The H19 non-coding RNA is essential for human tumor growth

Abstract

Background: Mutations and epigenetic aberrant signaling of growth factors pathways contribute to carcinogenesis. Recent studies reveal that non-coding RNAs are controllers of gene expression. H19 is an imprinted gene that demonstrates maternal monoallelic expression without a protein product; although its expression is shut off in most tissues postnatally, it is re-activated during adult tissue regeneration and tumorigenesis. Moreover, H19 is highly expressed in liver metastasis derived from a range of carcinomas. The objective of this study is to explore the role of H19 in carcinogenesis, and to determine its identification as an anti-tumor target.

Methodology/principle findings: By controlling oxygen pressure during tumor cell growth and H19 expression levels, we investigated the role of H19 expression in vitro and in vivo in hepatocellular (HCC) and bladder carcinoma. Hypoxia upregulates the level of H19 RNA. Ablations of tumorigenicity of HCC and bladder carcinomas in vivo are seen by H19 knockdown which also significantly abrogates anchorage-independent growth after hypoxia recovery, while ectopic H19 expression enhances tumorigenic potential of carcinoma cells in vivo. Knocking-down H19 message in hypoxic stress severely diminishes p57(kip2) induction. We identified a number of potential downstream targets of H19 RNA, including angiogenin and FGF18.

Conclusions: H19 RNA harbors pro-tumorigenic properties, thus the H19 gene behaves as an oncogene and may serve as a potential new target for anti-tumor therapy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. H19 RNA is largely induced in response to hypoxic stress and moderately by hypoxia-mimicking condition triggered by CoCl2 in Hep3B.

Hep3B cells were cultured under normal culture conditions for 24 hours before hypoxic or CoCl2 manipulation. Cells were either placed into an aneoropack rectangular jar to create a hypoxic condition within an hour, or left under normal culture conditions. Incubation lasted for 24 hours before RNA extraction. (A) Shown are RT-PCR products of H19 gene (28 PCR cycles) cultured under normal conditions-lane 1, or hypoxic conditions-lane 2 (lane M indicates the marker, and C is a PCR blank without a target). (B) PCR for Histone H3.3 as a positive control for RT-PCR integrity. Shown also are RT-PCR products of both the H19 gene (32 PCR cycles) (C), the GAPDH gene (D) for untreated Hep3B (lane1) and for 50, 100, 200, 300 and 400 µM CoCl2 treated cells (lanes 2, 3, 4, 5 and 6, respectively). Incubation with the indicated concentrations of CoCl2 lasted for an additional 22 hours before RNA extraction. QPCR analysis for H19 RNA levels normalized to β-actin in Hep3B treated CoCl2 is shown in (E) where the numbers above the bars indicate the concentrations of CoCl2 used.

Figure 2. Effect of four different H19 siRNA duplexes on the expression level of H19 in the Hep3B cell line under normal culture condition (A) and hypoxia-mimicking CoCl2 treatment (B).

Shown is H19 RNA levels (34 PCR cycles) in (A): Hep3B cells transfected with unrelated siRNA duplex that targets luciferase gene (lane 1) and with 4 different H19 siRNA1 to H19 siRNA4 duplexes (lanes 2–5) and an equimolar mixture of the four siRNAs (lane 6); for controls, we performed transfection also with lipofectamine 2000 without siRNA (Mock) (lane 7) and C is PCR blank without a target. (B): Hep3B cells were transfected under normal medium with siRNA duplex that targets the luciferase gene (lanes 1 and 5) and with 3 different H19 siRNA duplexes (lanes 2–4). Twenty four hours post transfection, media was changed, and 100 µM CoCl2 containing media was added except for lane 5 which continued to grow under normal culture media; the incubation lasted for an additional 22 hours. (C): Shown are RT-PCR products of GAPDH gene as a positive control for RT-PCR integrity.

Figure 3. H19 RNA is induced by hypoxic stress in Hep3B cell line and siRNA directed against H19 very efficiently impedes its induction.

Hep3B cells were seeded and transfected either with H19 siRNA or luc siRNA . Twenty four hours post transfection, cells were either placed into an aneoropack rectangular jar , or left under normal oxygen concentration. Incubation lasted for 24 hours before RNA extraction. Shown are RT-PCR analyses for H19 RNA (28 PCR cycles). (A): Hep3B transfected with Luc siRNA (lanes 1, 2) and H19 siRNA (lanes 3, 4) both in normal (lanes 1, 3) and hypoxic (lanes 2, 4) culture conditions, respectively. PCR analysis of a house-keeping gene Histone H3.3 (B), and uPAR (C). In (D) QPCR analysis for H19 RNA levels normalized to β-actin in Hep3B cells is depicted.. A Quantitative SYBR Green RT–PCR was performed on human total RNA for both H19 and β-actin to estimate H19 RNA copy number under different manipulations and efficiency of knockdown under hypoxic conditions.

Figure 4. Transient H19 RNA knockdown in Hep3B cells inhibited tumorigenicity in vivo.

Hep3B cells were transiently transfected with H19 siRNA3 or Luc siRNA. Forty eight hours post transfection, cells were washed twice with PBS, trypsinized and counted. Equal numbers of cells (1.5×106) were injected subcutaneously into the dorsal part of CD-1 nude mice (n = 7 for both, and 4 for mock transfected). Palpable tumors were observed 15 days post inoculation in mice inoculated with Hep3B, transiently transfected with Luc siRNA. Tumor volumes were followed up and measured using a caliper until day 30 post inoculation, after which mice were sacrificed. Significant (p<0.03) reductions of about 82% of both mean tumor weights (A) (± standard error) and mean tumor volumes (p<0.03) (B) (± standard error) were observed. Values represent end-points just before and after sacrificing animals. Shown are also representative features of tumors in 2 mice of each group (mice 1 and 2 are the H19 siRNA3 treated Hep3B cells, and mice 3 and 4 are the Luc siRNA ) before tumor surgical exposure (C), and after exposure of their internal tumors (D).

Figure 5. The effect of over-expression of H19 RNA on the tumorigenic potential of bladder carcinoma cells in vivo.

Equal amounts (2×106) of TA31H19high and TA11H19-ve cells were implanted subcutaneously to CD-1 mice (n = 5, each). Two weeks later, palpable tumors appeared and tumor volumes were measured for an additional two weeks . Shown are end point measurements of the mean tumor volumes of the two groups (upper left pannel), their mean tumor volumes kinetics (upper right), and a representative gross morphology of tumors derived from the TA11H19-ve (lower left) and TA31H19high cells(down and right).

Figure 6. The in vivo effect of H19 silencing on the tumorigenic potential of human bladder carcinoma cells-UMUC3.

One million UMUC3 cells were injected subcutaneously to athymic mice (n = 3 for GFP siRNA, and 5 for H19 siRNA ), 48 hours after transiently transfected with siRNAs. Palpable tumors were observed 6 weeks later in 2 out of 3 mice of the GFP siRNA group, while in none of the H19 siRNA group . Mice were sacrificed 8 weeks after inoculation. Mean tumor volumes (B, P

Figure 7. H19 RNA knockdown impedes p57Kip2 mRNA induction in response to hypoxic stress, in Hep3B cell line.

Hep3B cells were seeded and transfected either with H19 siRNA or Luc siRNA as described . Shown are RT-PCR analyses for H19 RNA (28 PCR cycles). (A): Hep3B transfected with luc siRNA (lanes 1, 2) and H19 siRNA (lanes 3, 4) both in normal (lanes 1, 3) and hypoxic (lanes 2, 4) culture conditions, respectively. And similar treatments are assessing the mRNA levels of: (B). p19INK4 (C). p57Kip2 (D). Histone.

Figure 8. Expression levels of selected genes in Hep3B cells transfected either with H19 and GFP siRNAs under normoxic and hypoxic conditions.

A few genes that showed variations as a result of H19 knockdown under different manipulations were selected, based on their potential importance in tumorigenesis, for further RT-PCR analysis to confirm the results of the microarray data. In the figure +denotes (+H19) and –(-H19). The genes chosen were (A)-FGF18; (B)-IGFBP4; (C)-ANG; (D)-LBR; and (E)-AKT1 . The knockdown, of H19 and the RT-PCR integrity are shown in Figure 7.