miR-185 inhibits hepatocellular carcinoma growth by targeting the DNMT1/PTEN/Akt pathway

Abstract

miRNAs have recently been implicated in hepatocarcinogenesis, although the actions and mechanisms of individual miRNAs remain incompletely understood. We examined the biological functions and molecular mechanisms of miR-185 in hepatocellular carcinoma (HCC). The expression of miR-185 is decreased in human HCC tissues compared with the nonneoplastic liver parenchyma. Quantitative RT-PCR showed a reduction of miR-185 in human HCC cells compared with primary hepatocytes. miR-185 overexpression in human HCC cells inhibited cell proliferation and invasion in vitro and prevented tumor growth in SCID mice. miR-185 overexpression inhibited DNMT1 3' untranslated region luciferase reporter activity in HCC cells; this effect was abolished when the miR-185 binding site was mutated. miR-185 mimic or overexpression decreased the level of DNMT1 protein in HCC cells. These findings establish DNMT1 as a bona fide target of miR-185 in HCC cells. The role of DNMT1 in miR-185-induced inhibition of HCC growth was further supported by the fact that DNMT1 overexpression prevented miR-185-induced inhibition of HCC cell proliferation/invasion. miR-185 mimic or overexpression reduced PTEN promoter DNA methylation and enhanced PTEN expression, leading to the inhibition of Akt phosphorylation; these effects were partially reversed by DNMT1 overexpression. These results provide novel evidence that miR-185 inhibits HCC cell growth by targeting DNMT1, leading to PTEN induction and Akt inhibition. Thus, reactivation or induction of miR-185 may represent a novel therapeutic strategy for HCC treatment.

Figure 1

Down-regulation of miR-185 in human HCC. A: Representative in situ hybridization for miR-185 in human HCC and normal liver tissues. The expression of miR-185 was decreased in HCC cells compared with nonneoplastic hepatocytes. Boxed areas on the left correspond to higher-magnification images on the right. B: miR-185 staining intensity in different types of tissue specimens [normal liver, cirrhotic liver, hepatitis B virus (HBV)–associated HCC, and hepatitis C virus (HCV)–associated HCC]. Staining intensities were compared and are depicted in a box plot. C: RT-qPCR showed a lower level of miR-185 expression in human HCC cells (Huh7, HepG2, and Hep3B) compared with human primary hepatocytes (HHs). Data are given as means ± SD. ^∗P < 0.05, ^∗∗P < 0.01. Original magnification: ×40 (A, left panels); ×400 (A, right panels).

Figure 2

miR-185 targets DNMT1 in HCC cells. Huh7 (A) and HepG2 (B) cells were transiently transfected with miR-185 mimic and miRNA scramble control oligonucleotide. The cell lysates were obtained for Western blot analysis to measure DNMT1 protein levels. DNMT1 protein expression was analyzed with β-actin as an internal control. The experiments were repeated five times (N = 5). Normalized fold expression of DNMT1 in Huh7 cells transfected with miR-185 mimic was compared with scramble control as depicted in bar graphs. Data are given as means ± SD. ^∗P < 0.05.

Figure 3

miR-185 targets DNMT1 and inhibits HCC proliferation and invasion in vitro. A: Real-time PCR quantification of miR-185 showed a significant increase in Huh7 cells stably transduced with L/miR-185 compared with cells stably transduced with L/Control. B: Western blot analysis for DNMT1 in Huh7 cells with or without stable overexpression of miR-185. Representative Western blots and average densitometry data. The level of DNMT1 protein is decreased in Huh7 cells stably expressing miR-185 (gray bars) compared with control cells (white bars). C: Cell proliferation was measured at 24, 48, and 72 hours in Huh7 stable cells after plating 1 × 10³ per well in 6-well plates. miR-185 overexpression decreased cell proliferation at all the time points. D: A cell invasion assay was performed in Huh7 stable cells. Huh7 miR-185 stable cells showed an approximately 50% reduction in the number of invaded cells on the matrix membrane surface compared with control after 24 hours. The images depict invaded cells. Data are given as means ± SD. N = 5 (B). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 4

miR-185 decreases DNMT 3′-UTR luciferase reporter activity in HCC cells. A: The putative miR-185 binding site in DNMT1 3′-UTR is depicted in the top sequence pairs. The original strand of DNMT1 3′-UTR contains the 7-Bp seed sequences that bind to miR-185; the DNMT1 3′-UTR mutant strand shows deletion of the 7-Bp seed sequence. B:DNMT1 3′-UTR luciferase reporter acidity assay in Huh7 stable cells transfected with wild-type or mutant DNMT1 3′-UTR reporter construct. Data are given as means ± SD. N = 3. ^∗P < 0.05.

Figure 5

Restoration of DNMT1 prevents miR-185–induced inhibition of HCC cell proliferation and invasion. A: Western blot analysis for DNMT1, PTEN, p-Akt, and Akt in Huh7 stable cells with or without DNMT1 overexpression. B: Cell proliferation analysis of Huh7 stable cells with or without DNMT1 overexpression. The experiments were performed 72 hours after plating 1 × 10³ per well in 6-well plates. The experiments were repeated three times. C: Cell invasion assay of Huh7 stable cells with or without DNMT1 overexpression. The numbers of invaded cells were counted 24 hours after cell plating. The images depict invaded cells. Data are given as means ± SD. N = 5 (A); N = 3 (B and C). ^∗P < 0.05.

Figure 6

miR-185 decreases PTEN promoter methylation and enhances PTEN expression in Huh7 cells. A: Huh7 cells stably infected with miR-185 or control lentivirus were subjected to methylation-specific PCR assay. miR-185 overexpression decreased methylation in the PTEN CpG island promoter region. B: Huh7 cells were transiently transfected with miR-185 mimic and scramble control oligonucleotide, and the cell lysates were obtained for Western blot analysis to determine the levels of PTEN, p-Akt, and Akt. Data are given as means ± SD. N = 5. ^∗P < 0.05.

Figure 7

miR-185 overexpression inhibits HCC growth in SCID mice. An Huh7 stable cell suspension mixed in Matrigel solution was inoculated into SCID mice via direct liver injections. A: Gross images depict liver tumors that originated from miR-185 overexpressed or control cells. Local liver tumors that originated from miR-185–transduced Huh7 cells are significantly smaller (11-fold decrease) in tumor volume compared with control. B: Ki-67 immunostaining showed a lower proliferative rate in miR-185 overexpressed tumors compared with the control. C: Western blot analysis for DNMT1, PTEN, and p-Akt in miR-185 overexpressed or control tumors recovered from the livers of SCID mice. Normalized fold expressions for DNMT1, PTEN, and p-Akt are depicted in bar graphs. D: Schematic illustration of miR-185–induced inhibition of HCC growth. Data are given as means ± SD. N = 12 (A); N = 5 (C). ^∗P < 0.05.