Although a few chemotherapeutic drugs were applied in AML treatment, aged AML patients still suffer from poor prognosis and low long-term overall survival rate . AML occurrence and development are affected by multiple factors, such as radiation, chemical degradation, viral infection, and genes’ susceptibility . In recent years, increasing evidence revealed that lncRNAs play vital biological functions in tumorigenesis [19, 20]. For example, Fang et al. found that XIST promotes cell proliferation, migration, and invasion through epigenetically suppressing the KLF2 family of proteins . Chen et al. revealed that CCAT1 acts as a molecular sponge of miR-181a-5p, regulates HOXA1 expression and promotes multiple myeloma progression . Therefore, it is imperative to explore the potential mechanism of lncRNAs in hematological malignancies.
Emerging evidence showed that CRNDE regulates cancer progression [23–26]. For example, Zheng et al. confirmed that CRNDE is the most upregulated lncRNA in glioma, where it promotes its progression by inhibiting the miR-384/PIWIL4/STAT3 axis . Xu et al. revealed that CRNDE promotes melanoma migration and invasion via the miR-205/CCL18 axis . CRNDE expression has been shown to be upregulated in the bone marrow tissues of AML patients and negatively correlated patients’ overall survival . However, the underlying mechanisms of CRNDE in AML remain unexplored. In this study, we confirmed that the expression of CRNDE was significantly upregulated in AML samples and cell lines. CRNDE promoted AML cells’ proliferation ability, decreases AML cells’ apoptotic rate and arrested AML cells at the G1 phase.
The hypothesis of ceRNA was first presented by Salmena et al., and explained how messenger RNAs, transcribed pseudogenes, and long non-coding RNAs communicate with each other using microRNA response elements (MREs). They proposed that the “competing endogenous RNA” (ceRNA) could bind and negatively regulate the target . Moreover, the function of lncRNAs was found to be closely related to its subcellular location . In this study, the lncLocator that predicts lncRNAs subcellular localizations was used to predict the location of CRNDE . We predicted that CRNDE was mainly located in the cytosol where it potentially acts as a ceRNA. Indeed, functional, and mechanistic experiments showed that CRNDE serves as a ceRNA for miR-136-5p and that it upregulates MCM5 expression by sponging miR-136-5p.
MiRNAs are a class of small single-stranded RNAs with a length of 18 to 25 nucleotides. Moreover, miRNAs were confirmed to be involved in cell growth, cell apoptosis, hematopoietic lineage differentiation and cell death [31–33]. Chen et al. revealed that miR-136-5p could inhibit cell growth, migration, and invasion of renal cell carcinoma cells . Gao et al. demonstrated that miR-136-5p may play an important role in regulating TC tumorigenesis via targeting MTDH . In the present study, the results of the bioinformatics software Starbase V2.0 showed that binding sites exist between CRNDE and miR-136-5p, which was proved by luciferase reporter assays. In addition, rescue assays also showed that CRNDE indeed influences MCM5 via acting on miR-136-5p.
Subsequently, we further explored the downstream target genes using the bioinformatics’ software Targetscan 7.0 and filtered out MCM5 as a miR-136-5p direct target. Minichromosome maintenance protein 5 (MCM5) is a member of the minichromosome maintenance (MCM) protein family, that is located on chromosome 22q13.1 . MCM proteins were first identified in the yeast Saccharomyces cerevisiae as mutants defective in the maintenance of minichromosomes, indicating their role in plasmid replication and cell cycle progression . MCM5 was well documented to play a role in tumorigenesis and cellular processes in various cancers. For example, Gong et al. revealed that MCM5 could be related to the prognosis and progression of renal cell carcinoma . Guida et al. found that targeting MCM proteins could be a used as a therapy for anaplastic thyroid carcinomas by disturbing their expression or obstructing their helicase function . It has also been proven that MCM5 had could be used a prognostic marker in various cancers [40–45]. In addition, MCM5 was confirmed to be overexpressed in AML by boutique microarrays, real-time PCR, and droplet digital PCR . In our study, we also verified that MCM5 was upregulated in AML and associated with poor prognostic. Luciferase reporter assays showed that MCM5 was a downstream target of miR-136-5p, and that MCM5 expression was downregulated when CRNDE was knocked down or when miR-136-5p was overexpressed.
In the present study, we demonstrated the potential mechanistic role of CRNDE in AML; however, there are still some limitations associated with the study. Firstly, we did not elucidate the cause of CRNDE upregulation in AML, which is necessary for a better understanding of CRNDE mechanism of action. Secondly, our research was performed at the cellular level and it is therefore, important to study its effect in vivo and the future.