Elsevier

Biomedicine & Pharmacotherapy

Volume 104, August 2018, Pages 181-192
Biomedicine & Pharmacotherapy

Long noncoding RNA MEG3 inhibits proliferation of chronic myeloid leukemia cells by sponging microRNA21

https://doi.org/10.1016/j.biopha.2018.05.047Get rights and content

Highlights

  • LncRNA MEG3 was downregulated and miR-21 was upregulated in advanced-stage CML.

  • MEG3 overexpression and knockdown of miR-21 inhibited proliferation and promoted apoptosis.

  • LncRNA MEG3 and miR21 may serve as novel therapeutic targets for CML blast crisis; demethylation drugs might also have potential clinical applications.

Abstract

The long noncoding RNA (lnc) maternally expressed 3 (MEG3) is downregulated in many types of cancers. However, the relationship between lncRNA MEG3, microRNA-21 (miR-21) and chronic myeloid leukemia (CML) blast crisis is unknown. This study examined bone marrow samples from 40 CML patients and 10 healthy donors. Proliferation and apoptosis assays, real-time polymerase chain reaction (PCR), bisulfite sequencing PCR, Western blotting, luciferase assay, RNA pull-down, RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP) and Chromatin immunoprecipitation (ChIP) were performed. We found that MEG3 and PTEN expression were down-regulated, whereas, MDM2, DNMT1 and miR-21 were up-regulated in the accelerated and blast phases of CML. Treated with 5-azacytidine decreased the level of MDM2, DNMT1 and miR21, but increased the level of MEG3 and PTEN. Overexpression of MEG3 and silencing the expression of miR-21 inhibited proliferation and induced apoptosis. MEG3 overexpression and silencing the expression of miR21 influence the levels of MMP-2, MMP-9, bcl-2 and Bax. MEG3 was able to interact with MDM2 and EZH2. MDM2 could interact with DNMT1 and PTEN. MYC and AKT can interact with EZH2. ChIP-seq showed that the promoter of KLF4 and SFRP2 interacts with DNMT1. In conclusion, lncRNA MEG3 and its target miR21 may serve as novel therapeutic targets for CML blast crisis; and demethylation drugs might also have potential clinical application in treating CML blast crisis.

Introduction

Chronic myeloid leukemia (CML) is a hematological malignancy that is characterized by the Philadelphia chromosome, which forms the breakpoint cluster region (BCR)/Abelson murine leukemia (ABL) fusion gene, which encodes the P210 BCR/ABL1 protein [1,2]. Key pathways are associated with CML [3]. Typically, CML patients pass through three phases: a chronic phase (CP), an accelerated phase (AP) and a blast phase (BP) [4]. Although tyrosine kinase inhibitors (TKIs) treatment can improve the 5-year survival rate of CML patients, patients who are resistant to TKIs or who receive no drug treatment will eventually progress to the advanced phases (AP and BP) in approximately 3–5 years. Unfortunately, even if these patients receive standard chemotherapy regimens, their prognosis remains poor [5]. Thus, it is important to determine the underlying molecular and biological mechanism of CML blast crisis and important to find novel treatment targets for CML blast crisis.

Long noncoding RNAs (lncRNAs) have been reported to be key regulators of important biological processes that play roles in specific cancers and in other diseases [6]. Noncoding RNAs (ncRNAs) are generally classified into two groups according to their length, namely, small ncRNAs (sncRNAs) and lncRNAs. SncRNAs are less than 200 nt long, and lncRNAs are transcripts that are longer than 200 nucleotides; neither has protein-coding functions [7,8]. LncRNAs are differentially expressed in solid tumors and hematopoietic tumors [9,10]. LncRNA maternally expressed 3 (MEG3) is associated with many cancers, such as colorectal cancer [11], ovarian cancer [12], and hepatocellular carcinoma [13], MEG3 is involved in imatinib resistance in CML and possibly contributes to imatinib resistance by regulating miR-21 [14], however, the relationship between MEG3 and the molecular biological mechanism underlying the blastic transformation of CML is still not clear.

Epigenetics involves heritable changes in gene expression, such as DNA methylation and chromatin conformation, without any corresponding changes in the DNA sequence [14,15]. Abnormal DNA promoters are reported to be involved in tumorigenesis. MEG3 methylation has been reported to be associated with many types of cancer, such as ovarian cancers and meningiomas [16,17]; however, the relationship between MEG3 methylation and the blastic transformation of CML remains largely unknown. Studies of the mode of action of lncRNAs have shown that a number of lncRNAs can interact with chromatin to regulate gene expression. MEG3 has been reported to interact with polycomb repressive complex 2(PRC2) in many cancer cells lines [18]. The proteins that bind to MEG3 in CML crisis cell lines are unknown.

Similar to lncRNAs, microRNAs (miRNAs) do not encode proteins, but are less than 200 nt in length. MicroRNAs are small, endogenous and noncoding RNA that contribute to the regulation of essential cellular processes, such as proliferation, apoptosis, development, and differentiation [19]. Recent studies have shown that miRNAs are involved in tumorigenesis and function as tumor suppressor genes or oncogenes [20,21]. MiR-21 involved in the progression of lung cancer and may be a novel therapeutic target for the disease [22]. However, the role of miR-21 in the progression of CML is largely unknown.

Section snippets

Specimen collection

Bone marrow samples were collected from 40 CML patients, who were admitted to the Department of Hematology of the Second Hospital of Hebei Medical University between May 2015 and June 2017 (Table 1). Bone marrow samples from 10 healthy donors were selected to serve as controls. Bone marrow mononuclear cells were isolated via lymphocyte separation. This study was approved by the Ethics Committee of the Department of Hematology of the Second Hospital of Hebei Medical University, and each patient

The mRNA expression levels in different phases of CML and healthy donors

MEG3, MDM2, DNMT1, PTEN and miR-21 mRNA levels in patients in different phases of CML and in healthy donors were detected using RT-PCR. The results revealed that MEG3 and PTEN mRNA expression was lower in AP and BP of CML patients than in chronic-phase CML patients and healthy donors (Fig. 1A; P < 0.05). The expression of DNMT1, MDM2 and miR21 was higher in AP and BP of CML patients as compare to that of chronic-phase CML patients and healthy donors (Fig. 1A; P < 0.05).

Protein levels of DNMT1,

Discussion

In this study, we examined the expression of MEG3, miR-21, DNMT1, MDM2 and PTEN in the three phases of CML. We found that lower levels of MEG3 and PTEN and higher levels of miR-21, DNMT1 and MDM2 were found in the advanced stage of CML. Both overexpression MEG3 and silencing miR-21 inhibited proliferation and promoted apoptosis. EZH2 and MDM2 regulated MEG3, DNMT1 and PTEN might regulate MDM2, MYC and AKT might regulate EZH2.ChIP-seq indicated that the promoter of KLF4 and SFRP2 interacts with

Author contributions

ZY L, L Y, XJ L and JM L designed the experiment. ZY N collected the specimens. ZY L performed the experiments and wrote the paper.

Funding

This study was founded by Natural Science Fund of Hebei Province.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgment

None.

References (28)

  • R. Hehlmann et al.

    Chronic myeloid leukemia: a model for oncology

    Ann. Hematol.

    (2005)
  • Y. Chen et al.

    Critical molecular pathways in cancer stem cells of chronic myeloid leukemia

    Leukemia

    (2010)
  • K. Tano et al.

    Long non-coding RNAs in cancer progression

    Front. Genet.

    (2012)
  • J.R. Alvarez-Dominguez et al.

    Long noncoding RNAs during normal and malignant hematopoiesis

    Int. J. Hematol.

    (2014)
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