Hepatitis B virus mRNA-mediated miR-122 inhibition upregulates PTTG1-binding protein, which promotes hepatocellular carcinoma tumor growth and cell invasion - PubMed (original) (raw)

. 2013 Feb;87(4):2193-205.

doi: 10.1128/JVI.02831-12. Epub 2012 Dec 5.

Yanzhong Wang, Saifeng Wang, Bo Wu, Junli Hao, Hongxia Fan, Ying Ju, Yuping Ding, Lizhao Chen, Xiaoyu Chu, Wenjun Liu, Xin Ye, Songdong Meng

Affiliations

Hepatitis B virus mRNA-mediated miR-122 inhibition upregulates PTTG1-binding protein, which promotes hepatocellular carcinoma tumor growth and cell invasion

Changfei Li et al. J Virol. 2013 Feb.

Abstract

As the most abundant liver-specific microRNA, miR-122 is involved in diverse aspects of hepatic function and neoplastic transformation. Our previous study showed that miR-122 levels are significantly decreased in hepatitis B virus (HBV)-infected patients, which may facilitate viral replication and persistence (S. Wang, L. Qiu, X. Yan, W. Jin, Y. Wang, L. Chen, E. Wu, X. Ye, G. F. Gao, F. Wang, Y. Chen, Z. Duan, and S. Meng, Hepatology 55:730-741, 2012). Loss of miR-122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G1-modulated P53 activity.). In this study, we provide evidence that all HBV mRNAs harboring an miR-122 complementary site act as sponges to bind and sequester endogenous miR-122, indicating that the highly redundant HBV transcripts are involved in HBV-mediated miR-122 suppression. We next identified pituitary tumor-transforming gene 1 (PTTG1) binding factor (PBF) as a target of miR-122 and demonstrated that HBV replication causes an obvious increase in PBF levels. Furthermore, we observed that the miR-122 levels were decreased and PBF was upregulated in chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). Overexpression and knockdown studies both revealed that PBF enhances proliferation and invasion of HCC cells, and silencing PBF resulted in a dramatic reduction of HCC tumor growth in vivo. Mechanistic analysis demonstrated that PBF interacts with PTTG1 and facilitates PTTG1 nuclear translocation, subsequently increasing its transcriptional activities. Therefore, we identified a novel HBV mRNA-miR-122-PBF regulatory pathway that facilitates malignant hepatocyte growth and invasion in CHB which may contribute to CHB-induced HCC development and progression. Our work underscores the reciprocal interplay of host miRNA sequestration and depletion by viral mRNAs, which may contribute to chronic-infection-related cancer.

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Figures

Fig 1

Fig 1

Suppression of miR-122 by HBV replication. (A and B) After transfection with the HBV replication vector pHBV1.3 or empty pcDNA3.1 plasmid (mock), miR-122 expression (A) and the expression of miR-21 and miR-181a (B) in Huh-7 cells were quantified by real-time PCR. The results were normalized to a U6 endogenous control, RNU6B. (C and D) Detection of miR-122 expression (C) and cyclin G1 expression (D) in the liver of HBV transgenic BALB/c mice (n = 5) and control BALB/c mice (n = 5) by real-time PCR. P < 0.05 (*) and P < 0.01 (**) compared to the control or mock treatment.

Fig 2

Fig 2

Inhibition of miR-122 by HBV mRNAs. (A) miR-122 promoter activity in response to pHBV1.3 transfection. Huh-7 cells were cotransfected with pHBV1.3 or the empty pcDNA3.1 vector (mock transfection), pGL-122 with a luciferase reporter under the miR-122 promoter, and pRL-TK. pGL-122 and renilla luciferase activities were measured using a dual-luciferase assay kit at the indicated time. (B) After transfection with pHBV1.3 or pcDNA3.1, pri-miR-122 expression in Huh-7 cells was quantified with real-time PCR. (C) Detection of the mRNA levels of miR-122 transcription factors after transfection with pHBV1.3 or pcDNA3.1 for 72 h in Huh-7 cells. (D, top) Predicted miR-122 binding sequence located in the HBV genome at nt 1684 to 1709. Perfect matches are indicated by a line. Mutations were made in the seed region of the miR-122 binding sites (HBV-mut) without changing the amino acid sequence of the X protein. (Lower) A putative miR-122 complementary region was found in all four HBV mRNAs. 293T cells were cotransfected with an miR-122 mimic or a randomized oligonucleotide (mock transfection) and a firefly luciferase reporter plasmid with a 3′-UTR containing either the wild-type (HBV-wt) or mutant (HBV-mut) miR-122 binding sequence. The firefly luciferase and renilla luciferase activities were measured using a dual-luciferase assay kit. (E) Huh-7 cells were transfected with the luciferase reporter plasmid with either the HBV-wt or HBV-mut 3′-UTR, and miR-122 levels were measured by real-time PCR 24 h after transfection. (F and G) Huh-7 cells were transfected with the wild-type (pHBV1.3-wt) or mutant (pHBV1.3-mut) HBV plasmid or with pcDNA3.1 (mock transfection). (F) At 24 h after transfection, miR-122 levels were assessed by real-time PCR (left) and Northern blotting (right). (G) The expression of cyclin G1 was detected at 48 h by Western blotting. (H) Huh-7 cells were transfected with pYMHD1.3 or pcDNA3.1, and at 24 h after transfection miR-122 levels were measured by real-time PCR. (I) Huh-7 cells were transfected with the indicated amount of pHBV1.3, and miR-122 levels and total HBV mRNA levels were analyzed by real-time PCR. (J) Correlation analysis between miR-122 levels and intrahepatic HBV mRNA levels in CHB by Spearman analysis. Values of the correlation coefficient (R) and P are shown. P < 0.05 (*) and P < 0.01 (**) compared to mock transfection.

Fig 3

Fig 3

PBF, an miR-122 target, is upregulated in patients with CHB and HCC. (A) Detection of miR-122 expression in liver biopsy specimens from CHB, HCC (the tumor), and healthy controls (HC) by real-time PCR. The results were normalized to a U6 endogenous control, RNU6B. The miR-122 levels in HC were arbitrarily set to 1.0. (B, top) Perfect matches are indicated by a line between the 3′-UTR of PBF (PBF-UTR) and miR-122. Mutations (PBF-UTRm) were made in the seed region of the miR-122 binding site for the reporter gene assay. (Lower) HepG2 cells were cotransfected with an miR-122 mimic or a randomized oligonucleotide as a mock transfection and an EGFP reporter plasmid carrying either the wild-type (EGFP-PBF-UTR) or mutant (EGFP-PBF-UTRm) 3′-UTR of PBF. The GFP levels were measured by Western blotting. (C and D) Western blot analysis of the expression of PBF in HepG2, SK-Hep-1, Huh-7, and L02 cells transfected with an miR-122 mimic or inhibitor (C) or transfected with the wild-type (pHBV1.3-wt) and mutant (pHBV1.3-mut) HBV replication vector (D). (E) Immunohistochemistry analysis of PBF expression in the liver samples from CHB, HCC, and HC. (F) Distribution of the miR-122 levels in CHB patients with PBF overexpression and nonoverexpression in liver tissue. Data are expressed as the medians and ranges (from the 10th percentile to the 90th percentile). *, P < 0.05.

Fig 4

Fig 4

PBF enhances the growth of liver cancer cells. (A to C) Huh-7 (A), HepG2 (B), and SK-Hep-1 (C) cells were transfected with PBF siRNA (or the control siRNA as a mock transfection) or with the PBF expression vector pPBF (or pcDNA3.1-Myc-his as a mock transfection). At 24, 48, and 72 h after transfection, cell growth was assessed by CCK-8 assay. (D and E) HepG2 cells were transfected with an miR-122 mimic or a randomized oligonucleotide as a mock transfection and PBF siRNA (D), and Huh-7 cells were transfected with an miR-122 inhibitor or a randomized oligonucleotide and PBF siRNA (E). At 24, 48, and 72 h after transfection, cell growth was measured by CCK-8 assay. (F to I) Huh-7 cells were transfected with pHBV1.3-wt or pcDNA3.1 (F) or with pHBV1.3-mut or pcDNA3.1 (G), or they were cotransfected with pHBV1.3 or pcDNA3.1, along with an miR-122 mimic (H) or PBF siRNA (I). At 24, 48, and 72 h after transfection, cell growth was measured by CCK-8 assay. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) compared to mock transfection.

Fig 5

Fig 5

PBF enhances the invasion of liver cancer cells. (A) Huh-7, HepG2, and SK-Hep-1 cells were transfected with pPBF and/or pPTTG1 or pcDNA3.1-Myc-his (mock transfection). The cell invasion activity was then assessed using a Matrigel-coated Boyden chamber assay. Representative images of invading cells (stained with DAPI) were photographed with a fluorescence microscope. (B) The mean numbers of invading cells are shown; results are presented as means ± SD from three independent experiments. (C and D) Huh-7 cells were transfected with pHBV1.3-wt or pcDNA3.1 (C) or with pHBV1.3-mut or pcDNA3.1 (D). The cell invasion ability was then measured, and the mean numbers of invading cells are shown. (E and F) Huh-7 cells were cotransfected with pHBV1.3 or pcDNA3.1, along with an miR-122 mimic (E) or PBF siRNA (F). The mean numbers of invading cells are shown. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) compared to the mock transfection.

Fig 6

Fig 6

Effect of PBF knockdown on liver tumor growth in nude mice. (A) Western blot analysis of PBF expression in SK-Hep-1 cells stably transfected with PBF siRNA (SK-Hep-1-PBFi) and luciferase siRNA-transfected cells (SK-Hep-1-Luci) as a mock transfection. Actin was used as a loading control. (B) SK-Hep-1-PBFi (PBF siRNA) or SK-Hep-1-Luci (mock) cells were s.c. injected into female BALB/c-nu/nu mice. Tumor diameters were measured twice a week for 6 weeks. (C) Tumor weight was measured when the mice were sacrificed at week 6. The results are presented as means ± SD from five mice. (D) Paraffin tumor tissue sections were stained with anti-PBF polyclonal antibody and counterstained with hematoxylin. *, P < 0.05 compared to mock treatment. Data are representative of two independent experiments.

Fig 7

Fig 7

PBF promotes PTTG1 transcriptional activity by facilitating PTTG1 nuclear translocation. (A) HepG2 and Huh-7 cells were cotransfected with pEGFP-PTTG1 and pPBF or the empty pcDNA3.1-Myc-His vector as a mock transfection. At 48 h after transfection, cells were fixed and PTTG1 was directly detected by green fluorescence. DAPI staining (blue) indicates the nucleus. (B) HepG2 cells were transfected with pPBF (or pcDNA3.1-Myc-his) or PBF siRNA (or control siRNA). At 48 h after transfection, the mRNA levels of VEGF, FGF-2, Sp1, c-myc, and MMP-2 were measured by real-time PCR. The data are presented as the mean ratios (± SD) compared to those for mock transfection. Results are presented from three independent experiments. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) compared to the mock transfection. (C) Schematic of how HBV-miR-122-PBF-PTTG1 may mediate liver cell growth and invasion. Inhibition (⊥) or stimulation (↓) was determined according to how CHB affects miR-122 expression and how miR-122 may contribute to HCC development. In CHB patients, miR-122 in hepatocytes is inhibited by viral mRNAs and/or chronic inflammation. Loss of miR-122 expression leads to upregulation of its target PBF, which initiates PTTG1 nuclear translocation, promoting PTTG1 transcriptional activity and thus enhancing cell growth and invasion. (D) HBV mRNAs act as sponges to block miR-122-mediated inhibition of endogenous target genes. In hepatocytes with no HBV infection, target mRNAs of miR-122 (black) are repressed by the miR-122/RISC complex (green rectangles). miR-122-induced target mRNA degradation is shown by black dashed lines. After HBV infection, viral mRNAs (red) are expressed at a high level and sequester the miR-122 complexes, rescuing the expression of target mRNAs.

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