Qiubai Li

MicroRNA and transcription factor co-regulatory network analysis reveals miR-19 inhibits CYLD in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy. The understanding of its gene expression regulation and molecular mechanisms still remains elusive. Started from experimentally verified T-ALL-related miRNAs and genes, we obtained 120 feed-forward loops (FFLs) among T-ALL-related genes, miRNAs and TFs through combining target prediction. Afterwards, a T-ALL miRNA and TF co-regulatory network was constructed, and its significance was tested by statistical methods. Four miRNAs in the miR-17–92 cluster and four important genes (CYLD, HOXA9, BCL2L11 and RUNX1) were found as hubs in the network. Particularly, we found that miR-19 was highly expressed in T-ALL patients and cell lines. Ectopic expression of miR-19 represses CYLD expression, while miR-19 inhibitor treatment induces CYLD protein expression and decreases NF-κB expression in the downstream signaling pathway. Thus, miR-19, CYLD and NF-κB form a regulatory FFL, which provides new clues for sustained activation of NF-κB in T-ALL. Taken together, we provided the first miRNA-TF co-regulatory network in T-ALL and proposed a model to demonstrate the roles of miR-19 and CYLD in the T-cell leukemogenesis. This study may provide potential therapeutic targets for T-ALL and shed light on combining bioinformatics with experiments in the research of complex diseases.

miR-146b-5p within BCR-ABL1–Positive Microvesicles Promotes Leukemic Transformation of Hematopoietic Cells

Evidence is accumulating that extracellular microvesicles (MV) facilitate progression and relapse in cancer. Using a model in which MVs derived from K562 chronic myelogenous leukemia (CML) cells transform normal hematopoietic transplants into leukemia-like cells, we defined the underlying mechanisms of this process through gene-expression studies and network analyses of transcription factors (TF) and miRNAs. We found that antitumor miRNAs were increased and several defense pathways were initiated during the early phases of oncogenic transformation. Later, oncomiRs and genes involved in cell cycle, DNA repair, and energy metabolism pathways were upregulated. Regulatory network analyses revealed that a number of TFs and miRNAs were responsible for the pathway dysregulation and the oncogenic transformation. In particular, we found that miR-146b-5p, which was highly expressed in MVs, coordinated the regulation of cancer-related genes to promote cell-transforming processes. Notably, treatment of recipient cells with MV derived from K562 cells expressing mimics of miR-146b-5p revealed that it accelerated the transformation process in large part by silencing the tumor-suppressor NUMB High levels of miR-146b-5p also enhanced reactive oxygen species levels and genome instability of recipient cells. Taken together, our finding showed how upregulation of oncogenic miRNAs in MVs promote hematopoetic cells to a leukemic state, as well as a demonstration for TF and miRNA coregulatory analysis in exploring the dysregulation of cancers and discovering key factors. Cancer Res; 76(10); 2901-11. ©2016 AACR.

Transcription factor and miRNA co-regulatory network reveals shared and specific regulators in the development of B cell and T cell

The maturation process of lymphocyte was related to many blood diseases, such as lymphoma and lymphoid leukemia. Many TFs and miRNAs were separately studied in the development of B and T cells. In this study, we aim to discover the TF and miRNA co-regulation and identify key regulators in the B and T cells maturation. We obtained the candidate genes, miRNAs and TFs for each stage of their maturation, then constructed the TF-miRNA-gene feed-forward loops (FFLs) for each stage by our previous methods. Statistical test for FFLs indicated their enrichment and significance. TF-miRNA co-regulatory networks for each stage were constructed by combining their FFLs. Hub analysis revealed the key regulators in each stage, for example, MYC, STAT5A, PAX5 and miR-17u2009~u200992 in the transition of pro-B cells into pre-B cells. We also identified a few common regulators and modules in two stages of B cell maturation (e.g. miR-146a/NFKB1/BCL11A) and two stages of T cell maturation (e.g. miR-20/CCND2/SORL1), as well as some shared regulators in the early stages of both B and T cell development. Our network will help to increase understanding of mature process of B and T cell, as well as the related blood diseases.

MicroRNA regulatory pathway analysis identifies miR-142-5p as a negative regulator of TGF-β pathway via targeting SMAD3.

MicroRNAs (miRNAs) are non-coding RNAs with functions of posttranscriptional regulation. The abnormally expressed miRNAs have been shown to be crucial contributors and may serve as biomarkers in many diseases. However, determining the biological function of miRNAs is an ongoing challenge. By combining miRNA targets prediction, miRNA and mRNA expression profiles in TCGA cancers, and pathway data, we performed a miRNA-pathway regulation inference by Fishers exact test for enrichment analysis. Then we constructed a database to show the cancer related miRNA-pathway regulatory network (http://bioinfo.life.hust.edu.cn/miR_path). As one of the miRNAs targeting many cancer related pathways, miR-142-5p potentially regulates the maximum number of genes in TGF-β signaling pathway. We experimentally confirmed that miR-142-5p directly targeted and suppressed SMAD3, a key component in TGF-β signaling. Ectopic overexpression of miR-142-5p significantly promoted tumor cell proliferation and inhibited apoptosis, while silencing of miR-142-5p inhibited the tumor cell proliferation and promoted apoptosis in vitro. These findings indicate that miR-142-5p plays as a negative regulator in TGF-β pathway by targeting SMAD3 and suppresses TGF-β-induced growth inhibition in cancer cells. Our study proved the feasibility of miRNA regulatory pathway analysis and shed light on combining bioinformatics with experiments in the research of complex diseases.

Prognostic Significance of KIT Mutations in Core-Binding Factor Acute Myeloid Leukemia: A Systematic Review and Meta-Analysis

The prognostic significance of KIT mutations in core-binding factor acute myeloid leukemia (CBF-AML), including inv(16) and t(8;21) AML, is uncertain. We performed a systematic review and meta-analysis of the effect of KIT mutations on the complete remission (CR) and relapse rates and overall survival (OS) of CBF-AML. PubMed, Embase, Web of Science, and the Cochrane Library were searched and relevant studies were included. Negative effect was indicated on relapse risk of CBF-AML (RR [relative risk], 1.43; 95%CI [confidence interval], 1.20–1.70) and t(8;21) AML (RR, 1.70; 95% CI, 1.31–2.21), not on OS of CBF-AML (RR, 1.09; 95% CI, 0.97–1.23), CR (OR [odds ratio], 0.95; 95% CI, 0.52–1.74), relapse risk (RR, 1.12; 95% CI, 0.90–1.41) or OS (RR, 1.03; 95% CI, 0.90–1.18) of inv(16) AML. Subgroup analysis of t(8,21) AML showed negative effect of KIT mutations on CR (OR, 2.03; 95%CI: 1.02–4.05), relapse risk (RR, 1.89; 95%CI: 1.51–2.37) and OS (RR, 2.26; 95%CI: 1.35–3,78) of non-Caucasians, not on CR (OR, 0.61; 95%CI: 0.19–1.95) or OS (RR, 1.12; 95%CI: 0.90–1.40) of Caucasians. This study indicates KIT mutations in CBF-AML to be included in the initial routine diagnostic workup and stratification system of t(8,21) AML. Prospective large-scale clinical trials are warranted to evaluate these findings.

Extracellular Vesicle-functionalized Decalcified Bone Matrix Scaffolds with Enhanced Pro-angiogenic and Pro-bone Regeneration Activities

Vascularization is crucial for bone regeneration after the transplantation of tissue-engineered bone grafts in the clinical setting. Growing evidence suggests that mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are potently pro-angiogenic both in vitro and in vivo. In the current study, we fabricated a novel EV-functionalized scaffold with enhanced pro-angiogenic and pro-bone regeneration activities by coating decalcified bone matrix (DBM) with MSC-derived EVs. EVs were harvested from rat bone marrow-derived MSCs and the pro-angiogenic potential of EVs was investigated in vitro. DBM scaffolds were then coated with EVs, and the modification was verified by scanning electron microscopy and confocal microscopy. Next, the pro-angiogenic and pro-bone regeneration activities of EV-modified scaffolds were evaluated in a subcutaneous bone formation model in nude mice. Micro-computed tomography scanning analysis showed that EV-modified scaffolds with seeded cells enhanced bone formation. Enhanced bone formation was confirmed by histological analysis. Immunohistochemical staining for CD31 proved that EV-modified scaffolds promoted vascularization in the grafts, thereby enhancing bone regeneration. This novel scaffold modification method provides a promising way to promote vascularization, which is essential for bone tissue engineering.

Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34(+) Cells.

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+ cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

Development of an angiogenesis-promoting microvesicle-alginate-polycaprolactone composite graft for bone tissue engineering applications

One of the major challenges of bone tissue engineering applications is to construct a fully vascularized implant that can adapt to hypoxic environments in vivo. The incorporation of proangiogenic factors into scaffolds is a widely accepted method of achieving this goal. Recently, the proangiogenic potential of mesenchymal stem cell-derived microvesicles (MSC-MVs) has been confirmed in several studies. In the present study, we incorporated MSC-MVs into alginate-polycaprolactone (PCL) constructs that had previously been developed for bone tissue engineering applications, with the aim of promoting angiogenesis and bone regeneration. MSC-MVs were first isolated from the supernatant of rat bone marrow-derived MSCs and characterized by scanning electron microscopic, confocal microscopic, and flow cytometric analyses. The proangiogenic potential of MSC-MVs was demonstrated by the stimulation of tube formation of human umbilical vein endothelial cells in vitro. MSC-MVs and osteodifferentiated MSCs were then encapsulated with alginate and seeded onto porous three-dimensional printed PCL scaffolds. When combined with osteodifferentiated MSCs, the MV-alginate-PCL constructs enhanced vessel formation and tissue-engineered bone regeneration in a nude mouse subcutaneous bone formation model, as demonstrated by micro-computed tomographic, histological, and immunohistochemical analyses. This MV-alginate-PCL construct may offer a novel, proangiogenic, and cost-effective option for bone tissue engineering.

Microvesicles as Potential Biomarkers for the Identification of Senescence in Human Mesenchymal Stem Cells

Senescence in human mesenchymal stem cells (MSCs) not only contributes to organism aging and the development of a variety of diseases but also severely impairs their therapeutic properties as a promising cell therapy. Studies searching for efficient biomarkers that represent cellular senescence have attracted much attention; however, no single marker currently provides an accurate cell-free representation of cellular senescence. Here, we studied characteristics of MSC-derived microvesicles (MSC-MVs) that may reflect the senescence in their parental MSCs. We found that senescent late passage (LP) MSCs secreted higher levels of MSC-MVs with smaller size than did early passage (EP) MSCs, and the level of CD105+ MSC-MVs decreased with senescence in the parental MSCs. Also, a substantially weaker ability to promote osteogenesis in MSCs was observed in LP than EP MSC-MVs. Comparative analysis of RNA sequencing showed the same trend of decreasing number of highly-expressed miRNAs with increasing number of passages in both MSCs and MSC-MVs. Most of the highly-expressed genes in LP MSCs and the corresponding MSC-MVs were involved in the regulation of senescence-related diseases, such as Alzheimers disease. Furthermore, based on the miRNA profiling, transcription factors (TF) and genes regulatory networks of MSC senescence, and the datasets from GEO database, we confirmed that expression of miR-146a-5p in MSC-MVs resembled the senescent state of their parental MSCs. Our findings provide evidence that MSC-MVs are a key factor in the senescence-associated secretory phenotype of MSCs and demonstrate that their integrated characteristics can dynamically reflect the senescence state of MSCs representing a potential biomarker for monitoring MSC senescence.

Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis

Microvesicles (MVs) in body fluids participate in a variety of physical and pathological processes, and are regarded as potential biomarkers for numerous diseases. Flow cytometry (FCM) is among the most frequently used techniques for MV detection. However, different handling methods unavoidably cause pre-analytical variations in the counts and sizes of MVs determined by FCM. The aim of the present study was to investigate the effect of centrifugation, storage conditions and anticoagulant on MV measurements. Blood samples were obtained from 13 healthy donors, including 4 women and 9 men. Calcein-AM staining was used to label MVs and assess the impact of pre-analytical preparation, including centrifugation, and storage conditions on MV measurements obtained using FCM. The range of factors investigated for comparison included: Platelet-free plasma (PFP) stored at −80°C for 1 or 4 weeks; MVs stored at 4°C for 3–4 days or 1 week; MVs frozen at −80°C for 1 or 4 weeks; and anticoagulants, either heparin or ethylenediaminetetraacetic acid (EDTA). No statistically significant differences in MV counts were detected between the two centrifugation speeds (16,000 and 20,500 × g) or among the three centrifugation times (15, 30 and 60 min) investigated. Similarly, no significant differences were noted in MV counts between the two anticoagulants tested (heparin and EDTA). However, the storage of PFP or MVs in heparin-anticoagulated plasma for different periods markedly affected the detected MV counts and size distribution. The counts and sizes of MVs from EDTA-anticoagulated plasma were only affected when the MVs were frozen at −80°C for 4 weeks. In conclusion, calcein-AM is able to efficiently identify MVs from plasma and may be an alternative to Annexin V for MV staining. EDTA preserves the MV counts and size more accurately compared with heparin under calcein-AM staining. PFP centrifuged at 16,000 × g for 15 min is sufficient to isolate MVs, which enables the batch processing of samples. PFP, rather than MVs alone, appears to be the preferable mode of sample storage, as MVs stored in PFP were less affected by storage temperature and duration. The present study provides a methodology for MV collection, storage and isolation, to facilitate further investigation of MVs as biomarkers in disease.