Open Access

Long non‑coding RNA H19 regulates viability and metastasis, and is upregulated in retinoblastoma

  • Authors:
    • Li Li
    • Wei Chen
    • Yuchuan Wang
    • Luosheng Tang
    • Mei Han
  • View Affiliations

  • Published online on: March 29, 2018     https://doi.org/10.3892/ol.2018.8385
  • Pages: 8424-8432
  • Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Retinoblastoma is the most common type of intraocular pediatric malignant tumor, which typically affects children <6 years of age. However, the underlying molecular mechanisms of retinoblastoma progression remain unclear. The aim of the present study was to investigate the function of long non‑coding RNA (lncRNA) H19 in retinoblastoma clinical samples and cell lines, using reverse transcription‑quantitative polymerase chain reaction, western blotting, colony formation, MTT, fluorescence activated cell sorting, cell invasion and migration, and in vivo growth assays. The results demonstrated that H19 may serve a critical oncogenic function in the progression of retinoblastoma, as lncRNA H19 levels were markedly increased in retinoblastoma cells and tissues compared with corresponding controls. In addition, patients with retinoblastoma with increased lncRNA H19 expression experienced poorer survival time compared with those with decreased lncRNA H19 levels. Knockdown of lncRNA H19 significantly suppressed retinoblastoma cell proliferation, migration and invasion in vitro and in vivo. Furthermore, lncRNA H19 expression was also associated with multiple proteins, including cyclin‑dependent kinase 1, B‑cell lymphoma‑associated X protein, apoptosis regulator, tumor protein p53, vimentin, cadherin 13 and matrix metallopeptidase 9. In conclusion, lncRNA H19 may serve an important function in tumorigenesis and may be a potential target for therapy and prognosis in retinoblastoma.

Introduction

Retinoblastoma is a type of embryonic malignant tumor of the retina of the eye, which originates from primitive stem cells (1). Retinoblastoma primarily occurs in childhood, with an average incidence of between 1/15,000 and 1/20,000 live births, typically arising in infants <6 years of age, often prior to the age of 2 years (2). Advanced retinoblastoma is able to rapidly cover the eye, invade the optic nerve and eventually spread to the central nervous system. Previous evidence from molecular, cellular and cytogenetic studies has indicated that retinoblastoma is typically caused by a mutation in the RB transcriptional co-repressor 1 (RB1) gene on chromosome 13 (35). RB1 inactivation results in mitotic defects, contributing to genomic instability, which manifests as aneuploidy and chromosomal rearrangements (6,7). At present, non-metastatic retinoblastoma is not considered a fatal childhood cancer and is effectively treated by enucleation, dependent on an early diagnosis and accurate prognosis of the disease (8). Dalgard et al (9) observed that the retinoblastoma cell lines Y79 and Weri-Rb1, and retinoblastoma tumor samples, presented with aberrant microRNA (miR)-34a and miR-34b expression. In addition, this study provided indicated that knockdown of miR-34a may result in increased retinoblastoma cell proliferation and chemotherapeutic resistance.

The aim of the present study was to investigate long non-coding RNA (lncRNA) H19-mediated regulation of the development of retinoblastoma in clinical samples and cell lines. LncRNAs are non-protein-coding transcripts, which are >200 nucleotides in length, and regulate gene expression transcriptionally or post-transcriptionally. These intergenic transcripts are involved in diverse cellular processes, including proliferation, migration, invasion, apoptosis and the reprogramming of stem cell pluripotency (1013). LncRNA H19 has been considered as an oncogenic lncRNA in multiple types of cancer, including hepatocellular, bladder carcinoma, breast cancer, bladder tumor and glioma (1417). Furthermore, previous studies reported that H19 regulates the development of gliomas via interactions with miR-675, which in addition contributes to the proliferation of gastric cancer cells and was associated with tumor metastasis (1820). Emerging evidence has also demonstrated that H19 expression is upregulated, and is involved, in carcinogenesis and cancer metastasis via the promotion of cell cycle progression (21). However, the function of H19 in retinoblastoma remains unclear.

In the present study, the clinical characteristics, biological function and potential underlying molecular mechanisms of lncRNA H19 in retinoblastoma were investigated. The results indicated that H19 levels were markedly increased in retinoblastoma cells and tissues. Furthermore, patients with retinoblastoma with increased lncRNA H19 expression experienced poorer survival time compared with patients with lower levels of lncRNA H19 expression. Furthermore, lncRNA H19 expression was also associated with several proteins, including cyclin-dependent kinase 1 (CDK1), B-cell lymphoma 2-associated X, apoptosis regulator (Bax), tumor protein p53 (p53), vimentin, cadherin 13 (CDH13) and matrix metalloproteinase 9 (MMP9). The oncogenic function of H19 suggests that it may serve as a potential target for retinoblastoma therapy and prognostic prediction.

Materials and methods

Cell lines and tumor tissues

The present study was approved by the Research Ethics Committee of Tianjin Eye Hospital (Tianjin, China). Written informed consent for all biological procedures was obtained from each patient, or their parents, and specimens for the present study were anonymized. Patients who had received treatment prior to surgery were excluded from the present study. The tumor samples were collected between June 2011 until November 2015 and were extracted from enucleated eyes and immediately snap-frozen in liquid nitrogen and stored at −80°C. A total of 80 freshly frozen retinoblastoma tissue samples (44 males and 36 females; the age distribution: 40 patients ≥2 years and 40 patients <2 years), and five normal retina samples (3 males and 2 females; the age distribution: 2 patients ≥2 years and 3 patients <2 years) obtained from ruptured globes, were collected at Tianjin Eye Hospital (Tianjin, China).

Cell culture

The cell lines used in the present study were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The human retinal pigment epithelial cell line ARPE-19 was cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin (Invitrogen, Thermo Fisher Scientific, Inc.), and 100 mg/ml streptomycin (Invitrogen, Thermo Fisher Scientific, Inc.). The human retinal microvascular endothelial cell line (HRMEC) was maintained in Endothelial-Cell Growth medium (EGM-2 SingleQuot Kit Supplement & Growth Factors; Lonza Group, Ltd., Basel, Switzerland), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin (Invitrogen, Thermo Fisher Scientific, Inc.), and 100 mg/ml streptomycin (Invitrogen, Thermo Fisher Scientific, Inc.). The retinoblastoma cell lines Weri-Rb1 and Y79 were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin (Invitrogen, Thermo Fisher Scientific, Inc.), and 100 mg/ml streptomycin (Invitrogen, Thermo Fisher Scientific, Inc.). All cell cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RNA was purified and then reverse-transcribed into cDNA using a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturers protocol. qPCR was performed using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol, and run on an ABI Prism 7000 Sequence Detection System. Relative expression of lncRNA H19 (primer: Forward, 5′-ATCGGTGCCTCAGCGTTCGG-3′; and reverse, 5′-CTGTCCTCGCCGTCACACCG-3) was normalized to the expression of GAPDH (primer: Forward, 5′-AGCCACATCGCTCAGACAC-3′ and reverse, 5′-GCCCAATACGACCAAATCC-3′). Relative gene expression levels were quantified using the 2−ΔΔCq method (22). The thermocycling conditions for lncRNA H19 quantification were as follows: 95°C for 10 min; 40 cycles of 95°C for 15 sec and 60°C for 1 min. Each sample was examined in triplicate.

Small interfering (si)RNA transfection

RNA interference was conducted using synthetic siRNA duplexes. Two synthetic siRNA duplexes (si-H19) corresponding to the H19 mRNA sequences, 5′-CCCACAACAUGAAAGAAAU-3′ (forward) and 5′-GCUAGAGGAACCAGACCUU-3′ (reverse), were used to inhibit H19 RNA expression. si-H19 and si-negative control (NC) were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells were cultured in 6-well plates and maintained until they reached ~60% confluence, prior to transfection with siRNA duplexes (25 nM) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Transfection efficiency to assess the inhibition of H19 after 48 h of transfection in Y79 cells was performed using RT-qPCR as previously outlined.

Flow cytometric analysis

Cell apoptosis was examined by flow cytometry. Weri-Rb1 or Y79 cells were seeded at a density of 1×104 cells/well in 96-well plates. In brief, cells were transfected with si-H19 or si-NC as aforementioned and, 2 days post-transfection, cells were trypsinized by 0.25% Trypsin (Gibco; Thermo Fisher Scientific, Inc.), followed by centrifugation at 350 × g for 2 min. and washed with PBS. Cells were then resuspended in PBS and fixed with 100% ethanol at 4°C overnight, and apoptosis was detected using a dual-staining method with annexin V-fluorescein isothiocyanate in combination with propidium iodide (D Pharmingen, San Diego, CA, USA). Apoptosis was analyzed using a FACScan flow cytometer and Cell Quest analysis software version 5.2 (BD Biosciences, Franklin Lakes, NJ, USA). Each experiment was run in quadruplicate for each condition.

Cell proliferation analysis

Weri-Rb1 or Y79 cells were transfected with si-H19 or si-NC for 24 h, and then seeded in 6-well plate with a density of 0.5×103 cells per well and cultured for 7–10 days with RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin (Invitrogen, Thermo Fisher Scientific, Inc.), and 100 mg/ml streptomycin (Invitrogen, Thermo Fisher Scientific, Inc.). The medium was replenished every two days. Colonies were then fixed for 5 min at room temperature with 10% formaldehyde, stained with 1.0% crystal violet for 30 sec at room temperature and counted under a light microscope (Olympus, Tokyo, Japan) in five predetermined fields of interest at ×200 magnification. An MTT assay was performed using the Cell Proliferation kit I (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's protocol. Briefly, 0.5 mg/ml MTT and 100% dimethylsulfoxide reagent were added to the cells at the indicted times. The optical density was measured at 590 nm using the Tecan SpectraFluor Microplate Reader (Tecan Group, Ltd., Mannedorf, Switzerland). Experiments were performed three times.

Cell migration and invasion assays

Cell migration and invasion assays were performed using Transwell insert chambers (Costar; Corning Incorporated, Corning, NY, USA), In brief, Weri-Rb1 or Y79 cells were seeded at a density of 1×104 cells/100 ml RPMI-1640 medium, without FBS, on a fibronectin-coated polycarbonate membrane insert in a Transwell apparatus (Costar; Corning Incorporated) and the lower chamber was filled with 500 µl RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% FBS, 100 U/ml penicillin (Invitrogen, Thermo Fisher Scientific, Inc.), and 100 mg/ml streptomycin (Invitrogen, Thermo Fisher Scientific, Inc.). Cells were incubated for 12 h at 37°C in a humidified atmosphere containing 5% CO2. And then the cells adhering to the lower surface were fixed with 100% methanol for 10 min at room temperature, stained with Giemsa solution at room temperature for 10 min and washed with PBS three times. The cells colonies were then counted under a light microscope (Olympus Corporation, Tokyo, Japan) in five predetermined fields of interest at ×200 magnification. The cell invasion assay was performed as aforementioned, with the exception that the Transwell membranes were precoated with 24 mg/ml Matrigel (R&D Systems, Inc., Minneapolis, MN, USA). Cells were counted as aforementioned. Experimental data are representative of a minimum of three independent repeats.

In vivo growth assay

A total of 40 female nude mice (18–20 g, Balb/C-nu/nu athymic; 4–5 weeks old) were purchased from the Animal Center of the Cancer Institute of Chinese Academy of Medical Science (Beijing, China). The mice were maintained in a facility under specific pathogen-free conditions and an allowed free access to pathogen free laboratory chow, allowed free access to autoclaved water in an air-conditioned room with a 12 h light/dark cycle. Mice were divided into an si-H19 group and an si-NC group (with each group containing 20 mice). Suspensions of 1×107 cells (Y79 cells transfected with siRNA duplexes) were subcutaneously injected into the upper back of the female nude mice. Mice were monitored daily. Tumor samples were carefully removed 21 days after injection and the size was calculated on the basis of width (x) and length (y): x2y/2, where x<y. The humane endpoints for the present study was that the mean diameter of the tumor did not exceed 1.2 cm in mice. Ethical approval for the animal experiments was granted by the Animal Ethical and Welfare Committee of Tianjin Medical University (Tianjin, China).

Western blotting

Total protein was extracted from transfected cells with radioimmunoprecipitation lysis buffer (http://www.keygentec.com.cn/; KenGEN, Nanjing, Jiangsu, China) for 30 min at 4°C, and centrifuged at 12,000 × g for 10 min at 4°C. The supernatant fraction was collected and then quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Protein lysates (30 mg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany) at 30 V overnight. The membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and gentle shaking, and then probed with antibodies against epithelial (E-)cadherin (cat. no. ab15148, dilution 1:1,000; Abcam, Cambridge, UK), MMP9 (cat. no. sc-10737, dilution 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), CDH13 (cat. no. sc-7940, dilution 1:200; Santa Cruz Biotechnology, Inc.), vimentin (cat. no. sc-5565, dilution 1:300; Santa Cruz Biotechnology, Inc.) and GAPDH (cat. no. sc-25778, dilution 1:2,500; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following washing 3 times with TBS 0.1% Tween 20, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat. no. 7074, Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:5,000 for 1 h at room temperature. Band detection was performed using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Statistical analysis

Statistical analyses were performed using the SPSS software package 17.0 (SPSS, Inc., Chicago, IL, USA). Results are expressed as the mean ± standard deviation. Data analysis was performed using Student's t-test (two-tailed) and one-way-analysis of variance followed by Tukey's multiple comparisons test as a post-hoc test. Univariate and multivariate relative risks were calculated using the Cox proportional hazards regression. P<0.05 was considered to indicate a statistically significant difference.

Results

lncRNAH19 is overexpressed in retinoblastoma tissues and cell lines

To explore whether lncRNA H19 had an effect on the malignant phenotype of retinoblastoma, the expression pattern of H19 in retinoblastoma tissues and cell lines was analyzed. The results from the present study indicated that H19 expression was significantly increased in retinoblastoma tissues compared with non-tumor retinal tissues (P<0.001; Fig. 1A). In addition, lncRNA H19 expression was upregulated in Weri-Rb1 and Y79 retinoblastoma cells compared with ARPE-19 and HRMECs (P<0.05; Fig. 1B).

lncRNA H19 is associated with retinoblastoma progression

To investigate the association between lncRNA H19 and retinoblastoma further, the overall survival times of patients with retinoblastoma were analyzed in accordance with H19 expression levels. According to classification by Motoyama et al (23), 80 retinoblastoma tissue samples were divided into the high-expression group (n=38) or the low-expression group (n=42). As presented in Table I, the associations between lncRNA H19 expression and tumor size (<10 vs. ≥10 mm; P=0.007), optic nerve invasion (positive vs. negative; P=0.013) and choroidal invasion (positive vs. negative; P=0.043) were statistically significant. However, no significant associations in age (P=0.263), sex (P=0.750), laterality (P=0.626) or pathological grade (P=0.282) were observed. Overall survival analysis demonstrated that the level of lncRNA H19 expression was significantly associated with the overall survival rate of retinoblastoma patients, as patients with high lncRNA H19 levels had a poorer survival rate compared with those with low lncRNA H19 levels (P<0.001; Fig. 1C). Simultaneously, multivariate analysis was applied to evaluate the association between lncRNA H19 expression and prognostic factors for overall survival rates. The results presented in Table II demonstrated that increased lncRNA H19 expression was a poor independent prognostic factor for patients with retinoblastoma (P<0.040).

Table I.

Association between lncRNA H19 expression and clinicopathological characteristics in retinoblastoma.

Table I.

Association between lncRNA H19 expression and clinicopathological characteristics in retinoblastoma.

lncRNA H19 expression

CharacteristicNumberHighLowP-value
Age, years
  <24022 (55)18 (45)0.263
  ≥24016 (40)24 (60)
Sex
  Male4418 (40.9)26 (59.1)0.750
  Female3616 (44.4)20 (55.6)
Tumor size, mm
  <103311 (33.3)22 (66.7)0.007a
  ≥104730 (63.8)17 (36.2)
Choroidal invasion
  Negative4518 (40)27 (60)0.043a
  Positive3522 (62.9)13 (37.1)
Optic nerve invasion
  Negative4726 (55.3)31 (44.7)0.013a
  Positive4324 (72.7)9 (27.3)
Laterality
  Unilateral5627 (48.2)29 (51.8)0.626
  Bilateral2413 (54.2)11 (45.8)
Pathological grade
  Well-differentiated4324 (55.8)19 (44.2)0.282
  Poorly3725 (67.6)12 (32.4)

a P<0.05.

Table II.

Univariate and multivariate Cox's regression of prognostic factors for overall survival in retinoblastoma.

Table II.

Univariate and multivariate Cox's regression of prognostic factors for overall survival in retinoblastoma.

Univariate analysisMultivariate analysis


CharacteristicHR95% CIP-valueHR95% CIP-value
Age, years
  <2 vs. ≥21.0460.425–2.4330.875
Sex
  Male vs. female1.2040.569–2.3640.640
Size, mm
  <10 vs. ≥105.0122.011–12.040.001a2.010.715–7.1400.249
Choroidal invasion
  Negative vs. positive4.4971.978–10.013 <0.001a1.8480.695–4.8270.213
Optic nerve invasion
  Negative vs. positive4.0981.457–5.980.001a3.1261.321–6.0150.038a
Laterality
  Bilateral vs. unilateral1.6540.871–2.9230.397
Pathological grade
  Well-differentiated vs. poorly0.8180.353–1.8570.270
LncRNA-H19
  Low vs. high5.1242.324–11.532 <0.001a2.9121.021–8.4520.039a

a P<0.05; HR, hazard ratio; CI, confidence interval.

Knockdown of lncRNA H19 decreases retinoblastoma cell proliferation

To elucidate the function of H19 on retinoblastoma cell proliferation, loss-of-function analysis using retroviral transduction of H19 in Weri-Rb1 and Y79 cells was performed. qPCR analysis was performed to confirm that H19 expression was significantly inhibited following knockdown (Fig. 2A). The MTT assay results revealed that the viability of cells (Weri-Rb1 and Y79 cells) transfected with si-H19 was significantly decreased compared with controls, at 48- and 72 h post-transfection (Fig. 2B and C). In addition, the results of the cell colony assay demonstrated that inhibition of H19 expression resulted in decreased cell proliferation in Weri-Rb1 and Y79 cells compared with controls (Fig. 2D).

Knockdown of lncRNA H19 promotes cell apoptosis

As aforementioned, knocking down H19 significantly decreased retinoblastoma cell proliferation, and therefore the subsequent effects on cell cycle and apoptosis were investigated further. In accordance with the aforementioned results, cells transfected with si-H19 displayed an increased proportion of G1 phase cells and a decreased proportion of S phase cells, indicating that silencing H19 may induce G1/S phase arrest in Weri-Rb1 and Y79 cells (Fig. 3A). Furthermore, analysis of cell apoptosis in the si-H19 group was markedly increased compared with si-NC (P<0.05; Fig. 3B). Consistent with these changes, the expression of apoptosis-associated proteins, including cyclin D1, Bax and p53 was decreased in the si-H19 group compared with that in the si-NC group (Fig. 3C), which indicated that H19 may influence cell apoptosis in Weri-Rb1 and Y79 cells.

Knockdown of lncRNAH19 inhibits cells migration and invasion in vitro

The effect of lncRNA H19 on cell migration and invasion in Y79 cells was investigated further. The results of the present study of the Matrigel invasion assay revealed that the number of migratory cells in the si-H19 group was significantly decreased compared with that in the si-NC group (P<0.05; Fig. 4A). As presented in Fig. 4B, the migration of cells transfected with si-H19 was also significantly suppressed in Y79 cells (P<0.05; Fig. 4B). Taken together, these results support the hypothesis that silencing of lncRNA H19 inhibited the migration and invasion of Y79 cells. In addition, the effects of lncRNA H19 on the expression of the migration- and invasion-associated proteins MMP9, CDH13, E-cadherin and vimentin were analyzed. As presented in Fig. 4C, lncRNA H19 downregulation decreased the expression of MMP9, CDH13, vimentin and E-cadherin compared with that in the si-NC group.

Knockdown of lncRNA H19 suppresses the proliferation of retinoblastoma cells in vivo

To validate the effects of lncRNA H19 on the proliferation and invasion of Y79 cells in vivo, the volume of tumors that grew in a nude mice xenograft model, 3 weeks after subcutaneous injection of Y79/si-H19 cells, were analyzed. After 3 weeks, subcutaneous tumors were established in the right groin of 20 nude mice injected with Y79/si-H19 or Y79/si-NC cells. Furthermore, a significant decrease in tumor volume was only observed in the si-H19 treatment group, compared with corresponding controls (Y79/si-NC cells). The results of the present study demonstrated that lncRNA H19 inhibited the proliferation of retinoblastoma cells in vivo.

Discussion

LncRNAs, >200 nucleotides in length, are intergenic transcripts, which, although they do not exhibit a protein-coding function, have been identified to mediate structural and regulatory functions in the development and pathogenesis of cancer (2426). It is well-documented that lncRNAs exert regulatory functions via interactions with target genes, cell cycle regulators, transcription factors or chromatin-modifying complexes, including p53 (27), E2Fs (28), Enhancer of Zeste homolog 2 (29) and sex-determining region y-related high-mobility group-box-2 (30). It has been identified that aberrant lncRNA expression is associated with multiple diverse cancer processes, indicating that lncRNAs may have a marked function in cancer development. A well-documented lncRNA, namely E2F1-regulated inhibitor of cell death (ERIC) was transcriptionally upregulated upon activation, and inhibition of ERIC resulted in increased apoptotic cell death (31).

Previous studies have demonstrated that lncRNA-H19 is involved in the development of cancer pathogenesis and may serve as a potential tumor biomarker. For example, Luo et al (32) observed that the H19 expression pattern was markedly increased in patients with invasive bladder cancer and demonstrated pro-tumorigenic properties. Furthermore, the level of H19 was significantly upregulated in patients with gastric cancer (GC), and the increased expression promoted the differentiation of early-stage GC (33). Shi et al (18) also revealed that H19 expression was associated with tumor grade, and was able to mediate glioma cell invasion by directly regulating CDH13. Consistent with previous studies, the present study demonstrated that H19 levels were markedly increased in retinoblastoma cells and tissues, and may serve a crucial function in retinoblastoma migration and invasion, as a potential novel target for therapeutic strategy and prognostic prediction.

In order to investigate the function of H19 further, an in vivo murine model in which female nude mice were injected with Y79/si-H19 cells was established. At 3 weeks post-injection, mice exhibited subcutaneous tumors and tumor volumes were significantly decreased in the si-H19 treatment group compared with controls, indicating that H19 may be involved in the development of retinoblastoma. Furthermore, the survival rates of patients with retinoblastoma with varying H19 expression levels were determined, and it was revealed that patients with high H19 levels had a poorer survival rate compared with those with low H19 levels. Therefore, the results of the present study indicated that patients with high H19 levels had a shorter survival time compared with patients with low levels. Collectively, the results support the hypothesis that H19 knockdown significantly increased the proliferation and inhibited the apoptosis of retinoblastoma cells.

Previous studies have demonstrated that ectopic expression of H19 partly contributed to increased cell proliferation, cancer migration and invasion by targeting miRNA (18,19). The results of the present study demonstrated that H19 promoted the proliferation, migration and invasion of Weri-Rb1 and Y79 cells, whereas silencing H19 significantly suppressed cell proliferation, migration and invasion in vitro. Furthermore, H19 regulates several proteins, including vimentin, CDK1, p53, CDH13 and E-cadherin. For example, H19 functioned as a competing endogenous RNA for miR-138 and miR-200a, antagonized their functions and led to the de-repression of their endogenous targets vimentin, E-cadherin and CDH13 (18,32,34,35). In the present study, the expression pattern of several corresponding proteins was investigated, and it was revealed that inhibition of H19 repressed MMP9, CDH13, vimentin and E-cadherin expression. However, the precise association between H19 and the corresponding proteins requires further study.

In conclusion, the results of the present study indicated that H19 may serve an oncogenic function in retinoblastoma cells and tissues, suggesting that inhibition of H19 may be a potential target mechanism for retinoblastoma therapy.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Foundation of Premature Retinopathy Genetic Screening and Clinical Screening Study (grant no. 2011KR17).

Availability of data and materials

The data generated in the present study are available from the corresponding author upon reasonable request.

Author's contributions

LL and LST performed the experiment. WC and YCW analyzed the experimental data and patients data. MH designed the experiment and wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Research Ethics Committee of Tianjin Eye Hospital (Tianjin, China) and written informed consent was obtained from all patients.

Consent for publication

Written informed consent was obtained from all patients.

Competing interests

The authors declare that they have no competing interests.

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June-2018
Volume 15 Issue 6

Print ISSN: 1792-1074
Online ISSN:1792-1082

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Spandidos Publications style
Li L, Chen W, Wang Y, Tang L and Han M: Long non‑coding RNA H19 regulates viability and metastasis, and is upregulated in retinoblastoma. Oncol Lett 15: 8424-8432, 2018
APA
Li, L., Chen, W., Wang, Y., Tang, L., & Han, M. (2018). Long non‑coding RNA H19 regulates viability and metastasis, and is upregulated in retinoblastoma. Oncology Letters, 15, 8424-8432. https://doi.org/10.3892/ol.2018.8385
MLA
Li, L., Chen, W., Wang, Y., Tang, L., Han, M."Long non‑coding RNA H19 regulates viability and metastasis, and is upregulated in retinoblastoma". Oncology Letters 15.6 (2018): 8424-8432.
Chicago
Li, L., Chen, W., Wang, Y., Tang, L., Han, M."Long non‑coding RNA H19 regulates viability and metastasis, and is upregulated in retinoblastoma". Oncology Letters 15, no. 6 (2018): 8424-8432. https://doi.org/10.3892/ol.2018.8385