Transcription factor EgGRP2A regulates EgFATA expression and promotes oleic acid accumulation in oil palm (Elaeis guineensis).

The oil palm (Elaeis guineensis) is emerging as the world's most important and prolific oilseed crop, celebrated for its impressive oil yield. However, the molecular intricacies that govern lipid metabolism and fatty acid accumulation in oil palm fruits remain relatively underexplored. This study reveals a significant correlation between the expression of EgGRP2A, a transcription factor, and the expression of EgFATA in the oil palm. Yeast one-hybrid analysis and electrophoretic mobility shift assays (EMSA) reveal and confirm the binding interactions between EgGRP2A and the promoter region of EgFATA. Subsequent experiments in oil palm protoplasts show that transient overexpression of EgGRP2A leads to a marked upregulation of EgFATA expression. Conversely, downregulation of EgGRP2A in transgenic oil palm embryoids leads to a significant reduction in EgFATA expression. Metabolite profiling in the transgenic embryoids reveals a significant reduction in unsaturated fatty acids, particularly oleic acid. These findings promise profound insights into the regulatory orchestration of EgFATA and the synthesis of fatty acids, particularly oleic acid, in the oil palm. Furthermore, the results lay the foundation for future breeding and genetic improvement efforts aimed at increasing oleic acid content in oil palm varieties.

EgMADS3 directly regulates EgLPAAT to mediate medium-chain fatty acids (MCFA) anabolism in the mesocarp of oil palm.

KEY MESSAGE: EgMADS3, a pivotal transcription factor, positively regulates MCFA accumulation via binding to the EgLPAAT promoter, advancing lipid content in mesocarp of oil palm. Lipids function as the structural components of cell membranes, which serve as permeable barriers to the external environment of cells. The medium-chain fatty acid in the stored lipids of plants is an important renewable energy. Most research on MCFA production in plant lipid synthesis is based on biochemical methods, and the importance of transcriptional regulation in MCFA synthesis and its incorporation into TAGs needs further research. Oil palm is the most productive oil crop in the world and has the highest productivity among the main oil crops. In this study, the MADS transcription factor (EgMADS3) in the mesocarp of oil palm was characterized. Through the VIGS-virus induced gene silencing, it was determined that the potential target gene of EgMADS3 was related to the biosynthesis of medium-chain fatty acid (MCFA). Transient transformation in protoplasts and qRT-PCR analysis showed that EgMADS3 positively regulated the expression of EgLPAAT. The results of the yeast one-hybrid assays and EMSA indicated the interaction between EgMADS3 and EgLPAAT promoter. Through genetic transformation and fatty acid analysis, it is concluded that EgMADS3 directly regulates the mid-chain fatty acid synthesis pathway of the potential target gene EgLPAAT, thus promotes the accumulation of MCFA and improves the total lipid content. This study is innovative in the functional analysis of the MADS family transcription factor in the metabolism of medium-chain fatty acids (MCFA) of oil palm, provides a certain research basis for improving the metabolic pathway of chain fatty acids in oil palm, and improves the synthesis of MCFA in plants. Our results will provide a reference direction for further research on improving the oil quality through biotechnology of oil palm.

Identification and Analysis of MADS-Box Genes Expressed in the Mesocarp of Oil Palm Fruit (Elaeis guineensis Jacq.).

Oil palm (Elaeis guineensis) is the most important tropical oil-bearing crop species worldwide. MADS-box proteins, which play crucial roles in plant growth and development and are involved in various physiological and biochemical processes, compose one of the largest families of plant transcription factors. In this study, 42 MADS-box genes were screened from the mesocarp transcriptome database of oil palm fruit, and their phylogenetic relationships with Arabidopsis thaliana MADS-box genes were analyzed. Based on the results, MADS-box genes from oil palm mesocarp were classified into four groups: MIKCc-type, MIKC*-type, Mα-type, and Mγ-type MADS-box genes. Members of the subfamilies were classified according to the presence of three specific protein motifs. To explore the differential expression of the MADS-box genes, the dynamic expression of all selected MADS-box genes in oil palm was measured by RNA-seq. The high expression of specific MADS-box genes in the mesocarp of oil palm during different developmental stages indicates that those genes may play important roles in the cell division of and metabolite accumulation in the fruit and could become important targets for fruit development and oil accumulation research in oil palm.

Characterization and functional analysis of the MADS-box EgAGL9 transcription factor from the mesocarp of oil palm (Elaeis guineensis Jacq.).

Oil palm (Elaeis guineensis Jacq.) is one of the most important oil crops in the world, and compared to all oil crops, it has the highest productive efficiency. In the present study, a MADS-box transcription factor of the AGAMOUS class, named EgAGL9, was identified by expression profile analysis in the different developmental stages of oil palm mesocarp. Real-time quantitative PCR results confirmed that the expression of EgAGL9 increased rapidly during the last stages of oil palm mesocarp development. Then, three downstream genes, including EgSAD (Stearoyl-ACP desaturase), EgTSA (Tryptophan synthase) and EgSDH (Succinate dehydrogenase), were screened by ChIP-Seq and data analysis. EMSA analysis verified that EgAGL9 interacted with the promoter regions of EgSAD, EgTSA and EgSDH. Moreover, the expression levels of EgSAD, EgTSA and EgSDH were downregulated in EgAGL9-overexpressing protoplasts and calli of oil palm. Compared to WT, the total lipid content and ratio of unsaturated fatty acids in transgenic calli (including oleic acid, linoleic acid and linolenic acid) were significantly decreased. Together, these results revealed that these three EgAGL9-regulated genes are involved in regulatory pathways in the oil palm mesocarp. Compared with previous studies, the present study provides a new research strategy for understanding of the molecular regulatory pathways of lipid metabolism in mesocarp of oil palm. The obtained results will bring a new perspective for a comprehensive understanding of the regulation of the metabolic accumulation in the oil palm mesocarp.

EgMYB108 regulates very long-chain fatty acid (VLCFA) anabolism in the mesocarp of oil palm.

KEY MESSAGE: EgMYB108 regulates VLCFA anabolism in oil palm. Very long-chain fatty acids (VLCFAs), which are fatty acids with more than 18 C, can not only be used as a form of triglyceride (TAG) but also provide precursors for the biosynthesis of cuticle wax, and they exist in plant epidermal cells in the form of wax in higher plants. However, which and how transcriptional factors (TFs) regulate this process is largely unknown in oil palm. In this study, a MYB transcription factor (EgMYB108) with high expression in the mesocarp of oil palm fruit was characterized. Overexpression of EgMYB108 promoted not only total lipid content but also VLCFA accumulation in oil palm embryoids. Subsequently, transient transformation in protoplasts and qRT-PCR analysis indicated that the EgKCS5 and EgLACS4 genes were significantly increased with the overexpression of EgMYB108. Furthermore, yeast one­hybrid assays, dual-luciferase assays and EMSAs demonstrated that EgMYB108 binds to the promoters of EgKCS5 and EgLACS4 and regulates their transcription. Finally, EgMYB108 interacts with the promoters of EgLACS and EgKCS simultaneously and finally improves the VLCFA and total lipid contents; a pathway summarizing this interaction was depicted.. The results provide new insight into the mechanism by which EgMYB108 regulates lipid and VLCFA accumulation in oil palm.

EgmiR5179 Regulates Lipid Metabolism by Targeting EgMADS16 in the Mesocarp of Oil Palm (Elaeis guineensis).

EgMADS16, one of the MADS-box transcription factors in oil palm, has a high expression level in the late fruit development of the oil palm fruit mesocarp. At the same time, it is also predicted to be the target gene of EgmiR5179, which has been identified in previous research. In this paper, we focused on the function and regulatory mechanism of the EgMADS16 gene in oil palm lipid metabolism. The results indicated that the transcription level of EgMADS16 was highest in the fourth stage, and a dual-luciferase reporter assay proved that the EgMADS16 expression level was downregulated by EgmiR5179. In both the OXEgMADS16 Arabidopsis seeds and oil palm embryonic calli, the total lipid contents were significantly decreased, but the contents of C18:0 and C18:3 in OXEgMADS16 lines were significantly increased. As expected, EgmiR5179 weakened the inhibitory effect of EgMADS16 on the oil contents in transgenic Arabidopsis plants that coexpressed EgmiR5179 and EgMADS16 (OXEgmiR5179-EgMADS16). Moreover, yeast two-hybrid and BiFC analyses suggested that there was an interaction between the EgMADS16 protein and EgGLO1 protein, which had been proven to be capable of regulating fatty acid synthesis in our previous research work. In summary, a model of the molecular mechanism by which miRNA5179 targets EgMADS16 to regulate oil biosynthesis was hypothesized, and the research results provide new insight into lipid accumulation and molecular regulation in oil palm.

A MADS-box gene, EgMADS21, negatively regulates EgDGAT2 expression and decreases polyunsaturated fatty acid accumulation in oil palm (Elaeis guineensis Jacq.).

KEY MESSAGE: EgMADS21 regulates PUFA accumulation in oil palm. Oil palm (Elaeis guineensis Jacq.) is the most productive world oil crop, accounting for 36% of world plant oil production. However, the molecular mechanism of the transcriptional regulation of fatty acid accumulation and lipid synthesis in the mesocarp of oil palm by up- or downregulating the expression of genes involved in related pathways remains largely unknown. Here, an oil palm MADS-box gene, EgMADS21, was screened in a yeast one-hybrid assay using the EgDGAT2 promoter sequence as bait. EgMADS21 is preferentially expressed in early mesocarp developmental stages in oil palm fruit and presents a negative correlation with EgDGAT2 expression. The direct binding of EgMADS21 to the EgDGAT2 promoter was confirmed by electrophoretic mobility shift assay. Subsequently, transient expression of EgMADS21 in oil palm protoplasts revealed that EgMADS21 not only binds to the EgDGAT2 promoter but also negatively regulates the expression of EgDGAT2. Furthermore, EgMADS21 was stably overexpressed in transgenic oil palm embryoids by Agrobacterium-mediated transformation. In three independent transgenic lines, EgDGAT2 expression was significantly suppressed by the expression of EgMADS21. The content of linoleic acid (C18:2) in the three transgenic embryoids was significantly decreased, while that of oleic acid (C18:1) was significantly increased. Combined with the substrate preference of EgDGAT2 identified in previous research, the results demonstrate the molecular mechanism by which EgMADS21 regulates EgDGAT2 expression and ultimately affects fatty acid accumulation in the mesocarp of oil palm.

Isolation and characterization of the EgWRI1 promoter from oil palm (Elaeis guineensis Jacq.) and its response to environmental stress and ethylene.

WRI1 is a plant-specific transcription factor that enhances the accumulation of oils through the upregulation of the expression of genes involved in glycolysis and fatty acid biosynthesis. In this study, the EgWRI1 promoter from oil palm was isolated and characterized in transgenic Arabidopsis. The sequence analysis results revealed that various putative plant regulatory elements are present in the EgWRI1 promoter region. The EgWRI1 promoter and beta-glucuronidase (GUS) reporter gene were transcriptionally fused and transformed into Arabidopsis thaliana. Histochemical analysis revealed that GUS staining was very strong in whole seedlings, especially the stems, leaves, and siliques. Moreover, GUS staining was strong in the silique coats but weak in the seeds. Furthermore, to detect whether EgWRI1 was induced by environmental stress, we detected the expression efficiency of the EgWRI1 promoter in transgenic Arabidopsis treated with low temperature, darkness, and exogenous ethylene. The results showed that the activity of the EgWRI1 promoter was induced by darkness but suppressed significantly when exposed to exogenous ethylene. When treated with low temperature, the activity of the EgWRI1 promoter was first reduced after 24 hours but recovered after 48 hours. Taken together, these results reveal the features of the EgWRI1 promoter from oil palm, which will be helpful for improving oil accumulation in oil palm via reasonable cultivation methods.

Genome-wide association analysis of the lipid and fatty acid metabolism regulatory network in the mesocarp of oil palm (Elaeis guineensis Jacq.) based on small noncoding RNA sequencing.

Oil palm (Elaeis guineensis Jacq.) is the highest oil-yielding crop in the plant kingdom and accumulates 90% of palm oil in the mesocarp. However, the regulatory mechanisms of lipid and fatty acid (FA) metabolism in oil palm are just beginning to be understood, and more studies are needed, especially in the understanding of small noncoding RNA (ncRNA) and mRNA. Based on the deep sequencing of small noncoding RNAs and the degradome in five developmental mesocarp stages, 452 microRNAs (miRNAs), including 170 conserved known-miRNAs (kn-miRNAs) and 282 novel-miRNA (nov-miRNAs), were identified. After predicting the targets of those miRNAs to 37 FA synthesis-related genes, we found that 22 kn-miRNAs and 14 nov-miRNAs might be involved in FA metabolism pathways. Among them, eg-miR156c, eg-miR397, eg-miR444b and nov-miR129 regulated FA synthesis in plastids and the transport of FA-ACP from plastids to the endoplasmic reticulum by targeting acetyl-CoA carboxylase 1 (ACC1), long-chain acyl-CoA synthetase 9 (LACS9), LACS4 and enoyl-ACP reductase (ENR), respectively. Nov-miR138 and nov-miR59 targeted glycerol-3-phosphate acyltransferase (GPAT), and nov-miR274 targeted phosphatidate phosphatase 1 (PAP1). Both target genes are involved in triacylglycerol synthesis in the endoplasmic reticulum. Eg-miR156e and eg-miR156j played pivotal roles by targeting ß-ketoacyl-CoA synthase 12 (KCS12), and nov-miR201 targets very-long-chain enoyl-CoA reductase (ECR). Several miRNAs were also predicted to indirectly regulate FA synthesis and lipid metabolism through the squamosa promoter-binding protein-like gene (SPL), NAC and MYB transcription factors. As a whole, indications of a complex and extensive miRNA-mRNA regulatory network associated with FA metabolism in the mesocarp of the oil palm is presented. The results help to broaden the knowledge of potential mechanisms that might be regulated by miRNAs through modulation of the expression of FA-related target gene metabolism in the oil palm.

Identification and characterization of a plastidial ω-3 fatty acid desaturase EgFAD8 from oil palm (Elaeis guineensis Jacq.) and its promoter response to light and low temperature.

In higher plants, ω-3 fatty acid desaturases are the key enzymes in the biosynthesis of alpha-linolenic acid (18:3), which plays key roles in plant metabolism as a structural component of both storage and membrane lipids. Here, the first ω-3 fatty acid desaturase gene was identified and characterized from oil palm. The bioinformatic analysis indicated it encodes a temperature-sensitive chloroplast ω-3 fatty acid desaturase, designated as EgFAD8. The expression analysis revealed that EgFAD8 is highly expressed in the oil palm leaves, when compared with the expression in the mesocarp. The heterologous expression of EgFAD8 in yeast resulted in the production of a novel fatty acid 18:3 (about 0.27%), when fed with 18:2 in the induction culture. Furthermore, to detect whether EgFAD8 could be induced by the environment stress, we detected the expression efficiency of the EgFAD8 promoter in transgenic Arabidopsis treated with low temperature and darkness, respectively. The results indicated that the promoter of EgFAD8 gene could be significantly induced by low temperature and slightly induced by darkness. These results reveal the function of EgFAD8 and the feature of its promoter from oil palm fruits, which will be useful for understanding the fuction and regulation of plastidial ω-3 fatty acid desaturases in higher plants.

Characterization and Functional Analysis of a Type 2 Diacylglycerol Acyltransferase (DGAT2) Gene from Oil Palm (Elaeis guineensis Jacq.) Mesocarp in Saccharomyces cerevisiae and Transgenic Arabidopsis thaliana.

Oil palm (Elaeis guineensis Jacq.) is the highest oil-yielding plant in the world, storing 90 and 60% (dry weight) oil in its mesocarp and kernel, respectively. To gain insights into the oil accumulation mechanism, one of the key enzymes involved in triacylglycerol (TAG) biosynthesis, a Type 2 diacylglycerol acyltransferase (DGAT2) from oil palm, was characterized for its in vivo activity. EgDGAT2 is highly expressed in mesocarp during the last two developmental stages while large amounts of oil are accumulated at the highest rate during ripening. Heterologous expression of EgDGAT2 in mutant yeast H1246 restored TAG biosynthesis with substrate preference toward unsaturated fatty acids (FAs) (16:1 and 18:1). Furthermore, seed-specific overexpression of EgDGAT2 in Arabidopsis thaliana enhanced the content of polyunsaturated FAs 18:2 and 18:3 (each by 6 mol%) in seed TAGs, when compared to that from wild-type Arabidopsis. In turn, the proportion of 18:0 and 20:0 FAs in seed TAGs from EgDGAT2 transgenic lines decreased accordingly. These results provide new insights into understanding the in vivo activity of EgDGAT2 from oil palm mesocarp, which will be of importance for metabolic enhancement of unsaturated FAs production.

Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.).

Coconut (Cocos nucifera L.) is a key tropical crop and a member of the monocotyledonous family Arecaceae (Palmaceae). Few genes and related metabolic processes involved in coconut endosperm development have been investigated. In this study, a new member of the WRI1 gene family was isolated from coconut endosperm and was named CoWRI1. Its transcriptional activities and interactions with the acetyl-CoA carboxylase (BCCP2) promoter of CoWRI1 were confirmed by the yeast two-hybrid and yeast one-hybrid approaches, respectively. Functional characterization was carried out through seed-specific expression in Arabidopsis and endosperm-specific expression in rice. In transgenic Arabidopsis, high over-expressions of CoWRI1 in seven independent T2 lines were detected by quantitative real-time PCR. The relative mRNA accumulation of genes encoding enzymes involved in either fatty acid biosynthesis or triacylglycerols assembly (BCCP2, KASI, MAT, ENR, FATA, and GPDH) were also assayed in mature seeds. Furthermore, lipid and fatty acids C16:0 and C18:0 significantly increased. In two homozygous T2 transgenic rice lines (G5 and G2), different CoWRI1 expression levels were detected, but no CoWRI1 transcripts were detected in the wild type. Analyses of the seed oil content, starch content, and total protein content indicated that the two T2 transgenic lines showed a significant increase (P < 0.05) in seed oil content. The transgenic lines also showed a significant increase in starch content, whereas total protein content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (C16:0) and linolenic acid (C18:3) increased significantly in the seeds of the transgenic rice lines, but oleic acid (C18:1) levels significantly declined.

Identification of a Δ12 fatty acid desaturase from oil palm (Elaeis guineensis Jacq.) involved in the biosynthesis of linoleic acid by heterologous expression in Saccharomyces cerevisiae.

Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. A Δ12-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from oil palm and their functions identified. The open reading frames (ORFs) of egFAD2 (GenBank accession: KT023602) consisted of 1176bp and code for 391 amino acids. Their deduced polypeptides showed 75-93% identity to microsomal Δ12-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. RT-PCR experiment indicated that the egFAD2 gene exhibited the highest accumulation in the mesocarp of fruits at 120-140 DAP (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage. Plasmid pYES2-egFAD2 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Yeast cells transformed with plasmid constructs containing egFAD12 produced an appreciable amount of linoleic acids (18:2(Δ9,)(12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ12-desaturase enzymes.

[HER2 protein testing in gastric cancer: a retrospective analysis of 1 471 cases during two different periods in a single medical center].

OBJECTIVE: To study the potential factors in influencing the performance of immunohistochemical testing for HER2 protein in gastric cancers. METHODS: The HER2 protein expression status of 1 471 surgically resected archival gastric cancer cases in Drum Tower Hospital collected during two different periods was retrospectively analyzed. The materials included 957 cases tested during the period from 2007 to 2009 (group 1) and 514 cases from 2012 to 2013 (group 2). The test procedures and results observed during these two periods were compared. RESULTS: The percentages of score 3 HER2 protein expression (14.4%, 74/514 versus 9.5%, 91/957) and score 2 or score 3 HER2 protein expression (27.2%, 140/514 versus 21.7%, 208/957) were both higher in group 2 than in group 1 (P < 0.05). In group 1, the cancer tissue was fixed in 10% formalin, stained manually with HER2 antibody A0485 (Dako) and assessed by different pathologists.In group 2, the tissue was fixed in 10% neutral buffered formalin (pH 7.2), stained using automated immunostaining system (Roche Benchmark XT) with HER2 antibody 4B5 (Ventana) and assessed by a specialized team of pathologists. CONCLUSION: The results of HER2 immunostaining in gastric cancer are influenced by a number of factors including type of fixative, clone number of primary antibody, staining methods and experience of pathologists.

Molecular cloning and characterisation of an acyl carrier protein thioesterase gene (CocoFatB1) expressed in the endosperm of coconut (Cocos nucifera) and its heterologous expression in Nicotiana tabacum to engineer the accumulation of different fatty acids.

Coconut (Cocos nucifera L.) contains large amounts of medium chain fatty acids, which mostly recognise acyl-acyl carrier protein (ACP) thioesterases that hydrolyse acyl-ACP into free fatty acids to terminate acyl chain elongation during fatty acid biosynthesis. A full-length cDNA of an acyl-ACP thioesterase, designated CocoFatB1, was isolated from cDNA libraries prepared from coconut endosperm during fruit development. The gene contained an open reading frame of 1254 bp, encoding a 417-amino acid protein. The amino acid sequence of the CocoFatB1 protein showed 100% and 95% sequence similarity to CnFatB1 and oil palm (Elaeis guineensis Jacq.) acyl-ACP thioesterases, respectively. Real-time fluorescent quantitative PCR analysis indicated that the CocoFatB1 transcript was most abundant in the endosperm from 8-month-old coconuts; the leaves and endosperm from 15-month-old coconuts had ~80% and ~10% of this level. The CocoFatB1 coding region was overexpressed in tobacco (Nicotiana tabacum L.) under the control of the seed-specific napin promoter following Agrobacterium tumefaciens-mediated transformation. CocoFatB1 transcript expression varied 20-fold between different transgenic plants, with 21 plants exhibiting detectable levels of CocoFatB1 expression. Analysis of the fatty acid composition of transgenic tobacco seeds showed that the levels of myristic acid (14 : 0), palmitic acid (16 : 0) and stearic acid (18 : 0) were increased by 25%, 34% and 17%, respectively, compared with untransformed plants. These results indicated that CocoFatB1 acts specifically on 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP, and can increase medium chain saturated fatty acids. The gene may valuable for engineering fatty acid metabolism in crop improvement programmes.

Effect of harvest date on the nutritional quality and antioxidant capacity in 'Hass' avocado during storage.

The effect of harvest date on nutritional compounds and antioxidant activity (AOC) in avocado (Persea americana Mill. cv Hass) fruit during storage was determined. The fruits were harvested at seven different dates and ripened at 25 °C following 21 or 35 days of cold storage. The results indicated that the phenolic and glutathione contents were increased and the ascorbic acid content was not significantly different in early harvested fruit (January to March), and the phenolic, ascorbic acid and glutathione contents were increased slightly and then decreased on late harvested fruit (April to June). Similar trends were observed in the changes of AOC. Furthermore, AOC in early harvested fruit after storage for 35 days was much higher than that in late harvested fruit after storage for 21 days. Therefore, avocado can be harvested earlier for economic benefits according to the market and can keep high nutritional value for human health benefits.

Upregulation of microRNA-125b contributes to leukemogenesis and increases drug resistance in pediatric acute promyelocytic leukemia.

BACKGROUND: Although current chemotherapy regimens have remarkably improved the cure rate of pediatric acute promyelocytic leukemia (APL) over the past decade, more than 20% of patients still die of the disease, and the 5-year cumulative incidence of relapse is 17%. The precise gene pathways that exert critical control over the determination of cell lineage fate during the development of pediatric APL remain unclear. METHODS: In this study, we analyzed miR-125b expression in 169 pediatric acute myelogenous leukemia (AML) samples including 76 APL samples before therapy and 38 APL samples after therapy. The effects of enforced expression of miR-125b were evaluated in leukemic cell and drug-resistant cell lines. RESULTS: miR-125b is highly expressed in pediatric APL compared with other subtypes of AML and is correlated with treatment response, as well as relapse of pediatric APL. Our results further demonstrated that miR-125b could promote leukemic cell proliferation and inhibit cell apoptosis by regulating the expression of tumor suppressor BCL2-antagonist/killer 1 (Bak1). Remarkably, miR-125b was also found to be up-regulated in leukemic drug-resistant cells, and transfection of a miR-125b duplex into AML cells can increase their resistance to therapeutic drugs, CONCLUSIONS: These findings strongly indicate that miR-125b plays an important role in the development of pediatric APL at least partially mediated by repressing BAK1 protein expression and could be a potential therapeutic target for treating pediatric APL failure.

Down-regulated miR-331-5p and miR-27a are associated with chemotherapy resistance and relapse in leukaemia.

Multidrug resistance (MDR) and disease relapse are challenging clinical problems in the treatment of leukaemia. Relapsed disease is frequently refractory to chemotherapy and exhibits multiple drug resistance. Therefore, it is important to identify the mechanism by which cancer cells develop resistance. In this study, we used microRNA (miRNA) microarray and qRT-PCR approaches to investigate the expression of miRNAs in three leukaemia cell lines with different degrees of resistance to doxorubicin (DOX) compared with their parent cell line, K562. The expression of miR-331-5p and miR-27a was inversely correlated with the expression of a drug-resistant factor, P-glycoprotein (P-gp), in leukaemia cell lines with gradually increasing resistance. The development of drug resistance is regulated by the expression of the P-gp. Transfection of the K562 and, a human promyelocytic cell line (HL) HL60 DOX-resistant cells with miR-331-5p and miR-27a, separately or in combination, resulted in the increased sensitivity of cells to DOX, suggesting that correction of altered expression of miRNAs may be used for therapeutic strategies to overcome leukaemia cell resistance. Importantly, miR-331-5p and miR-27a were also expressed at lower levels in a panel of relapse patients compared with primary patients at diagnosis, further illustrating that leukaemia relapse might be a consequence of deregulation of miR-331-5p and miR-27a.

MicroRNA patterns associated with clinical prognostic parameters and CNS relapse prediction in pediatric acute leukemia.

BACKGROUND: Recent reports have indicated that microRNAs (miRNAs) play a critical role in malignancies, and regulations in the progress of adult leukemia. The role of miRNAs in pediatric leukemia still needs to be established. The purpose of this study was to investigate the aberrantly expressed miRNAs in pediatric acute leukemia and demonstrate miRNA patterns that are pediatric-specific and prognostic parameter-associated. METHODOLOGY/PRINCIPAL FINDINGS: A total of 111 pediatric bone marrow samples, including 99 patients and 12 normal donors, were enrolled in this study. Of those samples, 36 patients and 7 normal samples were used as a test cohort for the evaluation of miRNA profiling; 63 pediatric patients and 5 normal donors were used as a validation cohort to confirm the miRNA differential expression. Pediatric ALL- and AML-specific microRNA expression patterns were identified in this study. The most highly expressed miRNAs in pediatric ALL were miR-34a, miR-128a, miR-128b, and miR-146a, while the highly expressed miRNAs in pediatric AML were miR-100, miR-125b, miR-335, miR-146a, and miR-99a, which are significantly different from those reported for adult CLL and AML. miR-125b and miR-126 may serve as favorable prognosticators for M3 and M2 patients, respectively. Importantly, we identified a "miRNA cascade" associated with central nervous system (CNS) relapse in ALL. Additionally, miRNA patterns associated with prednisone response, specific risk group, and relapse of ALL were also identified. CONCLUSIONS/SIGNIFICANCE: There are existing pediatric-associated and prognostic parameter-associated miRNAs that are independent of cell lineage and could provide therapeutic direction for individual risk-adapted therapy for pediatric leukemia patients.

Genome-wide analysis of small RNA and novel MicroRNA discovery in human acute lymphoblastic leukemia based on extensive sequencing approach.

BACKGROUND: MicroRNAs (miRNAs) have been proved to play an important role in various cellular processes and function as tumor suppressors or oncogenes in cancers including leukemia. The identification of a large number of novel miRNAs and other small regulatory RNAs will provide valuable insights into the roles they play in tumorgenesis. METHODOLOGY/PRINCIPAL FINDINGS: To gain further understanding of the role of miRNAs relevant to acute lymphoblastic leukemia (ALL), we employed the sequencing-by-synthesis (SBS) strategy to sequence small RNA libraries prepared from ALL patients and normal donors. In total we identified 159 novel miRNAs and 116 novel miRNA*s from both libraries. Among the 159 novel miRNAs, 42 were identified with high stringency in our data set. Furthermore, we demonstrated the different expression patterns of 20 newly identified and several known miRNAs between ALL patients and normal donors, suggesting these miRNAs may be associated with ALL and could constitute an ALL-specific miRNA signature. Interestingly, GO "biological process" classifications revealed that a set of significantly abnormally expressed miRNAs are associated with disease relapse, which implies that these dysregulated miRNAs might promote the progression of ALL by regulating genes involved in the pathway of the disease development. CONCLUSION/SIGNIFICANCE: The study presents a comprehensive picture of the expression of small RNAs in human acute lymphoblastic leukemia and highlights novel and known miRNAs differentially expressed between ALL patients and normal donors. To our knowledge, this is the first study to look at genome-wide known and novel miRNA expression patterns in in human acute lymphoblastic leukemia. Our data revealed that these deregulated miRNAs may be associated with ALL or the onset of relapse.