MicroRNA-based classification of hepatocellular carcinoma and oncogenic role of miR-517a

Abstract

Background & aims: Hepatocellular carcinoma (HCC) is a heterogeneous tumor that develops via activation of multiple pathways and molecular alterations. It has been a challenge to identify molecular classes of HCC and design treatment strategies for each specific subtype. MicroRNAs (miRNAs) are involved in HCC pathogenesis, and their expression profiles have been used to classify cancers. We analyzed miRNA expression in human HCC samples to identify molecular subclasses and oncogenic miRNAs.

Methods: We performed miRNA profiling of 89 HCC samples using a ligation-mediated amplification method. Subclasses were identified by unsupervised clustering analysis. We identified molecular features specific for each subclass using expression pattern (Affymetrix U133 2.0; Affymetrix, Santa Clara, CA), DNA change (Affymetrix STY Mapping Array), mutation (CTNNB1), and immunohistochemical (phosphor[p]-protein kinase B, p-insulin growth factor-IR, p-S6, p-epidermal growth factor receptor, β-catenin) analyses. The roles of selected miRNAs were investigated in cell lines and in an orthotopic model of HCC.

Results: We identified 3 main clusters of HCCs: the wingless-type MMTV integration site (32 of 89; 36%), interferon-related (29 of 89; 33%), and proliferation (28 of 89; 31%) subclasses. A subset of patients with tumors in the proliferation subclass (8 of 89; 9%) overexpressed a family of poorly characterized miRNAs from chr19q13.42. Expression of miR-517a and miR-520c (from ch19q13.42) increased proliferation, migration, and invasion of HCC cells in vitro. MiR-517a promoted tumorigenesis and metastatic dissemination in vivo.

Conclusions: We propose miRNA-based classification of 3 subclasses of HCC. Among the proliferation class, miR-517a is an oncogenic miRNA that promotes tumor progression. There is rationale for developing therapies that target miR-517a for patients with HCC.

Figure 1

miRNA-based molecular classification of HCC. (A) Heat map showing unsupervised hierarchical clustering of 89 HCV-related HCCs based on the expression levels of 358 human miRNAs. MiRNAs are in row, samples are in column. High and low expression levels are represented in red and blue, respectively. Molecular subclasses (A, B, C1, C2, C3) are indicated by colored bars. Up-regulation of C19MC family in subclass C2 is shown. Immunostaining for pEGFR, pAkt, pS6, pIGF1R, β-catenin localization, and the mutation status of TP53 and CTNNB1 genes are indicated in different colors (grey, positive; black, negative; white, missing value). Overlaps with transcriptomic-based molecular classes previously reported by our group are shown. Different colors correspond to distinct molecular classes. Chiang et al’s classification includes CTNNB1 class (blue), proliferation class (green), interferon-related class (pink), the Poly7 (polysomy of chromosome 7) class (orange), and unannotated samples (brown). Hoshida et al’s classification includes S1 class (blue), S2 class (green), and S3 class (yellow). (B) Summary of the proposed miRNA-based molecular classification of HCC. AFP, α-fetoprotein; VI, vascular invasion.

Figure 2

Validation of subclass C2 in an independent set of HCC. (A) Relative expression levels of miR-517a, miR-520g, and miR-516-5p were used as molecular surrogates of subclass C2 and evaluated in 165 formalin-fixed paraffin-embedded (FFPE) HCCs. High and low expression levels are indicated in gradient scale boxes. Black boxes, missing values. (B) Subclass association matrix for subclass C2 and other classes (non-C2) in the training (frozen tissues) and validation (FFPE sections) sets. Subclasses C2 in both HCC sets characterized by different tissue origin were significantly associated.

Figure 3

Molecular mechanisms involved in C19MC miRNAs overexpression. (A) Inferred copy number of chr19 for each sample in subclass C2. C19MC locus is indicated by an arrow. One sample (indicated by a red box) showed copy gain in the q arm of chr19 (inferred copy numbers in the 1 Mb surrounding 19q13.41 region, 2.97). (B) Genomic localization of C19MC miRNAs on chr19. A CpG island is located 15 kb upstream of the cluster. (C) Expression level of miR-517a after treatment with 5-aza-2′deoxycytidine (5-AZA-CDR) and 4-phenylbutyric acid (PBA) in Huh7 cells compared with untreated cells. Results are expressed as fold changes ± standard error of the mean. (D) Methylation-specific PCR of the CpG island located upstream of the C19MC locus. Presence of PCR product indicates methylated (lane M) or unmethylated (lane U) alleles. Huh7 (DNA from Huh7 without bisulfite conversion), negative control; SsI-Huh7 (enzymatically methylated DNA), positive control. HCC: subclass C2 (n = 6 samples); normal, 1 representative normal liver.

Figure 4

miR-517a and miR-520c promote cell proliferation, migration, and invasion in vitro. (A) Proliferation assay after transfection with control oligonucleotide, miR-517a, or miR-520c in Huh7 cells. Results are expressed as a percentage of ³H-thymidine incorporation (counts per million) ± standard error of the mean compared with control. (B) Transwell migration assay. Cells that migrated from the upper well of a Transwell chamber into the lower well were stained and counted. Magnification, ×100. (C) Cell invasion assays. Inserts coated with Matrigel were used to investigate the invasive potential of miR-517a and miR-520c transfected cells. Magnification, ×100. (D) Wound-healing assay. Cell monolayers were scratched and images were taken 0 and 24 hours after wounding. Results are expressed as distance (in μm) between the 2 edges ± standard error of the mean. Magnification, ×100.

Figure 5

miR-517a promotes tumorigenesis in vivo. (A) Luciferase-tagged Huh7 cells transduced with miR-517a or control vector were injected into the left lobe of the liver of nude mice. Representative images taken at day 1, and at 2, 4, 6, and 8 weeks are displayed. (B) Incidence of liver tumors in mice injected with Huh7 transduced with miR-517a or control vector. (C) Kaplan–Meier curves representing the decrease in survival of mice injected with miR-517a–expressing Huh7 compared with control-Huh7. (D) Multiple tumor nodules were observed in liver of mice administered with miR-517a–transduced Huh7.

Figure 6

miR-517a induces metastatic dissemination of Huh7 cells in vivo. (A) Bioluminescent imaging of dissected organs showed the presence of large liver tumors and metastases in lungs and kidneys of mice injected with miR-517a– expressing cells. Representative images are shown. (B) Histologic analysis showed the presence of vascular invasion and metastases in distant organs in mice administered with miR-517a– expressing Huh7.