The role of the novel LincRNA uc002jit.1 in NF-kB-mediated DNA damage repair in acute myeloid leukemia cells

https://doi.org/10.1016/j.yexcr.2020.111985Get rights and content

Highlights

  • The novel lincRNA uc002jit.1(D43770) is a target gene of RELA.

  • uc002jit.1 affects the stability of PARP1 mRNA, thus regulating DNA damage repair in AML cells.

  • uc002jit.1 has significant biological functions in cell proliferation and sensitivity to chemotherapeutic drugs.

Abstract

The roles and therapeutic potential of long noncoding RNAs (lncRNAs) in acute myeloid leukemia (AML) have attracted increased attention. However, many lncRNAs have not been annotated in AML, and their predictive value for AML therapy remains unclear. In this study, we identified a novel large intergenic noncoding RNA uc002jit.1 (D43770) from a lncRNA microarray. We first proved uc002jit.1 is a target gene of nuclear factor kappa B/RELA, RELA regulated uc002jit.1 transcription by binding to its promoter. Additionally, uc002jit.1 knockdown impaired the stability of poly (ADP-ribose) polymerase 1 (PARP1) mRNA, and then reduced PARP1 protein content and PARylation level upon DNA damage, thus inhibiting DNA damage repair in AML cells. Moreover, uc002jit.1 knockdown significantly inhibited AML cells proliferation and increased the sensitivity to chemotherapeutic drugs in vitro as well as in a mouse model in vivo. Overall, our study indicated that uc002jit.1 may be associated with the occurrence and prognosis of AML and could be a new diagnostic/prognostic biomarker and therapeutic target for AML.

Introduction

Long noncoding RNAs (lncRNAs) are arbitrarily defined as transcripts longer than 200 nt that possess no protein-coding capacity [1]. LncRNAs participate in almost all important regulatory processes by acting as guides [2], scaffolds [3], decoys [4], or signals [5], and their expression or dysfunction is also closely related to the prognosis, development and occurrence of disease [6]. However, due to the diversity and complexity of their mechanisms, there are still many unknowns that need to be further explored.

Acute myeloid leukemia (AML) is an aggressive malignant tumor, the drug resistance and recurrence remain prominent problems [7]. A number of individual lncRNAs, such as IRAIN, ZNF571-AS1, UCA1 are reported to be associated with risk stratification and clinic pathological features in AML [8]. However, there are still a large number of lncRNAs that have not been annotated in AML, and their predictive value for AML therapy remains unclear.

Most chemical drug treatments in the clinic achieve therapeutic effects by damaging tumor cell DNA [9]. Abnormal activation of the DNA repair ability of tumor cells is an important factor causing drug resistance in treatment involving DNA damaging agents [10,11]. Studies have shown that DNA damage induces obvious changes in lncRNA transcription, and these altered lncRNAs, such as lincRNA-p21, are involved in DNA damage repair [12]. LncRNAs can also affect the expression of cis or trans genes by binding to chromatin modifiers, protein complexes or other RNAs, thus serving as regulators in the DNA damage repair process [13,14]. Daunorubicin (DNR) is the main component of induction therapy for AML. We and others have shown that DNR exerts its efficacy by inducing tumor cell DNA damage and apoptosis, but also activates nuclear factor-kappaB (NF-κB) to promote tumor cell survival, thereby reducing drug efficacy and even producing resistance [15,16].

NF-κB is a transcription factor with multidirectional regulatory functions that is widely distributed in eukaryotic cells [17,18]. Stilmann M and others have shown that when tumor cell DNA is damaged, PARP1 senses DNA damage and recruits related proteins through PARylation to activate NF-κB [19]. Poly (ADP-ribose) polymerase 1 (PARP1) is a ribosyl transferase enzyme that is closely related to DNA repair and is involved in multiple DNA repair pathways, such as base excision repair (BER) [20], homologous recombination (HR) [21] and nonhomologous end joining (NHEJ) [22]. Our previous study showed that RELA, an important subunit of NF-κB, regulates DNA repair by binding to the PARP1 promoter and affecting PARP1 gene transcription. RELA knockdown or NF-κB inhibition reduced PARylation in DNR-damaged AML cells, conversely, PARP1 knockdown reduced NF-κB activity, indicating that RELA and PARP1 create a positive feedback loop in DNA repair. The effect of the dual inhibition of RELA and PARP1 was amplified by the positive feedback loop, resulting in a more significant DNA damage and cell proliferation inhibition effect of DNR [16].

Since NF-κB plays an important role in DNA repair, we knocked down RELA in KG1α cells and then analyzed whether lncRNAs play roles in DNA repair mediated by NF-κB, we found that a total of 748 lncRNAs were differentially expressed. However, how these lncRNAs participate in NF-κB-mediated DNA repair is unknown. Therefore, this study aimed to further explore the precise molecular mechanism of lncRNAs in NF-κB-mediated DNA repair, seeking a new prognostic biomarker and treatment strategy for AML.

Section snippets

Cell lines and reagents

KG1α, Kasumi-1, and HEK293 cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA), where they were characterized by DNA fingerprinting, mycoplasma detection, and cell vitality detection. These cell lines were immediately expanded and frozen. KG1α and Kasumi-1 were cultured in RPMI 1640 medium, HEK293 was cultured in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin.

uc002jit.1 plays a role in DNA repair regulated by RELA in AML cells

We used a lncRNA microarray to evaluate the impact of RELA knockdown on lincRNA expression in shCtrl and shRELA KG1α cells upon DNA damage, the results showed that a total of 748 lncRNAs were differentially expressed (fold change ≥ 1.5, P value < 0.05) (Fig. 1A). Of these lncRNAs, 551 were upregulated, and 197 were downregulated. The results of CNC network analysis showed that a total of 26 lncRNAs were positively co-expressed with RELA, 19 lncRNAs were positively co-expressed with PARP1, 11 of

Discussion

LncRNAs represent an interesting subgroup of noncoding RNAs that have been found to play powerful roles in various cancers, but most noncoding RNAs have unknown functions. They are generally classified into five categories: antisense lncRNA, intron transcript lncRNA, large intergenic noncoding RNA (lincRNA), promoter-related lncRNA, and untranslated region-related lncRNA [32]. uc002jit.1 (D43770) is a UCSC_known lincRNA, which has not been reported. We searched databases and confirmed that

Conclusions

In conclusion, we provide a new biomarker for AML diagnosis/prognosis and may pave the way for novel therapeutic interventions targeting the noncoding transcriptome.

CRediT authorship contribution statement

Ding Li: Conceptualization, Methodology, Software, Writing - original draft, Visualization, Data curation, Investigation, Formal analysis. Zelei Yu: Software, Visualization, Data curation, Investigation, Methodology. Tingting Wang: Methodology, Software, Validation. Yi Li: Methodology, Investigation, Formal analysis. Xianling Chen: Software, Resources. Lixian Wu: Investigation, Writing - review & editing, Project administration, Funding acquisition.

Declaration of competing interest

The authors consider that there are no actual or perceived conflicts of interest.

Acknowledgments

We are grateful to Mr. Jun-jin Lin and Zhi-hong Huang (Fujian Medical University, Fuzhou, China) for their excellent technical assistance. This work was supported by grants from the National Natural Science Foundation of China (81872898), Joint Funds for the Innovation of Science and Technology, Fujian Province (2016Y9057), Joint Research Program of Health and Education of Fujian Province (WKJ2016-2-33), Natural Science Foundation of Fujian Province (2018J01842 and 2017J01823), Startup Fund for

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    Ding Li and Zelei Yu contributed equally to this article.

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