Expression of Long Non-Coding RNA H19 in Acute Lymphoblastic Leukemia

Abstract

OObjective: Long non-coding RNA (lncRNA) H19 has essential roles in growth, migration, invasion, and metastasis of most cancers. H19 dysregulation is present in a large number of solid tumors and leukemia. However, the expression level of H19 in acute lymphoblastic leukemia (ALL) has not been elucidated yet. The current study aimed to explore H19 expression in ALL patients and cell lines.

Materials and methods: This experimental study was conducted in bone marrow (BM) samples collected from 25 patients with newly diagnosed ALL. In addition, we cultured the RPMI-8402, Jurkat, Ramos, and Daudi cell lines and assessed the effects of internal (hypoxia) and external (chemotherapy medications L-asparaginase [ASP] and vincristine [VCR]) factors on h19 expression. The expressions of H19, P53, c-Myc, HIF-1α and β-actin were performed using quantitative real-time polymerase chain reaction (qRT-PCR) method.

Results: There was significantly increased H19 expression in the B-cell ALL (B-ALL, P<0.05), T-cell ALL (T-ALL, P<0.01) patients and the cell lines. This upregulation was governed by the P53, HIF-1α, and c-Myc transcription factors. We observed that increased c-Myc expression induced H19 expression; however, P53 adversely affected H19 expression. In addition, the results indicated that chemotherapy changed the gene expression pattern. There was a considerable decrease in H19 expression after exposure to chemotherapy medications; nonetheless, hypoxia induced H19 expression through P53 downregulation.

Conclusion: Our findings suggest that H19 may have an important role in pathogenesis in ALL and may act as a promising and potential therapeutic target.

Keywords: Acute lymphoblastic leukemia; H19; Hypoxia; lncRNA.

Fig 1

Gene expression in patients with ALL. All gene expressions were detected by quantitative real time PCR. T and B cells were extracted from B-ALL (n=15) and T-ALL (n=10) patients and compared with healthy donors (n=20) as the control group. A. H19 expression in ALL patients and control group. B. HIF-1α, C. P53, and D. c-Myc expression levels in patient samples. Representative experiments performed in triplicate. The graphs demonstrate the standard deviation (SD). ALL; Acute lymphoblastic leukemia, B-ALL; B-cell acute lymphoblastic leukemia, T-ALL; T-cell acute lymphoblastic leukemia, PCR; Polymerase chain reaction, ns; Not significant, *; P<0.05, **; P<0.01, and ***; P<0.001.

Fig 2

H19 expression in ALL cell lines. Gene expression was assessed by q-RT PCR. A, B. H19 expression was analyzed in both B and T ALL cell lines and compared to normal B and T cells, respectively. C, D. Analysis of H19 expression in cell lines after 24 hours of exposure to hypoxic stress. E, F. Expression of H19 was evaluated after treatment with VCR and ASP. The data are from three independent experiments, each done in triplicate. ALL; Acute lymphoblastic leukemia, q-RT PCR; Quantitative real-time polymerase chain reaction, VCR; Vincristine, ASP; L-asparaginase, ns; Not significant, *; P<0.05, and **; P<0.01.

Fig 3

The effects of various doses of VCR and ASP on viability of ALL cell lines. A. MTT test was performed to investigate the survival of ALL cell lines at 48 hours after treatment with VCR and ASP. B. Staining with Annexin-V and PI assessed apoptosis. The histogram demonstrates the ratio of apoptotic or necrotic cells. Annexin V-/PI-, Annexin V+/PI-, Annexin V+, and PI+ represented live cells, early apoptotic cells, late apoptotic cells, necrotic cells, respectively. The data are from two independent experiments, each done in duplicate. VCR; Vincristine, ASP; L-asparaginase, ALL; Acute lymphoblastic leukemia, MTT; Dimethylthiazol diphenyl tetrazolium bromide, VCR; Vincristine, ASP; L-asparaginase, and PI; Propidium iodide.

Fig 4

Expression of H19-related genes in the ALL cell lines. Gene expression was carried out through quantitative real-time PCR and the treated cell lines were compared with untreated controls. A-C. Expression of c-Myc was evaluated in B- and T- ALL cell lines and its relative expression was measured after a 48 hours of treatment with VIN and ASP. D-F. The relative expression levels of P53 was measured in ALL cell lines and treated ones and compared to control group. The data are cultures from three independent experiments, each done in duplicate. ALL; Acute lymphoblastic leukemia, PCR; Polymerase chain reaction, VCR; Vincristine, ASP; L-asparaginase, ns; Not significant, *; P<0.05, **; P<0.01, and ***; P<0.001.

Fig 5

Expression of c-Myc and P53 genes in the ALL cell lines after hypoxic condition. A, B. Expression of HIF-1a was evaluated by qRT-PCR after the cell lines were put into hypoxic condition for 24 hours. C, D. Expression of P53 was evaluated after exposure to the hypoxic condition in comparison to normoxic culture conditions. The data are cultures from three independent experiments, each done in triplicate. ALL; Acute lymphoblastic leukemia, qRTPCR; Quantitative real time polymerase chain reaction, *; P<0.05, and **; P<0.00.