Volume 140, Issue 1, Pages 322-331 (January 2011) The MicroRNA miR-139 Suppresses Metastasis and Progression of Hepatocellular Carcinoma by Down-regulating Rho-Kinase 2  Carmen Chak–Lui Wong, Chun–Ming Wong, Edmund Kwok–Kwun Tung, Sandy Leung–Kuen Au, Joyce Man–Fong Lee, Ronnie Tung– Ping Poon, Kwan Man, Irene Oi–Lin Ng  Gastroenterology  Volume 140, Issue 1, Pages 322-331 (January 2011) DOI: 10.1053/j.gastro.2010.10.006 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Dysregulation of miRNA in human HCC. A q-PCR–based miRNA profiling analysis on 4 pairs of microdissected primary HCC. miRNA expression was normalized against endogenous control U6 by the −ΔCt(Target-U6) approach, and differential expression between HCC (T) and nontumorous livers (NT) was calculated by the ΔΔCt(NT-T) method. (A) Volcano plot illustrating the biological and statistical significances of differential miRNA expressions between human HCC and their corresponding nontumorous livers. X axis, average fold change (log2 T/NT), that is, ΔΔCt; Y axis, log10 P value (paired t test). miRNAs that were significantly underexpressed in human HCCs (ie, log2 T/NT < −1; P < .05) are highlighted in blue. On the other hand, miRNAs that were significantly up-regulated in human HCCs (ie, log2 T/NT > 1; P < .05) are marked in red. (B) Heat map diagram generated by unsupervised clustering analysis with 30 significantly dysregulated miRNAs in human HCCs. Hierarchical clustering was performed with average linkage and uncentered correlation. miRNA expression profile effectively segregated human HCC samples from their corresponding nontumorous livers. Gastroenterology 2011 140, 322-331DOI: (10.1053/j.gastro.2010.10.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Down-regulation of miR-139 in human HCC was associated with disease progression and venous metastasis. Expression of mature miR-139 in an expanded human HCC cohort and cell lines were determined by q-PCR and normalized against an endogenous control RNU44. (A) miR-139 expression in 67 pairs of primary HCCs (HCC) and their corresponding nontumorous livers (NT) (frozen samples). miR-139 expression levels were calculated by the miR-139/RNU44 expression ratio (ie, 2–ΔCt). The median miR-139 expression levels in normal liver (NL), nontumorous liver, and primary HCC were 0.041, 0.021, and 0.002, respectively. miR-139 expression was slightly decreased in nontumorous livers (P = .068, Mann–Whitney test), and was significantly underexpressed in primary HCC samples (P < .001). (B) Comparison of miR-139 expression level between primary HCCs and their corresponding nontumorous livers. A log2 fold change greater than +2 or less than −2 (ie, 4-fold) was considered as significant overexpression or down-regulation (dotted lines). miR-139 was found to be underexpressed in 76% (51 of 67) of the HCC cases examined (pink bars). (C) miR-139 expression in the immortalized normal liver cell line, LO2, HCC cell lines, BEL7402 and SMMC-7721, and metastatic HCC cell lines, H2M and MHCC97L, and the median values of miR-139 expression in NL, NT, and HCC. (D) Progressive decrease in miR-139 expression in HCC progression from normal livers (N = 9; median, 0.036) via nontumorous livers with chronic hepatitis (N = 26; median, 0.021) or cirrhosis (N = 36; median, 0.019), to early HCC (N = 28; median, 0.003) and eventually advanced HCC (N = 35; median, 0.001). (E) Left: H&E-stained paraffin section of a representing case consisting of nontumorous liver, primary HCC, and venous metastasis (VM) used for microdissection. Right: miR-139 expression in microdissected nontumorous livers, primary HCCs, and venous metastases. miR-139 down-regulation was found in 18 of the 20 primary HCCs and was decreased further (>2-fold) in 45% (9 of 20) of the venous metastases, including 2 HCC cases with only moderate miR-139 down-regulation in the primary HCCs. Values of miR-139 expressions (miR139/RNU44) were calculated as fold change relative to nontumorous tissues as 1. (F) Down-regulation of miR-139 in HCCs was associated with a poor prognosis. Patients were classified into 2 groups according to the median miR-139 expression in HCC. Patients with low miR-139 expression showed lower disease-free survival rates in the first 2 years after surgery (left) and lower 5-year overall survival rates (right). Gastroenterology 2011 140, 322-331DOI: (10.1053/j.gastro.2010.10.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Re-expression of miR-139–suppressed HCC cell migration. miR-139 was stably re-expressing in HCC cell lines BEL7402 and SMMC-7721 by lentiviral infection. (A) Successful re-expression of mature miR-139 was confirmed by q-PCR. Values of miR-139 expression (miR139/RNU44) were calculated as fold change relative to the vector control as 1. (B) Re-expression of miR-139 had no effect on the proliferation rate of HCC cells. (C) Re-expression of miR-139 significantly impeded cell migratory ability in BEL7402 and SMMC-7721 (P < .001, t test). (D) A total of 100 nmol/L of miR-139 inhibitor and miR inhibitor control were transiently transfected into LO2 cells. Inhibition of miR-139 in LO2 cells was verified by real-time q-PCR. (E) miR-139 inhibitor enhanced LO2 cell migration (P < .001, t test). Gastroenterology 2011 140, 322-331DOI: (10.1053/j.gastro.2010.10.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Re-expression of miR-139–suppressed HCC metastasis in vivo. MiR-139 was stably re-expressed in luciferase-labeled MHCC97L cells. (A) Successful re-expression of mature miR-139 was confirmed by q-PCR. Values of miR-139 expression (miR139/RNU44) were calculated as fold change relative to the vector control as 1. (B) Tumors derived from the vector and miR-139–infected cells were implanted orthotopically into the livers of nude mice. Tumor growth in the mice was monitored by a live imaging system detecting the luciferase signal (unit = photons/s/cm2/steradian). Both vector and miR-139–infected cells formed tumors effectively in nude mice. Representative live images from the MHCC97L vector and miR-139 groups are shown. The size of the miR-139–derived tumors was slightly smaller than that of the vector control group (mean luciferase signal = 8.846 and 3.099, respectively) but the difference was not statistically significant (P = .300, t test). (C) Lung metastasis of vector and miR-139–infected MHCC97L in nude mice at 7 weeks after orthotopic implantation. Lungs from the nude mice were dissected and the presence of HCC metastasis was detected by luciferase signals. (D) Representative H&E-stained sections of the lung tissues collected from vector and miR-139–infected groups are shown in the left panel. Arrow points to the tumor focus formed in the lung. Quantitative analysis was performed by counting the number of tumor foci in 10 randomly selected high-power fields under microscope (right panel). (E) Summary of pathologic analysis on MHCC97L vector and miR-139–derived tumors in the livers of the nude mice. Gastroenterology 2011 140, 322-331DOI: (10.1053/j.gastro.2010.10.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 miR-139 suppressed HCC cell migration via directly targeting ROCK2 protein expression. (A) Wild-type and mutant of putative miR-139 target sequences of ROCK2 3'UTR. (B) miRNA luciferase reporter assay. Two copies of wild-type and mutant miR-139 target sequences were fused with luciferase reporter and transfected into vector and miR-139 stably infected BEL7402 cells. miR-139 significantly suppressed the luciferase activity of wild-type ROCK2 3′UTR (*P = .034, **P = .009, ***P < .001, t test). (C) Re-expression of miR-139 in BEL7402 and SMMC-7721 attenuated ROCK2 protein expression, and transient expression of miR-139 inhibitor in LO2 cells moderately increased ROCK2 protein expression. (D) miR-139 down-regulation was significantly associated with ROCK2 protein overexpression in primary HCC (P = .030, Fisher exact test). (E) Knockdown of ROCK2 by Sh RNA significantly inhibited HCC cell migration (left panel) (P < .001, t test). Re-expression of miR-139 profoundly reduced cell migration in BEL7402 vector control cells (P < .001, t test) but failed to do so in BEL7402 Sh ROCK2 cells (P = .462, t test) (middle and right panels, respectively). Gastroenterology 2011 140, 322-331DOI: (10.1053/j.gastro.2010.10.006) Copyright © 2011 AGA Institute Terms and Conditions