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Comprehensive Analysis of microRNA-regulated Protein Interaction Network Reveals the Tumor Suppressive Role of microRNA-149 in Human Hepatocellular Carcinoma via Targeting AKT-mTOR Pathway

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Comprehensive Analysis of microRNA-regulated Protein Interaction Network Reveals the Tumor Suppressive Role of microRNA-149 in Human Hepatocellular Carcinoma via Targeting AKT-mTOR Pathway

Yanqiong Zhang et al. Mol Cancer.

Abstract

Background: Our previous study identified AKT1, AKT2 and AKT3 as unfavorable prognostic factors for patients with hepatocellular carcinoma (HCC). However, limited data are available on their exact mechanisms in HCC. Since microRNAs (miRNAs) are implicated in various human cancers including HCC, we aimed to screen miRNAs targeting AKTs and investigate their underlying mechanisms in HCC by integrating bioinformatics prediction, network analysis, functional assay and clinical validation.

Methods: Five online programs of miRNA target prediction and RNAhybrid which calculate the minimum free energy (MFE) of the duplex miRNA:mRNA were used to screen optimized miRNA-AKT interactions. Then, miRNA-regulated protein interaction network was constructed and 5 topological features ('Degree', 'Node-betweenness', 'Edge-betweenness', 'Closeness' and 'Modularity') were analyzed to link candidate miRNA-AKT interactions to oncogenesis and cancer hallmarks. Further systematic experiments were performed to validate the prediction results.

Results: Six optimized miRNA-AKT interactions (miR-149-AKT1, miR-302d-AKT1, miR-184-AKT2, miR-708-AKT2, miR-122-AKT3 and miR-124-AKT3) were obtained by combining the miRNA target prediction and MFE calculation. Then, 103 validated targets for the 6 candidate miRNAs were collected from miRTarBase. According to the enrichment analysis on GO items and KEGG pathways, these validated targets were significantly enriched in many known oncogenic pathways for HCC. In addition, miRNA-regulated protein interaction network were divided into 5 functional modules. Importantly, AKT1 and its interaction with mTOR respectively had the highest node-betweenness and edge-betweenness, implying their bottleneck roles in the network. Further experiments confirmed that miRNA-149 directly targeted AKT1 in HCC by a miRNA luciferase reporter approach. Then, re-expression of miR-149 significantly inhibited HCC cell proliferation and tumorigenicity by regulating AKT1/mTOR pathway. Notably, miR-149 down-regulation in clinical HCC tissues was correlated with tumor aggressiveness and poor prognosis of patients.

Conclusion: This comprehensive analysis identified a list of miRNAs targeting AKTs and revealed their critical roles in HCC malignant progression. Especially, miR-149 may function as a tumor suppressive miRNA and play an important role in inhibiting the HCC tumorigenesis by modulating the AKT/mTOR pathway. Our clinical evidence also highlight the prognostic potential of miR-149 in HCC. The newly identified miR-149/AKT/mTOR axis might be a promising therapeutic target in the prevention and treatment of HCC.

Figures

Figure 1
Figure 1
A schematic diagram of this comprehensive analysis.
Figure 2
Figure 2
Candidate miRNAs regulated protein interaction networks. (A) Interaction network of hub nodes in the candidate miRNAs regulated protein interaction networks. Nodes with yellow rings refer to the validated targets of hsa-miR-149, hsa-miR-302d, hsa-miR-184, hsa-miR-708, hsa-miR-122 and hsa-miR-124. (B) Five functional modules of interaction network of hub nodes in the candidate miRNAs regulated protein interaction networks.
Figure 3
Figure 3
MiRNA-149 directly targets AKT1 in HCC cells. (A) QRT-PCR analysis showing relative expression of miR-149 in HepG2 cells transfected with miR-149 mimics, miR-149 mimic control (negative control, NC), and blank control culture medium (mock). (B, C and F) Relative expression of AKT1, p-AKT1, mTOR and p-mTOR proteins in HepG2 cells transfected with miR-149 mimics, miR-149 mimic control (negative control, NC), and blank control culture medium (mock) detected by Western blot analysis. GAPDH was used as an internal loading control. (D) RNA sequence alignment showing the 3′-UTR of AKT1 mRNA contains a complementary site for the seed region of miR-149. AKT1mut is amutant with substitutions in the complementary region as a negative control. (E) Luciferase report assay was performed to verify whether AKT1 was a direct target of miR-149. The luciferase activity was detected after transfection of FLuci vector (3′-UTR-AKT1wt FLuci vector or 3′-UTR-AKT1mut FLuci vector) into the miR-149 mimic or miR-149 mimic control (negative control, NC) transfected HepG2 cells.
Figure 4
Figure 4
Reverse correlation between miR-149 and AKT1 mRNA expression in human HCC tissues and cells. (A and B) Relative expression of miR-149 and AKT1 mRNA in 130 self-pairs of HCC and adjacent nonneoplastic liver tissues; (C) Spearman Correlation analysis clearly showed a negative correlation between miR-149 and AKT1 mRNA expression in HCC tissues (rs = -0.639, P < 0.001); (D) Spearman Correlation analysis clearly showed a negative correlation between miR-149 and AKT1 protein expression in HCC tissues (rs = -0.716, P < 0.001); (E and F) Relative expression of miR-149 and AKT1 mRNA HCC (HepG2) and normal liver (HL-7702) cells.
Figure 5
Figure 5
MiR-149 inhibits cell proliferation, invasion and migration of HCC cells in vitro. (A) MTT assay showed that miR-149 overexpression could inhibit cell proliferation of HepG2 cells. (B and C) Transwell analysis showed miR-149 overexpression could inhibit invasion of HepG2 cells. (D and E) Scratch assays showed miR-149 overexpression could inhibit migration of HepG2 cells.
Figure 6
Figure 6
MiR-149 inhibits cell proliferation, invasion and migration of HCC cells in vitro by targeting AKT1/mTOR pathway. (A and B) Western blot analysis of AKT1, p-AKT1, mTOR and mTOR protein expression levels in HepG2 cells transfected with RNAi-AKT1 or RNAi-control vectors. (C-E) Overexpression of miR-149 fails to inhibit proliferation, invasion, and migration in AKT1 knockdown HepG2 cells.
Figure 7
Figure 7
Disease-free survival (A) and overall survival (B) curves for two groups defined by low and high expression of miR-149 in patients with HCC. The patients with low miR-149 expression had a significantly shorter 5-year overall and disease-free survival rate than those with high expression (all P < 0.001).

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