Distinct anti-oncogenic effect of various microRNAs in different mouse models of liver cancer

Abstract

Deregulation of microRNAs (miRNAs) is a typical feature of human hepatocellular carcinoma (HCC). However, the in vivo relevance of miRNAs along hepatocarcinogenesis remains largely unknown. Here, we show that liver tumors induced in mice by c-Myc overexpression or AKT/Ras co-expression exhibit distinct miRNA expression profiles. Among the downregulated miRNAs, eight (miR-101, miR-107, miR-122, miR-29, miR-365, miR-375, miR-378, and miR-802) were selected and their tumor suppressor activity was determined by overexpressing each of them together with c-Myc or AKT/Ras oncogenes in mouse livers via hydrodynamic transfection. The tumor suppressor activity of these microRNAs was extremely heterogeneous in c-Myc and AKT/Ras mice: while miR-378 had no tumor suppressor activity, miR-107, mir-122, miR-29, miR-365 and miR-802 exhibited weak to moderate tumor suppressor potential. Noticeably, miR-375 showed limited antineoplastic activity against c-Myc driven tumorigenesis, whereas it strongly inhibited AKT/Ras induced hepatocarcinogenesis. Furthermore, miR-101 significantly suppressed both c-Myc and AKT/Ras liver tumor development. Altogether, the present data demonstrate that different oncogenes induce distinct miRNA patterns, whose modulation differently affects hepatocarcinogenesis depending on the driving oncogenes. Finally, our findings support a strong tumor suppressor activity of miR-101 in liver cancer models regardless of the driver oncogenes involved, thus representing a promising therapeutic target in human HCC.

Keywords: AKT; HCC; Ras; c-Myc; mouse liver cancer.

Figure 1. c-Myc and AKT/Ras induced liver tumors exhibit distinct miRNA profiles

(A) Unsupervised hierarchical clustering analysis using 599 mouse miRNAs in normal livers from wild-type (WT) mice and liver tumors from c-Myc and AKT/Ras injected mice. (B) Scattering Plot of miRNAs in liver tumors from c-Myc and AKT/Ras injected mice. Red spots refer to miRNAs with significant difference (unpaired t-test, p < 0.05) between c-Myc and AKT/Ras injected mice. (C) Venn Diagram analysis of c-Myc signature and AKT/Ras signature. The signature was obtained from the miRNA profiling comparison of c-Myc-tumor (or AKT/Ras tumor) and non-tumor liver from wild-type mice. Unpaired t-test was used and miRNAs with p-value < 0.001 and fold change ≥ 2 or ≤ 0.5 were included in the signature.

Figure 2. AKT/Ras and c-Myc mouse liver tumor signatures are related to patients’ HCC prognosis

(A) Kaplan-Meier analysis of overall survival and disease-free survival in HCC cases (n = 227) based on the classification of group 1 and group 2 by the AKT/Ras signature. (B) Kaplan-Meier analysis of overall survival and disease-free survival in HCC cases (n = 227) based on the classification of group 1 and group 2 by the c-Myc signature. Cox-Mantel log-rank test was performed.

Figure 3. Overall diagram of study design to investigate tumor suppressor activity of miRNA in mice

Each of the eight miRNAs was co-expressed with c-Myc (A) or AKT/Ras (B) oncogenes in the mouse liver via hydrodynamic transfection. pT3-EF1α empty vector was also co-injected with c-Myc (c-Myc/pT3) or AKT/Ras (AKT/Ras/pT3) as a control. w.p.i: weeks post injection.

Figure 4. Overexpression of miR-375 strongly inhibits AKT/Ras but not c-Myc induced liver tumor formation in mice

(A) Macroscopic (upper panel) and microscopic (lower panel) appearance of livers from c-Myc/pT3 mice and c-Myc/miR-375 mice stained with H&E (100X), insets (400X). (B) Kaplan Meier survival curve of c-Myc/pT3 and c-Myc/miR-375 mouse cohort. (C) Macroscopic (upper panel) and microscopic (lower panel) appearance of livers from AKT/Ras/PT3 mice and AKT/Ras/miR-375 mice stained with H&E (100X). (D) Kaplan Meier survival curve of AKT/Ras/pT3 and AKT/Ras/miR-375 mouse cohort.

Figure 5. Overexpression of miR-101 efficiently inhibits c-Myc and AKT/Ras induced liver tumor development

(A) Macroscopic (upper panel) and microscopic (lower panel) appearance of livers from c-Myc/pT3 mice and c-Myc/miR-101 mice stained with H&E (100X), insets (400 X). (B) Kaplan Meier survival curve of c-Myc/pT3 and c-Myc/miR-101 mouse cohort. (C) Macroscopic (upper panel) and microscopic (lower panel) appearance of livers from AKT/Ras/PT3 mice and AKT/Ras/miR-101 mice stained with H&E (100X). (D) Kaplan Meier survival curve of AKT/Ras/pT3 and AKT/Ras/miR-101 mouse cohort.