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. 2023 Apr 15;13(4):1295-1309.
eCollection 2023.

microRNA-203 functions as a natural Ras inhibitor in hepatocellular carcinoma

Jun Guo  1 , Lei Li  2 , Nan Deng  2 , Yong Xu  2 , Guohua Wang  3 , Hongbo Luo  4 , Chuanrui Xu  2 , Xiaolei Li  5
Affiliations

microRNA-203 functions as a natural Ras inhibitor in hepatocellular carcinoma

Jun Guo et al. Am J Cancer Res. .

Abstract

microRNA-203 (miR203) plays an important role in the formation and development of multiple types of cancers. However, its role in hepatic carcinogenesis has not been well studied. Mitogen-activated protein kinase signaling is known to be activated in hepatocellular carcinoma (HCC), but there is a lack of effective drugs _targeting this pathway for HCC treatment. In this study, we investigated the role of miR203 in HCC and the underlying mechanism. We found that miR203 was significantly downregulated in HCC cell lines and patient tissues compared with a hepatocyte cell line (L02) or normal liver tissues. Restoration of miR203 inhibited HCC cell growth and induced cell cycle arrest and apoptosis. In primary and xenograft HCC mouse models, miR203 also significantly blocked HCC growth. Bioinformatic analysis indicated that miR203 directly binds to the 3'UTR of NRas mRNA, resulting in decreased expression of NRas and inactivation of mitogen-activated protein kinase (MAPK) signaling. Activation of MAPK signaling by ectopic NRas expression rescued the cell proliferation blocked by miR203. Together, our findings illustrate the fundamental role of miR203 as a natural inhibitor of RAS/MAPK signaling in hepatic carcinogenesis in vitro and in vivo. In light of the critical and universal activation of the MAPK pathway in HCC, miR203 has the potential to serve as a nucleotide drug for the treatment of HCC with activated MAPK signaling.

Keywords: Ras; hepatocellular carcinoma; microRNA-203; mitogen activated protein kinase.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
The expression of miR203 is downregulated in HCC. A. Reduced expression of miR203 in the livers of AKT/Ras-, AKT/h-Met-, and c-Myc-induced HCC mouse models compared with normal mice determined by using qRT-PCR. n = 5 in each group. B. Relative miR203 levels in human HCC cell lines with different genetic backgrounds compared with L02 cells detected using qRT-PCR. C. Downregulation of miR203 in human HCC tissues (n = 57) versus normal liver tissues (n = 57). Data were extracted from the GEO database (accession no: GSE67139). D, E. Survival analysis of HCC patients with high miR203 expression (n = 199) and patients with low miR203 expression (n = 500). Data were from the GEO database (GSE119221). The data were analyzed by Student’s t test or one-way ANOVA to evaluate the statistical significance, and the results are presented as the mean ± SD. *P < 0.05; **P < 0.01.
Figure 2
Figure 2
miR203 inhibits HCC cell proliferation in vitro. A. Upregulation of miR203 by lentivirus in human HCC cell lines with divergent backgrounds. B. Viability of HCC cells treated with miR203 lentivirus (LV3-miR203) or scrambled lentivirus (LV3-SC) using a CCK-8 kit. C. Cell proliferation curves of LV3-miR203-infected HCC cell lines compared to LV3-SC-infected cells. D. Soft agar colony formation of HepG2, Huh7, and Hep3B cells transfected with LV3-miR203 or LV3-SC. E. Cell cycle distribution in HCC cells treated with LV3-miR203 lentivirus or control lentivirus by flow cytometry (FCM). F. Expression of the cell cycle-related proteins cyclin D1 and cyclin B1 using Western blotting. Data are presented as the mean ± SD, n = 3 per group, **P < 0.01.
Figure 3
Figure 3
miR203 suppresses the growth of HCC xenograft tumors. A. Gross images of xenografts induced by Hep3B cells that were stably transfected with LV3-miR203 (n = 7) or LV3-SC (n = 7). B. Mouse body weight curve in two different groups. C, D. Weight and growth curve of HCC xenograft tumors in each group. E. miR203 level in each group by qRT-PCR. Data are presented as the mean ± SD, n = 7 per group, *P < 0.05, **P < 0.01.
Figure 4
Figure 4
miR203 overexpression inhibits the development of HCC induced by AKT/Ras and AKT/h-Met. A. Representative liver images of livers from AKT/Ras mice (n = 6) and AKT/Ras/miR203 mice (n = 6). B. Liver tumor numbers in mice injected with AKT/Ras (n = 6) or AKT/Ras/miR203 (n = 6). C. Kaplan-Meier survival curves of the AKT/Ras (n = 15) and AKT/Ras/miR203 (n = 15) mouse cohorts. D. miR203 level in each group by qRT-PCR. E. Body weight and liver/body weight ratio of AKT/Ras or AKT/Ras/miR203 mice (n = 6). F. Microscopic appearance and immunohistochemistry (IHC) staining of liver tissues from each group. G. Macroscopic appearance of livers from AKT/h-Met mice (n = 6) and AKT/h-Met/miR203 mice (n = 6). H. Survival curves of the AKT/h-Met and AKT/h-Met/miR203 mice. I. miR203 level in each group by qRT-PCR. J. Liver/body weight ratio of AKT/h-Met and AKT/h-Met/miR203 mice. Data represent mean ± SEM. *P < 0.05, **P < 0.01. Scale bar = 100 μm, magnification = 200 ×.
Figure 5
Figure 5
Microarray analysis indicates NRas as a downstream _target of miR203 in HCC. A. Heatmap of the differentially expressed genes with a fold-change > 2 and P < 0.05 between AKT/Ras and AKT/Ras/miR203 mouse liver tissues (n = 3, red = upregulation, green = downregulation). B. KEGG signaling pathway enrichment in AKT/Ras/miR203 mice versus AKT/Ras mice (n = 3) using DAVID analysis. C. Volcano plot of all differentially expressed genes in AKT/Ras/miR203 mice versus AKT/Ras mice (n = 3, orange = upregulation, green = downregulation).
Figure 6
Figure 6
NRas is the direct _target of miR203. A. Schematic representation of the conserved miR203 binding motifs within the 3’-UTRs of miR203. Complementary sequences to the seed regions of miR203 within the 3’-UTRs are highlighted in green. B. Luciferase reporter analysis containing either the WT or mutated 3’-UTRs of human NRas after transfection with LV3-miR203 or LV3-SC in Huh7 cells. C, D. Western blotting and qRT-PCR analysis of NRas and Ras/MAPK signaling pathway-related genes. E, F. Western blotting analysis of Ras/MAPK-related proteins in HCC cells or AKT/h-Met-induced HCC mice after miR203 overexpression.
Figure 7
Figure 7
Ectopic expression of NRas can restore the inhibitory effect of miR203 on the proliferation of Huh7 cells. A. Cell viability analysis of Huh7 cells treated with DMSO, miR203 lentivirus, NRas, and miR203 lentivirus+ NRas at different time points using a CCK-8 kit. B. Cell proliferation curves of Huh7 cells with different treatments. C. Soft agar colony formation of Huh7 cells in each group. Data are presented as the mean ± SD, n = 3 per group, *P < 0.05, **P < 0.01.
Figure 8
Figure 8
Schematic representation of the role of the Ras/MAPK signaling pathway in miR203-mediated repression of HCC development.
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