Volume 19, Issue 8, Pages 1521-1528 (August 2011) Tumor-specific Expression of MicroRNA-26a Suppresses Human Hepatocellular Carcinoma Growth via Cyclin-dependent and -independent Pathways  Lizao Chen, Jianming Zheng, Yan Zhang, Luxi Yang, Jiaqi Wang, Jian Ni, Daxiang Cui, Chaoqin Yu, Zailong Cai  Molecular Therapy  Volume 19, Issue 8, Pages 1521-1528 (August 2011) DOI: 10.1038/mt.2011.64 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 MicroRNA-26a (mir-26a) negatively regulates normal liver cell proliferation, and specific expression of miR-26a specifically suppresses tumor cell proliferation. (a) Real-time PCR detected the expression of mature miR-26a in L02 cells transfected with ph1MCG or its control vector ph1CG (normalized to U6 levels). (b) Compared with the control vector ph1CG, overexpression of miR-26a negatively regulated cell proliferation. (c) In the ph1MCG group, the percentage of cells in the G1 phase of the cell cycle increased by ∼20%. Accordingly, cell growth was reduced significantly. (d) Expression of mature miR-26a was detected by real-time PCR in Huh-7, SMMC-7721, and L02 cells transfected with pAT, pTM, pAM, and pATM. Relative expression was normalized to U6 levels. (e) Growth of Huh-7, SMMC-7721, and L02 cells after transfection with the indicated plasmids. The growth index was assessed at 0, 1, 2, 3, and 4 days. (f) Western blot analysis of cyclin D2 and cyclin E2 in Huh-7, SMMC-7721, and L02 cells transfected with pAT, pAM, pTM, and pATM. β-Actin served as an internal control. *P < 0.05, **P < 0.01. Molecular Therapy 2011 19, 1521-1528DOI: (10.1038/mt.2011.64) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 MicroRNA-26a (miR-26a) targets ERS1, and expression of estrogen receptor-α (ERα) and miR-26a in patient hepatocellular carcinoma (HCC) tissues (T) and pair-matched adjacent tissues (N) n = 12. (a) Western blot analysis demonstrates an abundance of ERα in pATM-transfected Huh-7 cells. The band intensities were normalized to β-actin levels. (b) Dual luciferase assay of Huh-7 cells co-transfected with the firefly luciferase constructs containing the ESR1 WT or Mut 3′-UTR and miR-26a mimics or scrambled oligonucleotides as the negative control. (c) Sequence and evolutionary conservation of the miR-26a binding site in the 3′-UTRs of transcripts encoding ERα (ESR1). The interaction sites between WT and Mut 3′-UTRs of ESR1 and miR-26a are indicated. (d) Western blot analysis of ERα protein in human HCC. β-Actin serves as an internal control (gray scale is represented as the mean ± SD). Expression of (e) mature miR-26a and (f) ESR1 was detected by real-time PCR. Relative expression was normalized to U6 and β-actin levels, respectively. *P < 0.05, **P < 0.01. Molecular Therapy 2011 19, 1521-1528DOI: (10.1038/mt.2011.64) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 MicroRNA-26a (miR-26a) downregulates the expression of estrogen receptor-α (ERα) and partly blocks the E2-ERα pathway. (a) Growth curve of Huh-7 cells treated with different concentrations of E2 at 0, 1, 2, and 3 days. (b) Proliferation index of Huh-7 cells stably transfected with or without pATM as well as transient transfected with pcDNA3.1-ERα/pcDNA3.1 and treated with E2 or Fulvestrant on day 2. (c) Immunofluorescence assay of Huh-7 cells stably transfected with or without pATM and treated with E2 or Fulvestrant at 8 hours to detect ERα expression (red) in the cell nucleus. DAPI (blue) was used to stain the nuclei. *P < 0.05, **P < 0.01. Molecular Therapy 2011 19, 1521-1528DOI: (10.1038/mt.2011.64) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 MicroRNA-26a (miR-26a) driven by the human α-fetoprotein (hAFP) and human telomerase reverse transcriptase (hTERT) dual promoter inhibits tumor cell growth in vivo. (a) Nude mice were inoculated with pATM- or pAT-transfected Huh-7 cells in their flanks. Two of the five mice are shown here. (b) Representative tumor growth 5 weeks after inoculation. Tumor volume was calculated. Data are represented as the mean ± SD. n = 5. (c) Expression of estrogen receptor-α (ERα) was measured by immunohistochemistry in the tissues of mice inoculated with pATM- or pAT-transfected Huh-7 cells. (d) Hematoxylin-eosin (HE) staining of tumor sections demonstrates the character of the hepatocellular carcinoma (HCC)-bearing nude mouse model generated by inoculation with HCC cell lines (×200). Molecular Therapy 2011 19, 1521-1528DOI: (10.1038/mt.2011.64) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Specific expression of microRNA-26a (miR-26a) for therapeutic experiments. (a) Tumor growth was observed in nude mice whose tumors were treated with the pATM or pAT mixture. (b) Hematoxylin-eosin (HE) staining of the hepatocellular carcinoma (HCC) mouse model SMMC-LTNM, which more closely models the clinical character of HCC (×200). (c) Representative tumor suppression 4 weeks after treatment. Tumor volume was calculated. Data are represented as the mean ± SD. n = 5. Molecular Therapy 2011 19, 1521-1528DOI: (10.1038/mt.2011.64) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Immunohistochemistry assay of tumor tissues treated with the pATM or pAT mixture, respectively. Proteins were divided into five groups: (a) tumor marker [α-fetoprotein (AFP) and CEA]; (b) cyclin-dependent pathway (cyclin D2 and cyclin E2); (c) cyclin-independent pathway [estrogen receptor-α (ERα), PR, and P53]; (d) proliferation index (PCNA and Ki-67) and (e) antioncogene (P53 and PTEN). The brown color seen in the cells denotes positive staining for the aforementioned proteins. The white frame indicates a representative ×400 magnification field. The original magnification is ×200. (f) Quantitative analysis of PCNA- and Ki-67-positive cells in each group. *P < 0.05. Molecular Therapy 2011 19, 1521-1528DOI: (10.1038/mt.2011.64) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions