Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype

Abstract

Alterations in microRNA (miRNA) expression in both human and animal models have been linked to many forms of cancer. Such miRNAs, which act directly as repressors of gene expression, have been found to frequently reside in fragile sites and genomic regions associated with cancer. This study describes a miRNA signature for human primary hepatitis B virus-positive human hepatocellular carcinoma. Moreover, two known oncomiRs--miRNAs with known roles in cancer--the miR-17-92 polycistron and miR-21, exhibited increased expression in 100% of primary human and woodchuck hepatocellular carcinomas surveyed. To determine the importance of these miRNAs in tumorigenesis, an in vitro antisense oligonucleotide knockdown model was evaluated for its ability to reverse the malignant phenotype. Both in human and woodchuck HCC cell lines, separate treatments with antisense oligonucleotides specific for either the miR-17-92 polycistron (all six members) or miR-21 caused a 50% reduction in both hepatocyte proliferation and anchorage-independent growth. The combination of assays presented here supports a role for these miRNAs in the maintenance of the malignant transformation of hepatocytes.

Figure 1

The 25 most up-regulated (A) and down-regulated (B) miRNAs in four hepatocellular carcinoma samples in respect to one normal liver sample as determined by small RNA cloning and sequencing. The ratio of relative cloning frequencies between hepatocellular carcinoma sample and normal liver sample are presented in log2 transformed for each individual patient sample. The average of these values as well as the 95% confidence interval is displayed. Bars above the figure indicate missing clones in either normal liver (A) or in any of the HCC samples (B). For statistical analysis, using a Bayesian framework, the clone counts for all hepatocellular samples were pooled and compared to the clone counts of normal liver taking into account also the total clone number obtained in the respective libraries. *P < 0.05 and **P < 0.001 of clone counts from the pooled HCC samples and liver being the same.

Figure 2

Conserved increase in expression of the miR-17–92 polycistron and miR-21 in both human and woodchuck HCCs. Northern blot analysis of the expression of miRNAs in human and woodchuck peri-tumor liver and primary HCCs. A: Human samples. B: Woodchuck samples. C samples = peri-tumor liver. T samples = primary HCC. Matching C samples are to the Left of T samples from the same liver. miRNAs are as labeled next to rows, miR-122, miR-92, miR-21, and miR-17. tRNA and U43 RNA were loading controls.

Figure 3

qRT-PCR analysis of the miR-17–92 polycistron and miR-21 expression in human HCC, and human cirrhotic livers as compared with normal liver. Expression data are reported as fold change (2^−ΔΔCt) above normal liver. Statistical significance was determined by a t-test comparing (ΔCt) of normal liver to either HCC or cirrhotic livers (ΔCt). *P < 0.05, **P ≤ 0.001, ***P < 0.0001.

Figure 4

Comparison of expression of the pri-miRNA for the miR17–92 polycistron in HCC cell lines. qRT-PCR analysis of the C13orf25 transcript expressed in HCC cell lines is reported as fold change (2⁻ΔΔCt) above normal liver.

Figure 5

Analysis of antisense oligonucleotide knockdown of selected miRNAs from the miR17–92 polycistron and miR-21. miRNA levels were assayed by qRT-PCR TaqMan assays at 48 hours (open bars) and 5 days (hatched bars) post transfection. The change in miRNA expression is reported as the (ΔΔCt) of ASO treated cells versus lipofectamine (control) alone treated cells, with U45 as reference. All knockdowns were statistically significant by t-test comparing (ΔCt) of control to ASO samples. *P < 0.05, **P ≤ 0.001, ***P < 0.0001.

Figure 6

Knockdown the miR-17–92 polycistron or miR-21 reduces HepG2 cell proliferation. Cell proliferation was measured by MTS, dose-response data from 0 to 100 nmol/L total ASO transfected, 72 hours after transfection. Data expressed as % of control (Lipofectamine only treatment). A: HepG2 cells treated with ASO against all six miRNAs in the miR17–92 polycistron (dark gray bars) or miR-21 (angle slash bars) or miR-122 control (light gray bars). T-test demonstrated that compared to control the reduction seen in all samples was significant P < 0.001. B: Effects of ASOs against individual miRNAs from the miR-17–92 cluster versus the mix of all six miRNAs. Measurement of cell proliferation measured 72 hours post-transfection of 250 nmol/L ASO. *P < 0.05, **P ≤ 0.001, ***P < 0.0001.

Figure 7

Knockdown of the miR-17–92 polycistron causes a retardation of the cell cycle. Fluorescence-activated cell sorter analysis (propidium iodide staining), was used to examine cell cycle progression 48 hours after transfection with ASO. A: HepG2 cells treated with ASO to the miR17–92 polycistron and a random scrambled sequence. B: HepG2 cells treated with ASO to individual members of the miR17–92 polycistron. Results presented as % of cells in phase; error bars on graph represent SEM. *P < 0.05, **P ≤ 0.001, ***P < 0.0001.

Figure 8

Knockdown of either the miR-17–92 polycistron or miR-21 reduces HepG2 anchorage independent growth. HepG2 cells were re-plated 24 hours after transfection into soft agar and allowed to grow for 5 days, on which time colonies were photographed and the area was measured using NIH Image J. A: HepG2 cells treated with either ASO against all six miRs in the miR17–92 polycistron, miR-21 or miR-122 alone. B: Microscopy images (magnification = original ×20) of HepG2 colonies in soft agar. Results presented as % control; error bars on graph represent mean SEM. *P < 0.05, **P ≤ 0.001, ***P < 0.0001.

Figure 9

Loss of miR-21 expression induces apoptosis. The effect of knockdown of the miR17–92 polycistron and miR-21 on apoptosis was assessed by determining the level active caspase-3 by absorption 5 days after transfection. Results presented as % control; error bars on graph represent mean SEM. *P < 0.05, **P ≤ 0.001, ***P < 0.0001.

Figure 10

The effect of ASO miR17–92 knockdown on E2F1 expression. A: qRT-PCR of E2F1 and E2F3 expression in HepG2 cells treated with either ASO to the miR17–92 polycistron as compared with lipofectamine treated cells 5 days after transfection. Values expressed as fold change (2^−ΔΔCt) above controls (lipofectamine alone) using GAPDH as reference. B: Western blot of E2F1 and COX IV (loading control) expression 5 days after transfection.