Functional role and epithelial to mesenchymal transition of the miR-590-3p/MDM2 axis in hepatocellular carcinoma

Abstract

Background: There is considerable evidence that microRNAs (miRNAs) regulate several key tumor-associated genes/pathways and may themselves have a dual regulatory function either as tumor suppressors or oncogenic miRNA, depending on the tumor type. MicroRNA-590-3p (miR-590-3p) is a small non-coding RNA involved in the initiation and progression of numerous tumors. However, its expression pattern and biological role in hepatocellular carcinoma (HCC) are controversial.

Results: In the current work, computational and RT-qPCR analysis revealed that HCC tissues and cell lines exhibited miR-590-3p downregulation. Forced expression of miR-590-3p attenuated HepG2 cells proliferation, migration, and repressed EMT-related gene expression. Bioinformatic, RT-qPCR, and luciferase assays revealed that MDM2 is a direct functional target of miR-590-3p. Moreover, the knockdown of MDM2 mimicked the inhibitory effect of miR-590-3p in HepG2 cells.

Conclusion: We have identified not only novel targets for miR-590-3p in HCC, but also novel target genes for miR590-3p/MDM2 pathway in HCC like SNAIL, SLUG, ZEB1, ZEB2, and N-cadherin. Furthermore, these findings demonstrate a crucial role for MDM2 in the regulatory mechanism of EMT in HCC.

Keywords: Colony formation; MDM2 knockdown; Transwell migration; miR-590-3p; miR-590-3p mimics.

Fig. 1

The downregulation pattern of hsa-miR-590-3p in hepatocellular carcinoma. A miR-590-3p differential expression between HCC (n = 370) relative to the normal liver tissues (n = 50). B MiR-590-3p is significantly lower in the SNU449 cell line compared to HepG2 cells, as shown by RT qPCR results. Data represents the mean ± SEM (N = 3). P-value was depicted using Student’s t-test. (** P < 0.01, **** P < 0.0001)

Fig. 2

miR-590-3p suppresses cellular clonogenicity, migration and EMT in HepG2 cells. A The transfection efficiency of miR-590-3p mimics compared to negative control B Overexpressing miR-590-3p significantly decreased the number of HepG2 colonies compared to the negative control. C Overexpressing miR-590–3 showed a low but significant reduction in the number of migrated cells as compared to the negative control in the HepG2 cell line. D overexpressing miR-590-3p caused a significant decrease in the mRNA levels of SNAIL, SLUG, ZEB1, and ZEB2. E N-cadherin levels were significantly lower in miR-590-3p mimics transfected cells, while Vimentin levels were not significantly changed. Images were taken at 20X magnification using a fluorescent microscope and counted using Fiji Image J software. Data represents the mean ± SEM. (N = 2 for qPCR, N = 3 for Transwell and colony assay). P-value was depicted using Student’s t test. (* P < 0.05, ** P < 0.01, *** P < 0.001)

Fig. 3

MDM2 is a novel direct target gene of miR-590-3p in HCC. A Differential expression of MDM2 between normal and HCC tissues as obtained from TCGA data using Pan-cancer analysis platform. B The mRNA levels of MDM2 in HepG2 cells transfected with miR-590-3p in mimic compared to the negative control. C The sequence alignment between MDM2 and miR-590-3p. D Relative luciferase activity of miR-590-3p mimic or negative control on the WT or MUT versions of MDM2. Data represents the mean ± SEM. (N = 3 for qPCR, N = 2 for luciferase assay). P-value was depicted using Student’s t-test. (* P < 0.05, ** P < 0.01, *** P < 0.001)

Fig. 4

Inhibition of MDM2 mimics the tumor suppressor effect of miR-590-3p. A RT-qPCR showing the expression of MDM2 after transfecting HepG2 cells with si-MDM2 or si-NC. B Western blot showing the successful depletion of MDM2 protein expression. Full-length blot is presented in Supplementary Figure S3(A&B). C Colony formation assay was performed to assess the effect of MDM2 knockdown on the ability of HepG2 cells to form colonies. D Transwell assay was employed to verify the effect of MDM2 silencing on the migration of HepG2 cells. E RT-qPCR showing the expression of SNAIL, SLUG, ZEB1, and ZEB2 following MDM2 knockdown, with GAPDH as an internal control. F RT-qPCR showing the transcript levels of N-cadherin and Vimentin in si-NTC and si-MDM2 transfected HepG2 cells, with GAPDH as an internal control. Data values are expressed as the mean ± SEM (N = 3) for all assays except for western blotting (N = 2). Statistically significant at *P < 0.05, ** P < 0.01, ***P < 0.001 (Student t-test, two-tailed). siMDM2-MDM2 siRNA, siNC- negative siRNA

Fig. 5

Hypothetical model for miR-590-3p/MDM2 axis role in HCC. MDM2 acts as an oncogene in HCC. miR-590-3p exerts tumor suppressive roles in HCC by directly targeting MDM2. The figure was created with Biorender.com