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Keywords:

  • cell proliferation;
  • gastric cancer;
  • H19;
  • lncRNA;
  • p53

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Long non-coding RNAs (lncRNAs) have been shown to have important regulatory roles in cancer biology, and the lncRNA H19 is up-regulated in hypoxic stress and in some tumors. However, the contributions of H19 to gastric cancer remain largely unknown. In this study, we assayed the H19 expression level in gastric cancer tissues by real-time PCR, and defined the biological functions by flow cytometry and RNA immunoprecipitation. We demonstrated that H19 levels were markedly increased in gastric cancer cells and gastric cancer tissues compared with normal controls. Moreover, ectopic expression of H19 increased cell proliferation, whereas H19 siRNA treatment contributed to cell apoptosis in AGS cell line. We further verified that H19 was associated with p53, and that this association resulted in partial p53 inactivation. These data suggest an important role for H19 in the molecular etiology of gastric cancer and potential application of H19 in gastric cancer therapy.


Abbreviations
H19

lncRNA 19

lncRNA

long non-coding RNA

MEG3

maternally expressed gene3

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Gastric cancer is one of the most frequent malignancies in East Asian countries [1,2]. Recurrence and metastasis are the biggest obstacles to treatment of gastric cancer. Despite efforts in multiple fields, there has been little success in improving the disease-free survival rate of patients. Advances in suitable therapy for the purpose of increasing survival rate have been limited because the pathophysiological mechanisms causing gastric cancer development are unknown. Therefore, revealing the molecular mechanism for gastric cancer development is indispensable for developing effective therapy.

Recently, a large-scale complementary DNA cloning project has identified that the majority of the mammalian genome is transcribed, although only minority of the transcripts represents protein-coding genes [3]. The function of the non-coding transcripts remains obscure, and their relevance to disease is undefined [4]. Although the role of protein-coding genes has been extensively investigated, the involvement of non-protein coding genes in tumorigenesis and tumor pathogenesis is less well characterized. Long non-coding RNAs (lncRNAs) such as HOTAIR, MALAT-1 and H19 have been implicated as playing a functional role in carcinogenesis or cancer growth [4–9]. Zhang et al. [5] demonstrated that expression of maternally expressed gene 3 (encoding MEG3) is associated with meningioma pathogenesis and progression. MEG3 suppresses DNA synthesis in meningioma cells by stimulating p53-mediated transactivation in these cell lines. HOTAIR expression levels are higher in cancerous tissues than corresponding non-cancerous tissues, and high HOTAIR expression correlated tightly with the presence of liver metastasis. Moreover, patients with high HOTAIR expression have a poorer prognosis [6,10]. Matouk et al. found that H19 RNA levels are up-regulated after exposure to hypoxia, and that H19 has pro-tumorigenic properties [11]. Ablation of tumorigenicity of hepatocellular carcinoma and gastric carcinomas by H19 knockdown was observed in vivo, which also significantly abrogates anchorage-independent growth after hypoxia recovery, while ectopic H19 expression enhances the tumorigenic potential of carcinoma cells in vivo [11].

H19 is a paternally imprinted gene located close to the telomeric region of chromosome 11p15.5, an area that is frequently involved in pediatric and adult tumors. While understanding of expression and imprinting of H19 RNA has progressed in recent years, its function remains enigmatic. H19 was initially proposed to possess tumor-suppressive properties based on its ability to inhibit tumorigenicity [12]. In contrast, recent research has shown that H19 RNA levels are up-regulated in tumors, and H19 possesses oncogenic properties [11,13,14]. Berteaux et al. demonstrated that H19 RNA is actively linked to E2F1 (E2F transcription factor 1) to promote cell-cycle progression of breast cancer cells [13].

Based on these findings, we tested whether H19 contributes to the biology of gastric cancer. We confirmed that H19 expression is markedly increased in gastric cancer tissues compared with adjacent normal tissues. Ectopic expression of H19 increased cell proliferation, whereas H19 siRNA treatment contributed to cell apoptosis in human gastric cancer cell lines. We further confirmed that H19 is associated with p53, and that this association results in partial p53 inactivation.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

lncRNA H19 levels are up-regulated in gastric cancer tissues

To assess the role of H19 in gastric cancer progression, we first examined the H19 expression levels in gastric cancer cells and gastric cancer tissues using quantitative real-time PCR. We verified that expression of H19 was increased in four gastric cancer cell lines relative to the expression in the human gastric epithelial mucosa cell line GES-1 (control) (Fig. 1A). We next assessed H19 expression in human gastric cancer tissues. The H19 levels were up-regulated in 77% of gastric cancer tissues compared with adjacent normal tissues (Fig. 1B). These data indicate that abnormal H19 expression may be related to gastric cancer progression.

image

Figure 1.  lncRNA H19 levels were up-regulated in gastric cancer cells and tissues. (A) Total RNA was extracted from cells using Trizol, and H19 expression was assessed by real-time PCR using SYBR Green in gastric cancer cell lines. H19 levels were normalized to β-actin levels in GES-1 cells. Asterisks indicate values that are significantly different from that in GES-1 (< 0.05). (B) H19 expression was assessed in human gastric cancer tissues and adjacent normal tissues. RNA was extracted from cancer tissues and adjacent normal tissues using Trizol. H19 expression was assessed by real-time PCR, and values were normalized to those for β-actin. Bars represent the ratio between expression in adjacent normal and gastric cancer tissues (log scale).

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H19 increased gastric cancer cell proliferation and inhibited cell apoptosis

To study the biological role of H19 in cell growth, gastric cancer cell lines treated with H19 were analyzed. H19 levels were significantly increased in human gastric cancer AGS cells transfected with H19, and up-regulation of H19 significantly increased AGS cell growth (Fig. 2A). Then AGS cells were treated with H19 siRNA to decrease H19 expression. Figure 2B shows that down-regulation of expression of H19 inhibited AGS cell growth. Similarly, down-regulated H19 expression in MKN45 cells suppressed cell growth (Fig. 3C). We also assessed whether down-regulated expression of H19 contributes to cell apoptosis. Figure 3 shows that down-regulation of H19 expression promoted AGS cell apoptosis. These data suggest that H19 positively regulates growth of gastric cancer cells.

image

Figure 2.  .H19 increased AGS cell proliferation. (A) AGS cells were transfected with pcDNA-H19, and H19 expression level was assayed after 48 h by real-time PCR. AGS cells were plated in a 24-well plate and transfected with pcDNA-H19, and the cell number was determined at the indicated time points using a CyQUANT cell proliferation assay. (B) AGS cells were treated with H19 siRNA, and the H19 expression level was assayed by real-time PCR. AGS cells were plated in a 24-well plate and transfected with H19 siRNA, and cell proliferation was determined at the indicated time points using a CyQUANT cell proliferation assay. (C) MKN45 cells were plated in a 24-well plate and transfected with H19 siRNA, and cell proliferation was determined at the indicated time points using a CyQUANT cell proliferation assay. Asterisks indicate values that are significantly different from controls (< 0.05).

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image

Figure 3.  Down-regulation of H19 expression promotes AGS cell apoptosis. AGS cells were treated with H19 siRNA and apoptosis inducers, and apoptosis was detected using flow cytometry. Down-regulated expression of H19 promoted AGS cell apoptosis.

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H19 was associated with p53 and suppressed p53 activation

To understand the molecular mechanism by which H19 increases gastric cancer cell growth, we examined whether H19 affects the function of the tumor suppressor p53, which potently inhibits cell growth by inducing a block of proliferation or by activating cell death programs [15]. A recent study demonstrated that lncRNAs play an important role in regulation of p53-mediated apoptosis [16]. Here, we performed RNA immunoprecipitation using an antibody against p53 from nuclear extracts of AGS cells. We found significant enrichment of H19 using the p53 antibody (Fig. 4A,B) compared with the IgG control antibody. To further confirm the association between H19 and p53, we performed RNA pulldown. Figure 4C shows a significant enrichment of p53 with H19 RNA compared with negative control RNA. These data suggest an association between H19 and p53. We then investigated whether H19 regulates p53 activation. AGS cells were co-transfected with the p53-responsive reporter plasmid and H19. Figure 4D shows that cells transfected with H19 show significantly decreased p53 activity. H19 also suppressed the protein level of the p53 target Bax (Fig. 4D). These data confirm that up-regulation of H19 expression contributes to tumorigenesis by regulating p53 activation in gastric cancer.

image

Figure 4.  H19 associates with p53. (A) RNA immunoprecipitation was performed using the p53 antibody to immunoprecipitate H19, and a primer was used to detect H19. (B) Quantification of H19 RNA levels. The asterisk indicates a significant difference compared with IgG (< 0.05). (C) RNA pulldown was performed as described in Experimental procedures. H19 RNA was incubated with nuclear extracts, and p53 protein was assayed by western blotting analysis. (D) Luciferase activity of p53 in AGS cells transfected with H19 or control, and western blotting analysis of Bax protein level after H19 over-expression in AGS cells. The asterisk indicates a significant difference compared with pcDNA-H19 (< 0.05).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

The human transcriptome is more complex than a collection of protein-coding genes and their splice variants [17–19]. With the advent of whole-genome and transcriptome sequencing technologies, it was determined that at least 90% of the genome is actively transcribed [19]. Although this was initially argued to be transcriptional noise, recent evidence suggests that this part of the genome may play a major biological role in cellular development and human diseases [20,21]. The newly discovered lncRNAs demonstrate developmental and tissue-specific expression patterns, and aberrant regulation in a variety of diseases, including cancer. However, the function of lncRNAs in tumor pathogenesis and growth is less well characterized, although recent studies have started to unravel their importance in tumorigenesis.

HOTAIR expression is increased in primary breast tumors and metastases, and the HOTAIR expression level in primary tumors is a powerful predictor of eventual metastasis and death [22]. Gupta et al. [10] demonstrated that enforced expression of HOTAIR in epithelial cancer cells induces genome-wide retargeting of Polycomb repressive complex 2 (PRC2) to an occupancy pattern that more closely resembles that of embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation and altered gene expression, and increased cancer invasiveness and metastasis. Conversely, loss of HOTAIR inhibits cancer invasiveness, particularly in cells that possess high PRC2 activity. Silva et al. identified 12 long stress-induced non-coding transcripts (LSINCTs) that show altered expression in cancer [23], and further demonstrated that LSINCT5 is over-expressed in breast and ovarian cancer cell lines and tumor tissues compared with normal tissues [24]. In addition, down-regulated expression of LSINCT5 in cancer cell lines caused a decrease in cellular proliferation [24]. These data suggest that lncRNAs may play an important role in the molecular etiology of human cancer.

H19, encoded by an imprinted gene, is an lncRNA. H19 was initially proposed to have tumor-suppressive properties based in its ability to inhibit tumorigenicity [8,12]. However, recent research has shown that H19 RNA levels are up-regulated in tumors, and H19 possesses oncogenic properties [11,13,14]. Using a mouse model for colorectal cancer, it was shown that mice lacking H19 show an increased polyp count compared to wild-type [25]. Barsyte-Lovejoy et al. [14] demonstrated that the c-Myc oncogene directly induces H19 expression by allele-specific binding, potentiating tumorigenesis. Several groups have studied the link between H19 and p53 [26,27]. Dugimont et al. [27] demonstrated that the H19 promoter is efficiently repressed by p53. Loss of parental methylation was observed at the insulin-like growth factor II (Igf2)/H19 loci in p53 knockout mice prior to tumor development [26]. In the present study, we demonstrated that H19 expression is markedly increased in gastric cancer cells and gastric cancer tissues compared with normal control. Ectopic expression of H19 increases cell proliferation, whereas H19 siRNA treatment contributes to cell apoptosis in AGS human gastric cancer cell lines. We further confirmed that H19 is associated with p53 and that this association results in partial p53 inactivation. In conclusion, our results suggest an important role for H19 in the molecular etiology of gastric cancer and potential application of H19 in gastric cancer therapy.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Cell lines and tumor tissues

Twenty-two specimens of gastric cancer tissues and adjacent benign tissues were collected at Changhai Hospital (Shanghai, China). Written informed consent for the biological studies was obtained from the patients involved in the study or their parents/guardians. The study was approved by the Ethics Committee of Changhai Hospital. The matched normal gastric tissue samples were obtained from tissues that were located 5 cm away from the tumor margin. The patients’ characteristics are detailed in Table 1.

Table 1.   Clinico-pathological characteristics of the patients with gastric cancer. All samples were obtained during surgical resection.
SampleAge (years)GenderT stageN stageM stage
 156MaleT2N1M0
 242MaleT1N1M0
 363MaleT4N2M0
 439FemaleT1N0M0
 570MaleT3N3M1
 648MaleT1N2M0
 760FemaleT2N3M1
 862FemaleT2N3M0
 955MaleT2N1M0
1064MaleT4N0M1
1147FemaleT1N0M0
1253MaleT3N1M1
1362MaleT4N3M1
1451MaleT1N2M0
1558MaleT2N2M0
1662FemaleT3N3M0
1770FemaleT4N3M1
1848MaleT2N0M0
1964MaleT2N1M0
2046FemaleT2N0M0
2173MaleT1N3M1
2278MaleT4N2M0

The human gastric cancer cell line AGS (p53 wild-type), MKN45 (p53 wild-type), MGC803 (p53 mutant) and SGC7901 (p53 mutant), and the immortalized human gastric epithelial mucosa cell line GES-1 were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained in RPMI-1640 Gibco with 10% FBS (North Andover, MA, USA) and cultured at 37 °C with 5% CO2.

Cell proliferation assay

For the cell proliferation assay, 5 × 104 cells were plated in a 24-well plate and transfected with pcDNA-H19 or H19 siRNA, and cell proliferation was determined using a CyQUANT cell proliferation assay (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The fluorescence intensity was measured using a fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA). Plasmid pcDNA-H19 was constructed by introducing a BamHI–EcoRI fragment containing the H19 cDNA into the same sites in pcDNA3.1. RNAi-mediated knockdown of H19 was performed as previously described [11]. The siRNAs used in this study were mixtures of three siRNAs, with the sequences: 5′-UAAGUCAUUUGCACUGGUUdTdT-3′ (H19-siRNA1), 5′-GCAGGACAUGACAUGGUCCdTdT-3′ (H19-siRNA2) and 5′-CCAACAUCAAAGACACCAUdTdT-3′ (H19-siRNA3).

Quantitative real-time PCR

Total RNA was extracted from gastric cancer tissues using Trizol reagent (Invitrogen), and reverse transcription reactions were performed using random primers and an M-MLV reverse transcriptase kit (Invitrogen). Real-time PCR was performed using a standard SYBR Green PCR kit (Toyobo, Osaka, Japan) and a Rotor-Gene RG3000A (Corbett Life Science, Mortlake, Australia) according to the respective manufacturers’ instructions. β-actin was used as a reference for lncRNAs. Each sample was analyzed in triplicate. The inline image method was used to quantify the relative levels of gene expression. The results represent the ratio between expression in gastric cancer tissues and adjacent normal tissues (log10 scale).

Flow cytometric analysis

AGS cells transfected with H19 siRNA (4 × 105) were plated in six-well plates, and apoptosis inducers A (Apopisa) and B (Apobid) (1:1000, Beyotime, Haining, China) were added to the culture. After 48 h incubation, the cultures were stained using annexin V–fluorescein isothiocyanate, and apoptosis rates were analyzed using a flow cytometer (FACSCalibur; BD Biosciences, Sparks, MD, USA).

RNA immunoprecipitation

RNA immunoprecipitation was performed using a Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. The p53 antibody used for RNA immunoprecipitation is ab2433 (Abcam, Cambridge, MA, USA). The co-precipitated RNAs were detected by RT-PCR. The primers used to detect H19 expression are 5′-GGGTCTGTTTCTTTACTTCCTCCAC-3′ (forward) and 5′-GATGTTGGGCTGATGAGGTCTGG-3′ (reverse). Total RNAs and controls were also assayed to demonstrate that the detected signals were from RNAs that specifically bind to p53.

RNA pulldown

RNA pulldown was performed as described previously [28]. Briefly, biotin-labeled RNAs were in vitro transcribed using a biotin RNA labeling mix (Roche, Indianapolis, IN, USA) and T7 RNA polymerase (Roche). Cell nuclear extract (1 mg) was mixed with biotinylated RNA biotin-labeled RNAs (100 pmol). Washed streptavidin–agarose beads (50 μL) were added to each binding reaction, and further incubated at room temperature for 1 h. Beads were washed briefly three times and boiled in SDS buffer for 30 minutes, and the retrieved protein was detected by standard western blotting technique [26].

Luciferase reporter assays

For analysis of luciferase reporters, AGS cells were plated in six-well plates, and transfected with pcDNA-H19 and p53-Luc using Lipofectamine 2000 (Invitrogen). Luciferase activity was determined using the luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions, and normalized to pRL-TK (Promega).

Statistical analysis

All data are expressed as means ± standard deviation (SD) from at least three separate experiments. The differences between groups were analyzed using Student’s t-test. The difference was deemed statistically significant at < 0.05.

References

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References
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