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. 2017 Jul 13;8(7):e2916.
doi: 10.1038/cddis.2017.309.

MicroRNA-148a deficiency promotes hepatic lipid metabolism and hepatocarcinogenesis in mice

Li Cheng  1   2 , Yahui Zhu  1   2 , Han Han  1   2 , Qiang Zhang  1   2 , Kaisa Cui  1   2 , Hongxing Shen  1   2 , Jinxiang Zhang  3 , Jun Yan  4   5 , Edward Prochownik  6 , Youjun Li  1   2
Affiliations

MicroRNA-148a deficiency promotes hepatic lipid metabolism and hepatocarcinogenesis in mice

Li Cheng et al. Cell Death Dis. .

Abstract

miRNAs are involved in many physiologic and disease processes by virtue of degrading specific mRNAs or inhibiting their translation. miR-148a has been implicated in the control of tumor growth and cholesterol and triglyceride homeostasis using in vitro or in vivo gene expression- and silencing-based approaches. Here miR-148a knockout (KO) mice were used to investigate the intrinsic role of miR-148a in liver physiology and hepatocarcinogenesis in mice. miR-148a downregulation was found to be correlated with poor clinical outcomes in hepatocellular carcinoma (HCC) patients. Under regular chow diet (RCD) or high fat diet (HFD), miR-148a deletion significantly accelerated DEN-induced hepatocarcinogenesis in mice. Mechanistically, miR-148a deletion promotes lipid metabolic disorders in mice. Moreover, restoration of miR-148a reversed these defects. Finally, miR-148a was found to directly inhibit several key regulators of hepatocarcinogenesis and lipid metabolism. These findings reveal crucial roles for miR-148a in the hepatic lipid metabolism and hepatocarcinogenesis. They further identify miR-148a as a potential therapeutic target for certain liver diseases, including cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
miR-148a is downregulated in HCC, and low expression predicts poor HCC patient outcomes. (a) miR-148a expression across mouse tissues of 2-week and 8-week old mice. Data present mean±S.D. (b) Expression of Pri/Pre-miR-148a and miR-148a-3p in 78 HCC samples and their adjacent noncancerous tissues. (a-b) Pri/Pre-miR-148a and miR-148a-3p RNA levels normalized for U6 were measured by stem-loop RT-qPCR in RNA purified from the above tissues. (c and d) Association of miR-148a-3p expression with clinical stages (c) and tumor differentiation (d) in sporadic HCC samples. (e) miR-148a-3p/5p expression in normal liver and HCC tissues from TCGA data set. (f) Association of miR-148a-3p/5p expression with clinical stages in HCC patients from TCGA data set. (b–f) Significance was performed using Wilcoxon signed rank test. The horizontal lines in the box plots represent the median, the boxes represent the interquartile range, and the whiskers represent the minimal and maximal values. (g) Survival of human HCC patients from TCGA data set with high versus low miR-148a-3p/5p expression. P-values and hazard ratios (HR) are indicated
Figure 2
Figure 2
MiR-148a deletion enhances DEN-induced hepatocarcinogenesis in mice. (a) Schematic overview of lentivirus containing Pri-miR-148a or control vector administration during DEN-induced hepatocarcinogenesis in mice fed with RCD or HFD. (b) RT-qPCR detection miR-148a-3p/5p RNA levels in tumors derived from (a). (c) Representative images of tumor nodules in the livers of mice (n=10). (d) Quantitation of tumor nodules in the liver of mice. (e) Tumor volumes in the liver of mice. Data present mean±S.D. in (b), (d) and (e). **P<0.01; ***P<0.001
Figure 3
Figure 3
Loss of miR-148a causes hepatic lipid accumulation. (a and b) Left: Representative images of ORO-stained liver histological sections from the WT and miR-148a KO mice fed a RCD or HFD with or without DEN treatment. Right: Quantifications of the lipid droplets (LDs) volumes from the areas of ORO-stained liver sections. (c-d) RT-qPCR analysis of the RNA levels of hepatic genes involved in lipogenesis (c) and fatty acid (FA) uptake (d) in the mice from (a and b). Data present mean±S.D. *P<0.05; **P<0.01; ***P<0.001
Figure 4
Figure 4
miR-148a deletion enhances cholesterol biosynthesis. (a and b) Serum (a) and hepatic (b) TC in the WT and miR-148a KO mice under RCD or HFD with or without DEN treatment (n=5–7). Significance was performed using Wilcoxon signed rank test. The horizontal lines in the box plots represent the median, the boxes represent the interquartile range, and the whiskers represent the minimal and maximal values. (c) RT-qPCR analysis of the expression of hepatic genes involved in cholesterol biosynthesis in the mice from (a). Data present mean±S.D. in (c). *P<0.05; **P<0.01; ***P<0.001
Figure 5
Figure 5
Pgc1α, Sirt7 and Hmgcr, Ybx1 are direct targets of miR-148a-3p and 5p, respectively. (a) RT-qPCR analysis of the expression of the predicted miR-148a-3p/5p targets in the hepatic tissues from the WT and miR-148a KO mice under RCD or HFD. (b) Western blot of the predicted miR-148a-3p/5p targets in the hepatic tissues from (a). (c) Luciferase activity of the reporter vector containing the WT or miR-148a-3p/5p-binding mutant 3′UTRs of the above predicted miR-148a-3p/5p targets was determined after co-transfection with Pri-miR-148a, miR-148a-3p sponge expression, miR-148a-5p inhibitor or control vectors in HEK293, HepG2 and Hep3B cells. (d and e) RT-qPCR analysis was performed to quantify the miR-148a-3p/5p (d) and their predicted targets RNA (e) levels incorporated into RISC in hepatic tissues from WT and miR-148a KO mice under RCD or HFD. Data present mean±S.D. in (a, c, d and e). **P<0.01; ***P<0.001
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