Systemic delivery of microRNA-101 potently inhibits hepatocellular carcinoma in vivo by repressing multiple targets

Abstract

Targeted therapy based on adjustment of microRNA (miRNA)s activity takes great promise due to the ability of these small RNAs to modulate cellular behavior. However, the efficacy of miR-101 replacement therapy to hepatocellular carcinoma (HCC) remains unclear. In the current study, we first observed that plasma levels of miR-101 were significantly lower in distant metastatic HCC patients than in HCCs without distant metastasis, and down-regulation of plasma miR-101 predicted a worse disease-free survival (DFS, P<0.05). In an animal model of HCC, we demonstrated that systemic delivery of lentivirus-mediated miR-101 abrogated HCC growth in the liver, intrahepatic metastasis and distant metastasis to the lung and to the mediastinum, resulting in a dramatic suppression of HCC development and metastasis in mice without toxicity and extending life expectancy. Furthermore, enforced overexpression of miR-101 in HCC cells not only decreased EZH2, COX2 and STMN1, but also directly down-regulated a novel target ROCK2, inhibited Rho/Rac GTPase activation, and blocked HCC cells epithelial-mesenchymal transition (EMT) and angiogenesis, inducing a strong abrogation of HCC tumorigenesis and aggressiveness both in vitro and in vivo. These results provide proof-of-concept support for systemic delivery of lentivirus-mediated miR-101 as a powerful anti-HCC therapeutic modality by repressing multiple molecular targets.

Figure 1. Analysis of miR-101 levels in human plasma samples by real-time PCR and Kaplan-Meier analysis for HCC patients DFS according to the plasma levels of miR-101.

A. Expression levels of miR-101 in human plasma samples from healthy donors (n = 50) and HCC patients with distant metastasis (n = 16) and without distant metastasis (n = 147). B. Kaplan-Meier analysis for HCC patients DFS according to plasma levels of miR-101. The levels of miR-101 was analyzed by real-time PCR, and ROC curve analysis was applied to determine the cutoff score for high expression (n = 88) and low expression of plasma miR-101 (n = 75).

Figure 2. Systemic delivery of lent-miR-101 suppresses tumor growth, angiogenesis and metastasis in the orthotopic liver implanted HCC model of mouse.

A. Upper panel, laser confocal microscopy showed efficient infection of lentivirus to the liver, the lung and tumor xenografts of mice, as indicated by coGFP expression. Down panel, the levels of miR-101 in the liver, the lung and tumor tissues were significantly higher in lent-miR-101 treated mice than that in both control mice (P<0.001). Data is presented as mean± SE. B. Representative tumor xenografts in the liver of mice in different (NaCl, lent-miR-ctr and lent-miR-101) treated groups. P, primary tumor; M, metastatic nodule. The average size of primary tumors in the liver was 10.75±3.25 mm in diameter in NaCl treated group (n = 8), 11.51±3.71 mm in lent-miR-ctr treated group (n = 8), and 2.78±1.25 mm in lent-miR-101 treated group (n = 7). C. Representative images of microvessel density (MVD) of implanted primary tumor examined by IHC staining of CD34 in lent-miR-101 treated and 2 control groups. D. Representative images of metastatic nodules in the liver, in the lung and in the mediastinum of mice in different treatment groups (indicated by arrows). E. The mean number of metastasis in lenti-miR-101 treated group (liver: 9.4±2.9; lung: 11.3±2.5, n = 7) was significantly larger than that in control NaCl (liver: 32.1±5.1; lung: 39.4±6.0, n = 8) and lent-miR-ctr (liver: 30.8±5.4; lung: 40.9±5.4, n = 8) treated groups (P<0.01). Data is presented as mean± SE. F. The difference in survival time of mice between the lenti-miR-101 group and 2 control (NaCl and lent-miR-ctr) groups was statistically significant (P<0.05).

Figure 3. ROCK2 is the target of miR-101.

A. Schematic of predicted miR-101-binding sites in the 3′UTR of ROCK2. B. MiR report constructs containing a wild-type and 2 mutated ROCK2 3’UTR were transfected into LM9 cells, respectively. Relative repression of firefly luciferase expression was standardized to a transfection control. The reporter assays were performed 3 times with essentially identical results. C. Left, the levels of miR-101 by Real-time PCR in the lenti-miR-101 and control MOCK and lent-miR-ctr treated LM9 cells. Right, real-time PCR examination of mRNA levels of ROCK2 between the lenti-miR-101 and control lent-miR-ctr treated LM9 cells. LM9 cells were infected with lent-miR-ctr or lent-miR-101 for 72 hours. Down, ectopic overexpression of miR-101 by lenti-miR-101 reduces the levels of ROCK2 proteins in LM9 cells, as compared to that in both MOCK and lent-miR-ctr treated LM9 cells. D. Left, the levels of miR-101 by Real-time PCR in the lenti-miR-101 and mock and lent-miR-ctr treated Huh7 cells. Right, real-time PCR examination of mRNA level of ROCK2 between the lenti-miR-101 and control lent-miR-ctr treated Huh7 cells. Huh7 cells were infected with lent-miR-ctr or lent-miR-101 for 72 hours. Down, ectopic overexpression of miR-101 by lenti-miR-101 reduces the levels of ROCK2 proteins in Huh7 cells, as compared to that in both Mock and lent-miR-ctr treated Huh7 cells. E. Upper, protein expression of ROCK2 is up-regulated in HCC HepG2 cells after the down-regulation of miR-101 by anti-miR-101, as compared to that in control Mock and anti-miR-NC HepG2 cells. Down, protein expressions of ROCK2 is up-regulated in lent-miR-101-LM9 cells after the down-regulation of miR-101 by anti-miR-101, as compared to that in control anti-miR-NC cells. F. IHC staining showing down-regulated expressions of ROCK2 in HCC tissues of mice treated with systemic delivery of lent-miR-101, as compared to that treated with NaCl or lent-miR-ctr.

Figure 4. Enforced overexpression of miR-101 inhibits HCC LM9 cells invasion and EMT in vitro and reduces metastasis in vivo.

A. The invasive properties of HCC LM9 cells transfected with lent-miR-ctr, lent-miR-101, si-STMN1, and si-ROCK2 were analyzed by an invasion assay using a Matrigel Invasion Chamber. Migrated cells were plotted as the average number of cells per field of view from 3 independent experiments (**, P<0.01). B. Expression levels of the epithelial markers E-cadherin, α-catenin and β-catenin and the mesenchymal markers fibronectin, N-cadherin and vimentin were analyzed by Western blot between lent-miR-101 and control lent-miR-ctr LM9 cells. C. IF staining (red signal) showing that the Epithelial markers E-cadherin, α-catenin, β-catenin were up-regulated and mesenchymal markers fibronectin, N-cadherin and vimentin were down-regulated in lent-miR-101 treated LM9 cells, as compare to that in lent-miR-ctr cells. D. The in vivo effects of miR-101 on HCC cell metastasis using an experimental metastasis assay, in which lent-miR-101, control lent-miR-ctr and mock LM9 cells were injected into the tail vein of SCID mice, respectively. Metastatic tumor growth in the liver and in the lung was assessed. Representative metastatic nodules and H&E staining of metastatic tumors in the liver and in the lung are indicated by arrows. E. The number of metastatic nodules in the liver and in the lungs of mice (n = 8 per group) 8 weeks after tail vein injection of let-miR-101 LM9 cells (mean±SE, liver: 2.8±0.8, lung: 9.8±4.7), mock LM9 cells (mean±SE, liver: 9.8±1.0, lung: 33.5±4.6) and lent-miR-ctr LM9 cells (mean±SE, liver: 9.5± 1.6, lung: 33.5± 6.7).

Figure 5. Ectopic overexpression of miR-101 inhibits stress fiber formation in vitro and angiogenesis in a CAM model, and a proposed regulatory loop of miR-101 in HCC tumorigenesis and metastasis.

A. Staining for F-actin demonstrated that the stress fiber formation in lent-miR-101 treated LM9 cells decreased when compared with that in lent-miR-ctr LM9 cells. B. Total and active forms of Rho-GTPases, including RhoA, Rac1, and cdc42 were compared between lent-miR-ctr and lent-miR-101 treated LM9 cells by Western blot analysis. GTP-bound (active) forms of RhoA, Rac1, and cdc42 were pulled down and examined by Western blot using corresponding antibodies. Active forms of RhoA, Rac1 and Cdc42 were lower in lent-miR-101 LM9 than that in lent-miR-ctr LM9 cells. C. Lent-miR-101 LM9 and Huh7 cells inhibits angiogenesis in a CAM model. Left panel, representative plugs from different (Mock, lent-miR-ctr and lent-miR-101 LM9 and Huh7 cells) treated groups. Right panel, the number of blood vessels was counted from 6 replicate experiments, and normalized to that of the lent-miR-ctr group as relative angiogenesis. The data were mean±SD. ** indicates the significant change with P<0.01. D. A proposed model in which miR-101 inhibits tumorigenesis and metastasis of HCC by repression of multiple target genes and tumor activators.