Hua-Lu Zhao

miR-29a and miR-142-3p downregulation and diagnostic implication in human acute myeloid leukemia

Expression profiling of microRNAs (miRNAs) in most diseases might be popular and provide the possibility for diagnostic implication, but few studies have accurately quantified the expression level of dysregulated miRNAs in acute myeloid leukemia (AML). In this study, we analyzed the peripheral blood mononuclear cells (PBMCs) from 10 AML patients (subtypes M1 to M5) and six normal controls by miRNA microarray and identified several differentially expressed miRNAs. Among them miR-29a and miR-142-3p were selectively encountered in Northern blot analysis and their significantly decreased expression in AML was further confirmed. Quantitative real-time PCR in 52 primarily diagnosed AML patients and 100 normal controls not only verified the expression properties of these 2 miRNAs, but also established that the expression level of miR-142-3p and miR-29a in PBMCs could be used as novel diagnostic markers. A better diagnostic outcome was achieved by combining miR-29a and miR-142-3p with about 90% sensitivity, 100% specificity, and an area under the ROC curve (AUC) of 0.97. Our results provide insights into the involvement of miRNAs in leukemogenesis, and offer candidates for AML diagnosis and therapeutic strategy.

Association of RFC1 A80G and MTHFR C677T polymorphisms with Alzheimer's disease

We examined polymorphisms in reduced folate carrier gene (RFC1) and methylenetetrahydrofolate reductase gene (MTHFR) for association with sporadic AD (SAD) in Chinese population. Significant associations of RFC1 A80G G allele and GG genotype with SAD (p=0.008, OR=1.312, 95%CI=1.072-1.605, and p=0.042, OR=1.383, 95%CI=1.012-1.890) were found. Further stratification of total samples by APOE epsilon4 carrier status, age/age at onset and gender revealed that RFC1 A80G G allele was an APOE epsilon4-independent risk factor for late-onset AD, and it might increase the risk of AD in females. No significant associations of MTHFR C677T allele and genotype with AD were observed in total samples, but significant associations of T allele and TT genotype with AD (p=0.031, OR=1.586, 95%CI=1.042-2.414, and p=0.028, OR=2.250, 95%CI=1.074-4.712) were identified in APOE epsilon4 carrier subgroup, suggesting that MTHFR 677 T allele and APOE epsilon4 allele may synergistically act to increase AD risk. No significant effect of RFC1 G80A and MTHFR C677T polymorphisms on plasma folate and homocysteine levels was detected.

Association analysis of brain-derived neurotrophic factor (BDNF) gene 196 A/G polymorphism with Alzheimer's disease (AD) in mainland Chinese

A functional polymorphism in the coding region of brain-derived neurotrophic factor (BDNF) gene (196 A/G, Met66Val) has recently been reported to be associated with Alzheimers disease (AD) and with an overrepresentation of G allele in AD patients, but different results have also been presented. We conducted a case-control study to analyze the association between the BDNF A/G polymorphism and sporadic AD in a sample composed of 203 AD patients and 239 controls from Mainland Chinese Han population. No association between the polymorphism and AD, no association between the polymorphism and age at onset in AD, and no significant interaction between BDNF and apolipoprotein E (APOE) genotype were detected in either the total or the male samples. However, a significantly high frequency of the GG genotype in the female controls compared with the female patients was detected. A postponed age at onset in the female patients with the GG genotype was also observed. These results suggest that the GG genotype has a protection effect from AD development in females. A significant low frequency of AD patients with the BDNF GG genotype in the AD APOEepsilon4 carriers compared with the frequency of the controls with the BDNF GG genotype in the control APOEepsilon4 carriers was also detected in the female individuals, suggesting that the BDNF GG genotype may reduce the effect of APOEepsilon4 on AD risk in females. Additionally, low frequencies of BDNF G allele and GG genotype were revealed in Chinese when compared with that in the other race populations so far reported.

PU.1-Regulated Long Noncoding RNA lnc-MC Controls Human Monocyte/Macrophage Differentiation through Interaction with MicroRNA 199a-5p

ABSTRACT Long noncoding RNAs (lncRNAs) are emerging as important regulators in mammalian development, but little is known about their roles in monocyte/macrophage differentiation. Here we identified a long noncoding monocytic RNA (lnc-MC) that exhibits increased expression during monocyte/macrophage differentiation of THP-1 and HL-60 cells as well as CD34+ hematopoietic stem/progenitor cells (HSPCs) and is transcriptionally activated by PU.1. Gain- and loss-of-function assays demonstrate that lnc-MC promotes monocyte/macrophage differentiation of THP-1 cells and CD34+ HSPCs. Mechanistic investigation reveals that lnc-MC acts as a competing endogenous RNA to sequester microRNA 199a-5p (miR-199a-5p) and alleviate repression on the expression of activin A receptor type 1B (ACVR1B), an important regulator of monocyte/macrophage differentiation. We also noted a repressive effect of miR-199a-5p on lnc-MC expression and function, but PU.1-dominant downregulation of miR-199a-5p weakens the role of miR-199a-5p in the reciprocal regulation between miR-199a-5p and lnc-MC. Altogether, our work demonstrates that two PU.1-regulated noncoding RNAs, lnc-MC and miR-199a-5p, have opposing roles in monocyte/macrophage differentiation and that lnc-MC facilitates the differentiation process, enhancing the effect of PU.1, by soaking up miR-199a-5p and releasing ACVR1B expression. Thus, we reveal a novel regulatory mechanism, comprising PU.1, lnc-MC, miR-199a-5p, and ACVR1B, in monocyte/macrophage differentiation.

A feedback loop consisting of microRNA 23a/27a and the β-like globin suppressors KLF3 and SP1 regulates globin gene expression.

ABSTRACT The developmental stage-specific expression of the human β-like globin genes has been studied for decades, and many transcriptional factors as well as other important cis elements have been identified. However, little is known about the microRNAs that potentially regulate β-like globin gene expression directly or indirectly during erythropoiesis. In this study, we show that microRNA 23a (miR-23a) and miR-27a promote β-like globin gene expression in K562 cells and primary erythroid cells through targeting of the transcription factors KLF3 and SP1. Intriguingly, miR-23a and miR-27a further enhance the transcription of β-like globin genes through repression of KLF3 and SP1 binding to the β-like globin gene locus during erythroid differentiation. Moreover, KLF3 can bind to the promoter of the miR-23a∼27a∼24-2 cluster and suppress this microRNA cluster expression. Hence, a positive feedback loop comprised of KLF3 and miR-23a promotes the expression of β-like globin genes and the miR-23a∼27a∼24-2 cluster during erythropoiesis.

miR‐199a‐5p inhibits monocyte/macrophage differentiation by targeting the activin A type 1B receptor gene and finally reducing C/EBPα expression

miRNAs are short, noncoding RNAs that regulate expression of target genes at post‐transcriptional levels and function in many important cellular processes, including differentiation, proliferation, etc. In this study, we observed down‐regulation of miR‐199a‐5p during monocyte/macrophage differentiation of HL‐60 and THP‐1 cells, as well as human CD34+ HSPCs. This down‐regulation of miR‐199a‐5p resulted from the up‐regulation of PU.1 that was demonstrated to regulate transcription of the miR‐199a‐2 gene negatively. Overexpression of miR‐199a‐5p by miR‐199a‐5p mimic transfection or lentivirus‐mediated gene transfer significantly inhibited monocyte/macrophage differentiation of the cell lines or HSPCs. The mRNA encoding an ACVR1B was identified as a direct target of miR‐199a‐5p. Gradually increased ACVR1B expression level was detected during monocyte/macrophage differentiation of the leukemic cell lines and HSPCs, and knockdown of ACVR1B resulted in inhibition of monocyte/macrophage differentiation of HL‐60 and THP‐1 cells, which suggested that ACVR1B functions as a positive regulator of monocyte/macrophage differentiation. We demonstrated that miR‐199a‐5p overexpression or ACVR1B knockdown promoted proliferation of THP‐1 cells through increasing phosphorylation of Rb. We also demonstrated that the down‐regulation of ACVR1B reduced p‐Smad2/3, which resulted in decreased expression of C/EBPα, a key regulator of monocyte/macrophage differentiation, and finally, inhibited monocyte/macrophage differentiation.

Hypoxic induction of human erythroid-specific δ-aminolevulinate synthase mediated by hypoxia-inducible factor 1.

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric basic helix-loop-helix transcription factor that regulates many key genes. δ-Aminolevulinate synthase (ALAS) catalyzes the first and rate-limiting reaction in the heme biosynthetic pathway. In this study, we show that hypoxia-induced expression of erythroid-specific ALAS2 is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased ALAS2 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ hematopoietic stem/progenitor cells. Enforced HIF1α expression increased the level of ALAS2 expression, while HIF1α knockdown by RNA interference decreased the level of ALAS2 expression. In silico analysis revealed three potential hypoxia-response elements (HREs) that are located 611, 621, and 741 bp downstream of the ALAS2 gene. The results from reporter gene and mutation analysis suggested that these elements are necessary for a maximal hypoxic response. Chromatin immunoprecipitation and polymerase chain reaction showed that the HREs could be recognized and bound by HIF1α in vivo. These results demonstrate that the upregulation of ALAS2 during hypoxia is directly mediated by HIF1. We hypothesize that HIF1-mediated ALAS2 upregulation promotes erythropoiesis to satisfy the needs of an organism under hypoxic conditions. This may be accomplished via increased heme levels and an interaction between ALAS2 and erythropoietin.

Association between Angiotensin-Converting Enzyme Gene Polymorphism and Alzheimer’s Disease in a Chinese Population

Angiotensin-converting enzyme has shown altered activity in patients with neurological diseases. An insertion/deletion (I/D) polymorphism of the DCP1 gene encoding angiotensin-converting enzyme has been reported to be associated with the risk for Alzheimer’s disease (AD), but ambiguous results have also been presented. We conducted a case-control study in a sample composed of 192 sporadic AD patients and 195 age- and sex-matched controls from Chinese Han population in Beijing and Xi’an districts to investigate the possible effect of the polymorphism. Our data revealed no association between the DCP1 polymorphism and AD risk in the total sample. There was no significant difference in the DCP1 allele or genotype frequencies between cases and controls when stratified by gender and APOE ε4 status. However, the D allele and D/D genotype were more frequent among AD patients between 66 and 70 years compared with controls (D allele: OR = 2.8, 95% CI = 1.5–5.2, p = 0.001; D/D genotype: OR = 5.9, 95% CI = 1.7–19.9, p = 0.002). Our results provided new proof that the DCP1 D allele was a probable risk factor for late-onset AD. Its role was independent and was limited to the population at a certain age.

The PU.1-Modulated MicroRNA-22 Is a Regulator of Monocyte/Macrophage Differentiation and Acute Myeloid Leukemia.

MicroRNA-22 (miR-22) is emerging as a critical regulator in organ development and various cancers. However, its role in normal hematopoiesis and leukaemogenesis remains unclear. Here, we detected its increased expression during monocyte/macrophage differentiation of HL-60, THP1 cells and CD34+ hematopoietic stem/progenitor cells, and confirmed that PU.1, a key transcriptional factor for monocyte/macrophage differentiation, is responsible for transcriptional activation of miR-22 during the differentiation. By gain- and loss-of-function experiments, we demonstrated that miR-22 promoted monocyte/macrophage differentiation, and MECOM (EVI1) mRNA is a direct target of miR-22 and MECOM (EVI1) functions as a negative regulator in the differentiation. The miR-22-mediated MECOM degradation increased c-Jun but decreased GATA2 expression, which results in increased interaction between c-Jun and PU.1 via increasing c-Jun levels and relief of MECOM- and GATA2-mediated interference in the interaction, and thus promoting monocyte/macrophage differentiation. We also observed significantly down-regulation of PU.1 and miR-22 as well as significantly up-regulation of MECOM in acute myeloid leukemia (AML) patients. Reintroduction of miR-22 relieved the differentiation blockage and inhibited the growth of bone marrow blasts of AML patients. Our results revealed new function and mechanism of miR-22 in normal hematopoiesis and AML development and demonstrated its potential value in AML diagnosis and therapy.

ZFP36L1 promotes monocyte/macrophage differentiation by repressing CDK6.

RNA binding proteins (RBPs)-mediated post-transcriptional control has been implicated in influencing various aspects of RNA metabolism and playing important roles in mammalian development and pathological diseases. However, the functions of specific RBPs and the molecular mechanisms through which they act in monocyte/macrophage differentiation remain to be determined. In this study, through bioinformatics analysis and experimental validation, we identify that ZFP36L1, a member of ZFP36 zinc finger protein family, exhibits significant decrease in acute myeloid leukemia (AML) patients compared with normal controls and remarkable time-course increase during monocyte/macrophage differentiation of PMA-induced THP-1 and HL-60 cells as well as induction culture of CD34+ hematopoietic stem/progenitor cells (HSPCs). Lentivirus-mediated gain and loss of function assays demonstrate that ZFP36L1 acts as a positive regulator to participate in monocyte/macrophage differentiation. Mechanistic investigation further reveals that ZFP36L1 binds to the CDK6 mRNA 3′untranslated region bearing adenine-uridine rich elements and negatively regulates the expression of CDK6 which is subsequently demonstrated to impede the in vitro monocyte/macrophage differentiation of CD34+ HSPCs. Collectively, our work unravels a ZFP36L1-mediated regulatory circuit through repressing CDK6 expression during monocyte/macrophage differentiation, which may also provide a therapeutic target for AML therapy.