Background As a common haematological malignancy, acute myeloid leukaemia (AML), particularly with extramedullary infiltration (EMI), often results in a high mortality rate and poor prognosis. Circular RNAs (circRNAs) regulate biological and pathogenic processes, suggesting a potential role in AML. We have previously described the overall alterations in circRNAs and their regulatory networks between patients with AML presenting with and without EMI. This study aims to find new prognostic and therapeutic targets potentially associated with AML. Methods qRT-PCR was performed on samples from 40 patients with AML and 15 healthy controls. The possibility of using circPLXNB2 (circRNA derived from PLXNB2) as a diagnostic and prognostic biomarker for AML was analysed with multiple statistical methods. In vitro, the function of circPLXNB2 was studied by lentivirus transfection, CCK-8 assays, flow cytometry, and Transwell experiments. Western blotting and qRT-PCR were performed to detect the expression of related proteins and genes. The distribution of circPLXNB2 in cells was observed using RNA fluorescence in situ hybridization (RNA-FISH). We also investigated the role of circPLXNB2 by establishing AML xenograft models in NOD/SCID mice. Results By analysing the results of qRT-PCR detection of clinical samples, the expression of the circPLXNB2 and PLXNB2 mRNAs were significantly increased in patients with AML, more specifically in patients with AML presenting with EMI. High circPLXNB2 expression was associated with an obviously shorter overall survival and leukaemia-free survival of patients with AML. The circPLXNB2 expression was positively correlated with PLXNB2 mRNA expression, as evidenced by Pearson’s correlation analysis. RNA-FISH revealed that circPLXNB2 is mainly located in the nucleus. In vitro and in vivo, circPLXNB2 promoted cell proliferation and migration and inhibited apoptosis. Notably, circPLXNB2 also increased the expression of PLXNB2, BCL2 and cyclin D1, and reduced the expression of BAX. Conclusion In summary, we validated the high expression of circPLXNB2 and PLXNB2 in patients with AML. Elevated circPLXNB2 levels were associated with poor clinical outcomes in patients with AML. Importantly, circPLXNB2 accelerated tumour growth and progression, possibly by regulating PLXNB2 expression. Our study highlights the potential of circPLXNB2 as a new prognostic predictor and therapeutic target for AML in the future.

SummaryAlternative splicing is associated with human disease. In acute promyelocytic leukaemia (APL) patients with the long (L)-type promyelocytic leukaemia-retinoic acid receptor a fusion gene (PML-RARA), three alternative splicing isoforms can be detected: E5(+)E6(+), E5(À)E6(+), and E5(À)E6(À). This study is the first to demonstrate that alternative splicing of L-type PML-RARA is associated with time to achieve complete remission (CR) in APL. Higher expression of the E5(À)E6(À) isoform, the short isoform, was related to longer time to achieve CR. Each isoform was constructed into recombinant lentiviral vector and transfected into U937 cells. Compared with the E5(À)E6(+) and E5(+)E6(+) groups, the U937 cells with E5(À) E6(À) showed lower sensitivity to all-trans-retinoic acid treatment.

Pyroptosis is an important factor affecting the proliferation, invasion, and metastasis of tumor cells. However, in multiple myeloma (MM), there are few studies on whether the occurrence of pyroptosis is related to the occurrence and prognosis of the disease. Based on the Gene Expression Omnibus and Cancer Genome Atlas database search dataset, this study identified pyroptosis-related genes with a specific prognosis, constructed and verified the prediction model by stepwise Cox regression analysis and time receiver operating characteristic curve analysis, and predicted specific functions by single-sample gene set enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes. Dataset analysis identified key genes, which were used to construct a risk scoring system for the prognosis of MM. The entire test set and external verification set verified the results. The expression levels of related genes in the clinical samples were detected using fluorescence quantitative PCR. A prognostic gene model based on six pyroptosis-related genes (CYCS, NLRP9, AIM2, NOD2, CHMP3, and GSDME) was constructed. The model has an excellent prognostic ability and can be popularized in the external validation set. The predictive prognostic nomogram integrating clinical information can effectively evaluate the risk score of each patient and predict their survival. After sample validation, our study found three potential key pyroptosis-related genes in multiple myeloma. GSDME, NOD2, and CHMP3 were significantly different between MM and healthy subjects, suggesting that they are pyroptosis-related protective genes. This study shows that the key pyroptosis-related gene in the model can be used as a marker for predicting the prognosis of myeloma, which may provide a basis for clinical individualized stratification therapy.

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outwardfacing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription. [BMB Reports 2013; 46(3): 163-168]

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BackgroundHypercalcemia induced by multiple myeloma (MM) affects the biological functions of excitable and non-excitable cells. However, red blood cells (RBCs) regulatory effect on calcium in hypercalcemia is still not fully understood.MethodsA total of 113 patients with MM osteolytic lesions were studied retrospectively. Flow cytometry and atomic absorption spectroscopy were used to detect calcium content. Immunofluorescence and Western blotting were used to investigate protein expression. GEO and miRNA databases were used to screen miRNAs. Exosomal miR-4261 migration was investigated by Transwell assay. Dual-luciferase assays confirmed the targeting relationship between miR-4261 and ATP2B4. An RBC oxidative stress model was constructed, and Omega-Agatoxin IVA was used to study the role of plasma membrane Ca2+-ATPase 4 (PMCA4) in RBCs.ResultsThe results showed that MM RBCs had calcium overload, and serum calcium levels increased as the number of RBCs decreased. The expression of PMCA4 in MM RBCs was significantly lower than in normal RBCs. The exosomal miR-4261 produced by MM cells could be transferred to RBCs to downregulate the expression of ATP2B4.ConclusionsStudies have confirmed that RBCs experience calcium overload in MM with osteolytic lesions, which is related to the downregulation of ATP2B4 by MM exosomal miR-4261.

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