration, and invasion while fostering apoptosis, which could be counteracted by upregulating AURKA. MiR-199b-3p repressed the tumor growth in vivo by targeting AURKA and affected PI3K/AKT signaling pathway.

Conclusion: To summarize, this study implied the regulatory mechanism of miR-199b-3p/AURKA axis in HCC, and supplied optional therapeutic targets for HCC patients.

Keywords: AURKA; hepatocellular carcinoma; invasion; miR-199b-3p; migration; proliferation.

FIGURE 1

High expression of AURKA in HCC cells. (A) Heat map of the top 20 differential genes screened by the combined analysis of the four groups of HCC mRNA chips in the GEO database. (B) The protein interaction network diagram of the differential gene. (C). Statistics of the core degree value (degree value) in the interaction network graph. Abscissa: degree value; Ordinate: gene name. (D) The expression of AURKA in TCGA‐LIHC dataset. Green: normal; Red: tumor. (E) AURKA mRNA expression in HCC tumor and adjacent normal tissue. Tumor: n = 30; Normal: n = 30. (F, G) AURKA mRNA and protein levels in human hepatic normal and HCC cell lines. *p < 0.05.

FIGURE 2

Patients with high AURKA expression have lower survival rate. (A) Survival curve of AURKA level on patient's prognosis in TCGA‐LIHC and GEO dataset. Red: high expression; Blue: low expression. (B) Multivariate Cox repression analysis of AURKA.

FIGURE 3

Overexpressed AURKA fosters the proliferation of HCC cells. (A) The transfection efficiency of AURKA in HCC cell lines HepG2 and SK‐HEP1. (B) The proliferation of oe‐NC and oe‐AURKA groups of HCC cell lines HepG2 and SK‐HEP1. (C) The cloning ability of HepG2 cells and SK‐HEP1 cells. *p < 0.05.

FIGURE 4

Overexpressed AURKA fosters the migration and invasion of HCC cells and restrains cell apoptosis. (A) The migration of HepG2 cells and SK‐HEP1 cells (40×). (B) The invasion of HepG2 cells and SK‐HEP1 cells (100×). (C) The apoptosis rate of HepG2 cells and SK‐HEP1 cells. *p < 0.05.

FIGURE 5

Overexpressed AURKA facilitates the phosphorylation of AKT. Western blot was applied to detect the expression of related proteins in the PI3K/AKT signaling pathway. *p < 0.05.

FIGURE 6

MiR‐199b‐3p is less expressed in HCC cells and negatively regulated AURKA. (A) Volcano map of the differential miRNAs in the HCC dataset. Red: upregulated miRNAs; Green: downregulated miRNAs. (B) Venn diagram of predicted upstream regulatory miRNAs of AURKA and downregulated differential miRNAs. (C) Pearson's correlation analysis between AURKA and miR‐199b‐3p. (D) MiR‐199b‐3p expression. Green: normal samples; Red: tumor samples. (E) MiRNA‐199p‐3b level in HCC tumor and adjacent normal tissue. Tumor: n = 30; Normal: n = 30. (F) MiR‐199b‐3p expression in human hepatic normal cell line HL‐7702 and HCC cell lines HepG2, Hep3B, SK‐HEP1, and Huh‐7. (G) Schematic diagram of bioinformatics analysis predicting the binding of AURKA‐WT and AURKA‐MUT to miR‐199b‐3p sequence. (H) The luciferase activity of HepG2 cells and SK‐HEP1 cells in NC‐mimic and miR‐mimic groups. (I) AURKA mRNA level in HepG2 cells and SK‐HEP1 cells. *p < 0.05.

FIGURE 7

Overexpressed miR‐199b‐3p represses the protein level of AURKA. Western blot determined the expression of AURKA protein. *p < 0.05.

FIGURE 8

MiR‐199b‐3p suppresses proliferation, migration, and invasion of HCC cells and stimulates apoptosis through suppressing AURKA. (A) AURKA expression in HepG2 cells in the NC‐mimic + oe‐NC, miR‐mimic + oe‐NC, and miR‐mimic + oe‐AURKA groups. (B) Proliferation of HepG2 cells. (C) Cloning ability of HepG2 cells. (D) The migration of HepG2 cells (40×). (E) Invasion of HepG2 cells (100×). (F) Apoptosis level of HepG2 cells. (G) p‐AKT and AKT protein levels in HepG2 cells. *p < 0.05.

FIGURE 9

MiR‐199b‐3p/AURKA axis inhibits malignant progression of HCC. (A) Image of mouse tumor. (B) The tumor growth was measured. Tumor volume was monitored every 3 days. (C) Changes of tumor weight within 3 weeks in nude mice. (D) AURKA level were performed by qRT‐PCR. (E) Levels of AURKA, p‐AKT, AKT at protein level were analyzed by western blot. (F) Representative images of immunochemistry staining of mice tumor sections. *p < 0.05.