A comprehensive overview of miRNA targeting drought stress resistance in plants / Uma visão geral abrangente do miRNA visando a resistência ao estresse hídrico em plantas

Abstract MicroRNAs (miRNAs) are essential nonprotein-coding genes. In a range of organisms, miRNAs has been reported to play an essential role in regulating gene expressions at post-transcriptional level. They participate in most of the stress responsive processes in plants. Drought is an ultimate abiotic stress that affects the crop production. Therefore understanding drought stress responses are essential to improve the production of agricultural crops. Throughout evolution, plants have developed their own defense systems to cope with the adversities of environmental stresses. Among defensive mechanisms include the regulations of gene expression by miRNAs. Drought stress regulates the expression of some of the functionally conserved miRNAs in different plants. The given properties of miRNAs provide an insight to genetic alterations and enhancing drought resistance in cereal crops. The current review gives a summary to regulatory mechanisms in plants as well as miRNAs response to drought stresses in cereal crops. Some possible approaches and guidelines for the exploitation of drought stress miRNA responses to improve cereal crops are also described.

Resumo MicroRNAs (miRNAs) são genes essenciais não codificadores de proteínas. Em uma variedade de organismos, foi relatado que miRNAs desempenham papel essencial na regulação da expressão gênica em nível pós-transcricional. Eles participam da maioria dos processos responsivos ao estresse nas plantas. A seca é um estresse abiótico final que afeta a produção agrícola. Portanto, compreender as respostas ao estresse da seca é essencial para melhorar a produção de safras agrícolas. Ao longo da evolução, as plantas desenvolveram seus próprios sistemas de defesa para lidar com as adversidades do estresse ambiental. Entre os mecanismos de defesa está a regulação da expressão gênica por miRNAs. O estresse hídrico regula a expressão de alguns dos miRNAs funcionalmente conservados em diferentes plantas. As propriedades dadas dos miRNAs fornecem uma visão das alterações genéticas e aumentam a resistência à seca nas safras de cereais. A revisão atual apresenta um resumo dos mecanismos regulatórios nas plantas, bem como a resposta dos miRNAs ao estresse hídrico nas plantações de cereais. Algumas abordagens e diretrizes possíveis para a exploração das respostas do miRNA ao estresse da seca para melhorar as safras de cereais também são descritas.

Regulation of Notch1 and Foxp1 by MiR-34a in psoriasis vulgaris / Regulación de Notch1 y Foxp1 por MiR-34ª en psoriasis vulgaris

SUMMARY: This study is to investigate the regulation of Notch1 and Foxp1 by miR-34a in the development of psoriasis vulgaris. RT-PCR was used to compare the levels of miR-34a in the skin lesions of 20 patients with psoriasis vulgaris and 20 normal skin tissues. Immunohistochemistry was used to detect the expression of Notch1 and Foxp1 in 51 patients with psoriasis vulgaris, which were further compared with that in 29 normal control tissues. In addition, in HaCaT cells, we used miR-34a mimics and inhibitors to overexpress and inhibit miR-34a, respectively, and detected the mRNA and protein levels of miR-34a, Notch1, and Foxp1. The level of miR-34a in the skin lesions of patients with psoriasis vulgaris was significantly higher than that in normal skin tissues (t=2.192, P<0.05). The positive rate of Notch1 in the skin lesions of patients with psoriasis vulgaris was 76.47 %, which was significantly higher than that in normal skin tissues (13.79 %) (t=29.215, P<0.01). The positive rate of FOXP1 in the psoriasis vulgaris group was 92.16 %, which was also significantly higher than that in the normal skin group (65.52 %) (t=9.087, P<0.01). In addition, overexpression of miR-34a significantly promoted the expression of Notch1 and Foxp1. However, inhibition of miR-34a significantly reduced Notch1 and Foxp1 levels. miR- 34a is highly expressed in the skin tissues of patients with psoriasis vulgaris, and may participate in the development of psoriasis vulgaris by regulating Notch1 and Foxp1.

RESUMEN: El objetivo de este estudio fue investigar la regulación de Notch1 y Foxp1 por miR-34a en el desarrollo de la psoriasis vulgar. Se utilizó RT-PCR con el fin de comparar los niveles de miR-34a en las lesiones cutáneas de 20 pacientes con psoriasis vulgar y 20 tejidos de piel normales. Se utilizó inmunohistoquímica para detectar la expresión de Notch1 y Foxp1 en 51 pacientes con psoriasis vulgar, que se compararon además con la de 29 tejidos normales control. Además, en las células HaCaT, usamos miméticos e inhibidores de miR-34a para sobreexpresar e inhibir miR-34a, respectivamente, y detectamos los niveles de ARNm y proteína de miR-34a, Notch1 y Foxp1. El nivel de miR- 34a en las lesiones cutáneas de pacientes con psoriasis vulgar fue significativamente mayor que en los tejidos normales de la piel (t=2,192, P<0,05). La tasa de positividad de Notch1 en las lesiones cutáneas de pacientes con psoriasis vulgar fue del 76,47 %, que fue significativamente mayor que la de los tejidos normales de la piel (13,79 %) (t=29,215, P<0,01). La tasa positiva de FOXP1 en el grupo de psoriasis vulgar fue del 92,16 %, que también fue significativamente mayor que la del grupo de piel normal (65,52 %) (t=9,087, P<0,01). Además, la sobreexpresión de miR-34a promovió significativamente la expresión de Notch1 y Foxp1. Sin embargo, la inhibición de miR-34a redujo de manera importante los niveles de Notch1 y Foxp1. miR-34a se expresa en gran medida en los tejidos de la piel en pacientes con psoriasis vulgar y puede participar en el desarrollo de la psoriasis vulgar mediante la regulación de Notch1 y Foxp1.

MicroRNA-204 Carried by Exosomes of Human Umbilical Cord-derived Mesenchymal Stem Cells Regulates the Polarization of Macrophages in a Mouse Model of Myocardial Ischemia-reperfusion Injury / 中国医学科学院学报

Objective To explore the role and mechanism of microRNA-204(miR-204) carried by the exosomes of human umbilical cord-derived mesenchymal stem cells(hUC-MSC) in regulating the polarization of macrophages in a mouse model of myocardial ischemia-reperfusion(I/R) injury. Methods After the hUC-MSCs were isolated,cultured,and identified,their adipogenic and osteogenic differentiation capabilities were determined.The exosomes of hUC-MSCs were separated by ultracentrifugation,and the expression of CD81,CD63,tumor susceptibility gene 101(Tsg101),and calnexin in the exosomes was determined by Nanoparticle Tracking Analysis software,transmission electron microscopy,and Western blotting.Three groups(hUC-MSC,miR-204 mimic,and negative control) were designed for the determination of the expression of miR-204 in the cells and their exosomes by qRT-PCR.The C57BL/6J mice were randomly assigned into a sham operation group,an I/R group,a hUC-MSC exosomes group,a negative control group,and a miR-204 mimic group.Except the sham operation group,the I/R model was established by ligating the left anterior descending artery.The echocardiography system was employed to detect the heart function of mice.HE staining was employed to observe the pathological changes of mouse myocardium.ELISA was employed to determine the levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),arginase 1(Arg-1),and IL-10 in the myocardial tissue.After the macrophages of mouse myocardial tissue were isolated,flow cytometry was employed to determine the expression of CD11c and CD206,and ELISA to measure the levels of IL-1β,TNF-α,Arg-1,and IL-10 in the macrophages. Results hUC-MSCs had adipogenic and osteogenic differentiation capabilities,and the exosomes were successfully identified.Compared with the negative control group,the miR-204 mimic group showed up-regulated expression of miR-204 in hUC-MSCs and their exosomes(P<0.001,P<0.001).Compared with the sham operation group,the modeling of I/R increased the left ventricular end-diastolic diameter(LVEDD)(P<0.001),left ventricular end-systolic diameter(LVESD)(P<0.001),myocardial injury score(P<0.001),and the levels of IL-1β(P<0.001),TNF-α(P<0.001),and CD11c(P<0.001).Meanwhile,it lowered the left ventricular ejection fraction(LVEF)(P<0.001),left ventricular fractional shortening(LVFS)(P<0.001),Arg-1(P<0.001),IL-10(P<0.001),and CD206(P<0.001).Compared with those in the I/R group,the LVEDD(P<0.001),LVESD(P<0.001),myocardial injury score(P<0.001),and the levels of IL-1β(P<0.001),TNF-α(P=0.010),and CD11c(P<0.001) reduced,while LVEF(P<0.001),LVFS(P<0.001),and the levels of Arg-1(P<0.001),IL-10(P=0.028),and CD206(P=0.022) increased in the hUC-MSC exosomes group.Compared with those in the negative control group,the LVEDD(P<0.001),LVESD(P<0.001),myocardial injury score(P=0.001),and the levels of IL-1β(P=0.048),TNF-α(P<0.001),and CD11c(P=0.007) reduced,while the LVEF(P<0.001),LVFS(P<0.001),and the levels of Arg-1(P<0.001),IL-10(P=0.001),and CD206(P=0.001) increased in the miR-204 mimic group. Conclusion The hUC-MSC exosomes overexpressing miR-204 can inhibit the polarization of macrophages in the I/R mouse model to M1-type and promote the polarization to M2-type.

Bioinformatics analysis of the microRNA expression profile in the peripheral blood lymphocytes of Kazakh patients with essential hypertension in Xinjiang / 生理学报

This study aimed to investigate the differential expression profiles of microRNAs (miRNAs) in peripheral blood lymphocytes between patients with essential hypertension and healthy individuals in Xinjiang Kazakh and to provide insight into the mechanism involved in the pathogenesis of hypertension in this ethnic group. From April 2016 to May 2019, 30 Kazakh patients with essential hypertension in the inpatient and outpatient departments of Cardiology, First Affiliated Hospital of Shihezi University were used as the hypertension group; 30 healthy Kazakh patients were used as the control group. The miRNA expression profiles in peripheral blood lymphocytes of 6 Kazakh hypertensive patients and 6 matched healthy individuals were compared, and the differentially expressed miRNAs were analyzed by cluster analysis, GSEA enrichment analysis, target gene prediction, target gene annotation and other bioinformatics analyses. In addition, qRT-PCR was used to verify the differentially expressed miRNAs. The results showed that compared with the control group, 73 differentially expressed miRNAs were identified in the hypertension group, of which 39 miRNAs were up-regulated and 34 miRNAs were down-regulated. A total of 11 miRNAs related to hypertension were screened by GSEA enrichment analysis, including hsa-miR-100-5p, hsa-miR-150-5p, hsa-miR-299-5p, hsa-miR-299-3p, hsa-miR-296-5p, hsa-miR-196b-5p, hsa-miR-503-5p, hsa-miR-628-5p, hsa-miR-874-3p, hsa-miR-543 and hsa-miR-940. qRT-PCR test found that the expression of hsa-miR-100-5p, hsa-miR-299-5p, hsa-miR-299-3p, hsa-miR-196b-5p, hsa-miR-503-5p, hsa-miR-628-5p and hsa-miR-543 was up-regulated, while the expression of hsa-miR-150-5p, hsa-miR-296-5p, hsa-miR-874-3p and hsa-miR-940 was down-regulated in the hypertension group compared with the control group. The expression trend in the gene chip was consistent with the results verified by qRT-PCR. Using online database to predict target genes of 11 miRNAs related to hypertension, we found that a total of 1 647 target genes might be regulated by these 11 miRNAs. GO function enrichment showed that (a) in biological processes, the predicted hypertension related target genes are mainly relevant to nervous system development, cellular localization, regulation of cellular metabolic process, generation of neurons and positive regulation of biological process; (b) In terms of cellular components, they are mainly related to membrane-bounded organelle, cytoplasm, intracellular membrane-bounded organelle, synapse part, neuron part, and nucleoplasm; (c) In terms of molecular function, they are mainly related to protein binding, transcription regulatory region DNA binding, RNA polymerase II regulatory region DNA binding, transcription regulator activity, and ion binding. KEGG enrichment analysis showed that the p53 signaling pathway, adrenergic signaling in cardiomyocytes, cAMP signaling pathway, TGF-β signaling pathway, endocrine and other factor-regulated calcium reabsorption, mTOR signaling pathway, and aldosterone-regulated sodium reabsorption may be related to the occurrence and development of hypertension. In conclusion, there are significant differences in the expression of miRNAs in peripheral blood lymphocytes between Kazakh patients with essential hypertension and healthy people. The differentially expressed miRNAs may be related to the occurrence and development of essential hypertension in Kazakh. However, the underlying mechanism needs to be further explored and verified.

Molecular biomarkers for gestational diabetes mellitus and postpartum diabetes / 中华医学杂志(英文版)

Gestational diabetes mellitus (GDM) is a growing public health problem worldwide that threatens both maternal and fetal health. Identifying individuals at high risk for GDM and diabetes after GDM is particularly useful for early intervention and prevention of disease progression. In the last decades, a number of studies have used metabolomics, genomics, and proteomic approaches to investigate associations between biomolecules and GDM progression. These studies clearly demonstrate that various biomarkers reflect pathological changes in GDM. The established markers have potential use as screening and diagnostic tools in GDM and in postpartum diabetes research. In the present review, we summarize recent studies of metabolites, single-nucleotide polymorphisms, microRNAs, and proteins associated with GDM and its transition to postpartum diabetes, with a focus on their predictive value in screening and diagnosis.

Association of miR-146a rs2910164 G/C polymorphism with its abnormal expression and risk of gastric cancer / 中华医学遗传学杂志

OBJECTIVE@#To assess the influence of rs2910164 G/C single nucleotide polymorphism (SNP) of the miR-146a gene on its expression and susceptibility to gastric cancer.@*METHODS@#Fifty three gastric cancer patients and six gastric cancer cell lines were selected for determining the miR-146a expression by Taqman quantitative PCR. A model was constructed to assess the influence of miR-146a overexpression on the growth of AGS gastric cancer cells. A case-control study involving 417 gastric cancer patients and 420 cancer-free individuals was then conducted, and the allelic and genotypic frequencies of the rs2910164 G/C SNP were compared. The genotypes of all subjects were determined by using a Taqman allelic discrimination assay. A Taqman assay was also used to quantify mature and pri-miR-146a transcripts among 65 gastric cancer patients with known genotypes.@*RESULTS@#The expression of miR-146a was down-regulated among the 53 gastric cancer patients and six gastric cancer cell lines. Over-expression of miR-146a has suppressed the growth of gastric cancer by inhibiting the G1/S-phase transition of AGS cells. The case-control study showed that subjects with GC/CC genotypes had significantly lower risk for gastric cancer compared with those with GG genotype. In addition, miR-146a G/C SNP has significantly increased the level of mature miR-146a in those with GC/CC genotype compared with GG genotype.@*CONCLUSION@#Down-regulation of miR-146a may play an important role in the pathogenesis of gastric cancer. The rs2910164 polymorphism of the miR-146a gene may reduce the risk of gastric cancer by influencing the processing of mature miR-146a.

Down-regulation of lncRNA TTTY15 targeting miR-4500 to inhibit the biological characteristics of A172 glioma cells / 中华医学遗传学杂志

OBJECTIVE@#To explore the effect and mechanism of down-regulating lncRNA TTTY15 targeting miR-4500 on the proliferation, apoptosis, migration and invasion of A172 glioma cells.@*METHODS@#The difference in TTTY15 expression between the glioma cells and tissue was determined with a qRT-PCR method. Complementary binding sites of TTTY15 and miR-4500 were predicted with Starbase software, and the targeting relationship was validated with a luciferase reporter system. A172 glioma cells were divided into Control, si-NC (transfected with control siRNA), si-TTTY15 (transfected with TTTY15 siRNA), si-TTTY15+Anti-miR-NC (co-transfected with TTTY15 siRNA and inhibitor control) and si-TTTY15+Anti-miR-4500 (co-transfected with TTTY15 siRNA and miR-4500 inhibitor) groups. Proliferation, apoptosis, migration and invasion, and the expression of Bax, Bcl-2, MMP-2 and MMP-9 proteins of the A172 glioma cells were respectively detected with CCK-8, flow cytometry, Transwell chamber and Western blotting assays.@*RESULTS@#The expression of TTTY15 in glioma cells and glioma tissues have both increased. The expression levels of TTTY15 and miR-4500 in glioma tissues were inversely correlated. TTTY15 and miR-4500 are mutually targeted. Compared with those of the Control and si-NC groups, the glioma cells in the si-TTTY15 group showed increased level of miR-4500, decreased survival rate, increased apoptosis rate, enhanced cell migration and invasion, increased expression of Bax protein, and decreased expression of Bcl-2, MMP-2 and MMP-9 proteins (P<0.05). Compared with those of the si-TTTY15+Anti-miR-NC group, the A172 glioma cells in the si-TTTY15+Anti-miR-4500 group showed decreased level of miR-4500, increased cell survival rate, decreased apoptosis rate, enhanced cell migration and invasion, decreased expression of Bax protein, and increased expression of Bcl-2, MMP-2, and MMP-9 proteins (P<0.05).@*CONCLUSION@#Down-regulating TTTY15 targeting miR-4500 can inhibit the proliferation, migration, invasion and induce apoptosis of the A172 glioma cells.

Effect of circ-SFMBT2 on the biological behavior of non-small cell lung cancer cells by targeting the miR-7-5p/ADAM10 axis / 中华医学遗传学杂志

OBJECTIVE@#To explore the effect of circ-SFMBT2 on the biological behavior of non-small cell lung cancer (NSCLC) cells and its regulatory role on the miR-7-5p/ADAM10 axis.@*METHODS@#qRT-PCR and Western blotting were used to determine the expression of circ-SFMBT2, miR-7-5p, and ADAM10 in NSCLC tissues and adjacent tissues. Pearson analysis was used to analyze the correlation between circ-SFMBT2 and miR-7-5p, and between miR-7-5p and ADAM10. In vitro cultured human bronchial epithelial-like cells (HBE) and lung cancer cell lines H1650, H460, A549, H1299. CCK-8 and EdU methods were used to assess the ability of cell proliferation. Plate experiment was used to detect the clone formation ability. Flow cytometry was used to detect the apoptosis rate. Transwell experiment was used to detect cell invasion ability. Dual luciferase reporter experiment detects the targeting relationship between circ-SFMBT2 and miR-7-5p, and between miR-7-5p and ADAM10. Transplanted tumor experiment in nude mice assessed the effect of knocking down circ-SFMBT2 on the growth of transplanted tumor. Immunohistochemical experiments were performed to detect the positive rates of ADAM10 and Ki67 proteins in transplanted tumor tissues.@*RESULTS@#The expression levels of circ-SFMBT2 and ADAM10 were increased in NSCLC tissues and cell lines, while decreased the expression of miR-7-5p. circ-SFMBT2 was negatively correlated with miR-7-5p, while miR-7-5p was negatively correlated with ADAM10. Silencing the overexpression of circ-SFMBT2 and miR-7-5p could inhibit cell proliferation, clone formation and invasion, and also promote apoptosis. circ-SFMBT2 could target miR-7-5p, and ADAM10 was the target gene of miR-7-5p. The combined effect of silencing circ-SFMBT2 and inhibition of miR-7-5p, as well as miR-7-5p overexpression and ADAM10 overexpression could promote cell proliferation, clone formation and invasion, and also suppress cell apoptosis. Silencing circ-SFMBT2 could inhibit the growth of transplanted tumors.@*CONCLUSION@#Silencing circ-SFMBT2 can suppress the proliferation, clone formation, invasion ability and induce apoptosis of NSCLC cells by regulating the miR-7-5p/ADAM10 axis.

Effect of BDNF-AS/miR-145-5p axis on renal tubular epithelial cell injury induced by high glucose / 中华医学遗传学杂志

OBJECTIVE@#To investigate the effect and possible mechanism of BDNF-AS on renal tubular epithelial cell injury induced by high glucose.@*METHODS@#Human renal tubular epithelial cells HK-2 were cultured in vitro and transfected with BDNF-AS small interfering RNA or miR-145-5p mimic, or co-transfected with BDNF-AS small interfering RNA and miR-145-5p inhibitor, respectively. The cells were then intervened with 30 mmol/L glucose for 24 hours. The expression of BDNF-AS and miR-145-5p were detected by RT-qPCR. Cell proliferation was detected by CCK-8, and apoptosis was detected by flow cytometry. The expression of Bcl-2 and Bax proteins were detected by Western blotting, and the levels of IL-1β and IL-6 in cell culture supernatant were detected by enzyme-linked immunosorbent assay. Dual luciferase reporter gene experiment was used to verify the regulatory relationship of BDNF-AS with miR-145-5p.@*RESULTS@#High glucose promoted the expression of BDNF-AS in HK-2 cells (P<0.05), but inhibited that of miR-145-5p (P<0.05). Interfering with BDNF-AS or overexpression of miR-145-5p decreased the inhibition rate, apoptosis rate and expression of Bax protein, IL-1β and IL-6 of HK-2 cells induced by high glucose (P<0.05), but promoted the expression of Bcl-2 protein (P<0.05). Interfering with miR-145-5p reversed the effect of interfering with BDNF-AS on the proliferation, apoptosis rate and the expression of IL-1β and IL-6 of HK-2 cells induced by high glucose. BDNF-AS could target and down-regulate miR-145-5p.@*CONCLUSION@#Interfering with BDNF-AS may promote the proliferation of renal tubular epithelial cells induced by high glucose and inhibit cell apoptosis and the expression of inflammatory factor by down-regulating miR-145-5p.

Research progress on role of traditional Chinese medicine in hepatic fibrosis based on miRNA-mediated activation of NLRP3 inflammasome / 中国中药杂志

In recent years, liver fibrosis has become a hotspot in the field of liver diseases. MicroRNA(miRNA)-mediated Nod-like receptor pyrin domain containing 3(NLRP3) inflammasome activation is pivotal in the pathogenesis of liver fibrosis. The present study mainly discussed the role of miRNA-mediated NLRP3 inflammasome activation in the pathogenesis of liver fibrosis. Different miRNA molecules regulated liver fibrosis by mediating NLRP3 inflammasome activation, including miRNA-350-3 p(miR-350-3 p)/interleukin-6(IL-6)-mediated signal transducer and activator of transcription 3(STAT3)/c-myc signaling pathway, miR-148 a-induced autophagy and apoptosis of hepatic stellate cells via hedgehog signaling pathway, miR-155-mediated NLRP3 inflammasome by the negative feedback of the suppressor of cytokine signaling-1(SOCS-1), miR-181 a-mediated downstream NLRP3 inflammatory pathway activation through mitogen-activated protein kinase kinase(MEK)/extracellular signal-regulated kinase(ERK)/nuclear transcription factor κB(NF-κB) inflammatory pathway, miR-21-promoted expression of NF-κB and NLRP3 of RAW264.7 cells in mice by inhibiting tumor necrosis factor-α inducible protein 3(A20), and miR-20 b-promoted expression of IL-1β and IL-18 by activating NLRP3 signaling pathway. Additionally, the anti-liver fibrosis mechanism of different active components in Chinese medicines(such as Curcumae Rhizoma, Glycyrrhizae Radix et Rhizoma, Aurantii Fructus, Polygoni Cuspidati Rhizoma et Radix, Moutan Cortex, Paeoniae Radix Alba, Epimedii Folium, and Cinnamomi Cortex) was also explored based on the anti-liver fibrosis effect of miRNA-mediated NLRP3 inflammasome activation.

Advances of miRNA-mediated regulatory roles in plant-microbe interaction / 生物工程学报

There are many bidirectional communication and crosstalk between microbes and host plants. The plant-pathogen interaction directly affects the survival of host plants, while the interaction between plants and their probiotics benefits both. Plant miRNA responds quickly to pathogenic or beneficial microbes when they enter the plant tissues, while microbes also produce miRNA-like RNA (milRNA) to affect plant health. These means miRNA or milRNA is an important fast-responding molecular mediator in plant-microbe interactions, and these internal mechanisms have been better understood in recent years. This review summarized the regulatory roles of miRNA in plant-pathogens and plant-probiotics interaction. The regulatory role of miRNA in disease resistance of host plants during plant-pathogens interaction, and the regulatory role of miRNA in promoting host growth and development during plant-probiotics interaction, as well as the cross-kingdom regulatory role of milRNA in host plants, were discussed in-depth.

Advances in the research of microRNA in Orchidaceae / 生物工程学报

As a class of small non-coding RNAs, microRNA (miRNA) is widely present and plays important regulatory roles in plant growth, development and stress response. Based on the mechanism of miRNAs in plants, we review the identification of miRNAs in some genera of Orchidaceae, the specific functions of several miRNAs and other relevant studies on miRNAs in the last decade, in order to provide a reference for better understanding function and regulatory network of small RNAs in orchids.

Sequencing and Proteomic Analysis of Exosomes from Apheresis Platelets in Different Storage Periods / 中国实验血液学杂志

OBJECTIVE@#To investigate the changes of gene sequencing and proteomics of apheresis platelet (AP) exosomes in different storage periods and predict the function of AP exosomes in different storage periods.@*METHODS@#Platelets at different storage periods of 0 day (D0), 3 day (D3) and 5 day (D5) were collected, exosomes were extracted with Gradient centrifugation; gene sequencing and proteomic analysis were used to analyze the exosomes, and biological functions of platelet exosomes were analyzed and predicted by bioinformatics. Liquid mass spectrometry (LMS) was used to detect the changes and function prediction of exosomes proteins. The small RNA sequencing library was prepared, and the constructed library was sequenced and bioinformatics technology was used for data analysis.@*RESULTS@#AP exosome iTRAQ protein analysis showed that AP exosomes stored in D3 with 55 up-regulated proteins and 94 down-regulated proteins (P<0.05, FC<0.83 or FC>1.2), while AP exosomes stored in D5 with 292 up-regulated proteins and 53 down-regulated proteins (P<0.05, FC<0.83 or FC>1.2) as compared with D0. KEGG pathway analysis showed that the proteins were mainly involved in transport and metabolism, immune system, cancer, membrane transport and other processes. There were statistically significant differences between AP exosome miRNAs in different storage days (P<0.01). The number of miRNA up-regulated and down-regulated was 374 and 255 as compared with the number of platelet exosomes miRNA stored in D3 and D0, while that was 297 and 242 in D5 and D0, and 252 and 327 in D5 and D3, respectively. The target genes of differential platelet exosome miRNAs were analyzed by GO enrichment. Target genes of differential miRNA were mainly involved in membrane composition, mainly played molecular functions binding to proteins, and participated in biological processes of transcriptional regulation.@*CONCLUSION@#The exosome differential proteins and miRNAs in D5 are significantly different from those in the D0 of APs, and they are involved in various biological processes.

Expression Level and Correlation of miR-211, miR-155, C-myc in Acute T Lymphocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression and correlation of miR-211, miR-155, and C-myc in acute T lymphocytic leukemia (T-ALL), aiming to provide evidence for the diagnosis and treatment.@*METHODS@#A total of 96 T-ALL patients who were diagnosed and treated in People's Hospital of Zhengzhou from June 2014 to June 2017 were selected, and 69 healthy volunteers who had a physical examination were selected as control group in the same period. Real-time fluorescent quantitative PCR (RT-PCR) was used to determine the expression levels of miR-211, miR-155, and C-myc in peripheral blood mononuclear cells in each group. Kaplan-Meier was used to analyze the survival of T-ALL patients and correlation of miR-211, miR-155, and C-myc with prognosis. Pearson correlation analysis was used to evaluate the correlation of miR-211, miR-155, and C-myc with disease risk.@*RESULTS@#The expression levels of miR-211 mRNA, miR-155 mRNA, and C-myc mRNA in T-ALL group were higher than those in the control group (P<0.01), those in non-remission group were higher than those in remission group (P<0.01), and those in high-risk group were also higher than those in low-risk group and intermediate-risk group (P<0.01). The survival time of T-ALL patients with low miR-211 expression was longer than that with high miR-211 expression (P<0.01), that with low miR-155 expression was longer than that with high miR-155 expression (P<0.01), and that with low C-myc expression was also longer than that with high C-myc expression (P<0.01). The high expression of miR-211, miR-155, and C-myc was linearly positively correlated with high risk of disease (r=0.749, 0.781, 0.804).@*CONCLUSION@#The expressions of miR-211, miR-155, and C-myc are up-regulated in T-ALL patients, closely related to prognosis, and linearly positively correlated with disease risk.

Mechanism of miR-155 Promoting Drug Resistance in Childhood Acute Lymphoblastic Leukemia by Regulating Wnt/β-Catenin Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the mechanism of miR-155 promoting drug resistance of children B-ALL to Ara-C by regulating Wnt/β-Catenin signaling pathway.@*METHODS@#The expression of miR-155 in bone marrow tissue and cell line of B-ALL was detected by PCR. The chemotherapy resistant strain REH/ Ara-C was constructed by using REH cells. REH/ Ara-C cells were transfected with miR-155 inhibitor. The proliferation of REH/Ara-C cells was detected by EdU. The apoptosis of REH/ Ara-C cells was detected by flow cytometry. The drug resistance of REH/Ara-C cells were analyzed by CCK-8 method and colony formation assay. The expression of Wnt/β-Catenin signaling pathway related proteins were determined by Western blot. MiR-155 inhibitor and Wnt activator agonist were used to transfect REH/Ara-C cells, and their effects on cell proliferation, apoptosis and drug resistance were determined.@*RESULTS@#Compared with normal tissues and cells, the expression level of miR-155 in B-ALL bone marrow tissue/cell line was increased (P<0.05); Compared with drug sensitive B-ALL tissues/cell lines, the expression level of miR-155 in drug resistant B-ALL tissues and cell lines was increased (P<0.05); Inhibition of miR-155 expression decreased the proliferation of REH/Ara-C cells (P<0.05), promoted apoptosis (P<0.05), enhanced the cytotoxicity of Ara-C (P<0.05), and inhibited Wnt/β-Catenin signaling pathway related protein and MDR1 gene expression (P<0.05), which could be reversed by activating Wnt expression (P<0.05).@*CONCLUSION@#The expression of miR-155 is up-regulated in bone marrow of children with B-ALL, which may be related to the activation of Wnt/β-Catenin signaling pathway promotes the proliferation of B-ALL cells and inhibits apoptosis, which leads to chemotherapy resistance.

Effect of Carvacrol on the Biological Behavior of Leukemia Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of carvacrol on the biological behavior of leukemia cells and its regulation to circ-0008717/miR-217 molecular axis.@*METHODS@#Human acute lymphoblastic leukemia cells Molt-4 were cultured in vitro, and different concentrations of carvacrol were added to the cells. si-NC and si-circ-0008717 were transfected into Molt-4 cells (si-NC group, si-circ-0008717 group). pcDNA, pcDNA-circ-0008717, anti-miR-NC, anti-miR-217 were transfected into Molt-4 cells and then added to carvacrol-treated cells (carvacrol+pcDNA group, carvacrol+pcDNA-circ-0008717 group, carvacrol+anti-miR-NC group, carvacrol+anti-miR-217 group). MTT, plate clone formation experiment, and flow cytometry were used to detect the viability of the cell, colony formation number, and apoptosis rate of cells, respectively. The RT-qPCR method was used to detect the expression levels of circ-0008717 and miR-217. The dual luciferase reporter gene experiment was used to detect the targeting relationship between circ-0008717 and miR-217.@*RESULTS@#After carvacrol treatment, the cell viability decreased significantly (r=-0.9405), expression level of circ-0008717 decreased (r=-0.9117), colonies formed number decreased (r=-0.9256), while the cell apoptosis rate increased (r= 0.8464), and the expression level of miR-217 increased (r=0.9468). Compared with the si-NC group, the expression level of miR-217 in si-circ-0008717 group increased (P＜0.001), the cell apoptosis rate increased (P＜0.001), while cell viability decreased (P＜0001), the number of colonies formed decreased (P＜0.001). Compared with the carvacrol+pcDNA group, the cell viability of the carvacrol+pcDNA-circ-0008717 group increased (P＜0.001), the number of colonies formed increased (P＜0.001), while the cell apoptosis rate decreased (P＜0.001). circ-0008717 could target miR-217. The cell viability of the carvacrol+anti-miR-217 group increased (P＜0.001), and the number of colonies formed increased (P＜0.001), while the cell apoptosis rate decreased (P＜0001) as compared with the carvacrol+anti-miR-NC group.@*CONCLUSION@#Carvacrol can promote the expression of miR-217 by down-regulating the expression of circ-0008717, thereby reducing the proliferation and cloning ability of leukemia cells and promoting cell apoptosis.

Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML / 中国实验血液学杂志

OBJECTIVE@#Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.@*METHODS@#The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.@*RESULTS@#In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.@*CONCLUSION@#The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

Expression Analysis of miRNA Profiles in Apheresis Platelets during Storage / 中国实验血液学杂志

OBJECTIVE@#To study the expression profiles changes of miRNA in apheresis platelets after 1, 3 and 5 days of storage.@*METHODS@#The apheresis platelets were collected from 20 volunteer blood donors. After mixing fully, the platelets were stored in a shaker with (22±2) ℃ horizontal oscillation. The samples were taken on the 1st, 3rd and 5th day, and used to sequence for miRNAs by DNA nanoball (DNB) sequencing technology, which were named as C_1, C_3 and C_5, respectively. The expression level of platelets miRNA was standardized by transcripts per kilobase million (TPM) algorithm. MiRNAs with P-value < 0.001 and the expression difference of more than two times were considered as significant difference between two groups. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (RT-qPCR).@*RESULTS@#By DNB sequencing, there were 688, 730, and 679 platelet miRNAs expressed in C_1, C_3 and C_5 group, respectively. Cluster analysis showed that the expression profile of miRNAs changed significantly. The expression level of the first 20 high abundance miRNAs was about 4/5 of the total amounts of expressed miRNAs in each group, which the top five miRNAs were miR-21-5p, miR-26a-5p, miR-199a-3p, miR-126-3p, and let-7f-5p. The correlation of high abundance platelet miRNAs among the three groups was high (R2=0.876, R2=0.979, R2=0.937, respectively) and the differences were not statistically significant (P>0.05). Compared with the differential expression of platelet miRNAs with more than 1 000 TPM in the C_3 and C_1 group, there were 6 differentially expressed miRNAs, including 3 up-regulated (miR-146a-5p, miR-379-5p, and miR-486-5p) and 3 down-regulated (miR-652-3p, miR-142-5p, and miR-7-5p). While in the C_5 and C_1 group, there were 4 differentially expressed miRNAs, including 2 up-regulated (miR-146a-5p and let-7b-5p) and 2 down-regulated (miR-30d-5p and miR-142-5p). Compared with the differentially expression of platelet miRNAs between 1-1 000 TPM in the C_3 and C_1 group, there were 133 differentially expressed miRNAs, in which 99 were up-regulated and 34 were down-regulated. While in the C_5 and C_1 group, there were 77 differentially expressed miRNAs, in which 31 were up-regulated and 46 were down-regulated. The six selected differentially expressed miRNAs verified by RT-qPCR were consistent with those of sequencing.@*CONCLUSION@#The expression profiles of platelets miRNAs change significantly among 1, 3, and 5 d of storage in vitro.

The Relationship between MicroRNA Expression Profiling in Imatinib-Resistant Cell Line K562/G and Potential Mechanism through FOXO3/Bcl-6 Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the drug resistant related FOXO3/Bcl-6 signaling pathway in K562/G cell line and its related microRNA(miRNA) mechanisms.@*METHODS@#The drug resistance potency of imatinib on K562/G was detected by MTT assay. The expression of FOXO3 and Bcl-6 proteins in K562 and K562/G cells was detected by Western blot. Real-time PCR (RT-PCR) was used to detect the expression of FOXO3 and Bcl-6 mRNA. The miRNA expression profiling in K562 and K562/G cells was analyzed by microarray technique, and the miRNA targeted to FOXO/Bcl-6 signaling pathway was identified.@*RESULTS@#The expression of FOXO3 and Bcl-6 protein was significantly increased in K562/G cells as compared with that in K562 cells (P<0.01), the expression level of Bcl-6 mRNA showed no increase in K562/G cells. However, FOXO3 mRNA was up-regulated in K562/G cells (P<0.05). MiRNA microarray results showed that 109 miRNAs were expressed differentially in K562 and K562/G cells. The expression of 81 miRNAs were up-regulated while 28 miRNAs were down-regulated. Through reverse prediction by bioinformatics, miR-6718-5p, miR-5195-5p, miR-4711-3p, miR-4763-5p, miR-4664-5p and miR-3176 were related to FOXO/Bcl-6 signaling pathway.@*CONCLUSION@#The FOXO3/Bcl-6 signaling pathway contributes to imatinib resistance in K562/G cell line, and the miRNA expression profiles showed significant differences between K562/G and K562 cells.

Expression of miR-106b-5p in children with primary immune thrombocytopenia and its correlation with T cells / 中国当代儿科杂志

OBJECTIVES@#To study the expression level of plasma miR-106b-5p in primary immune thrombocytopenia (ITP) and its correlation with the levels of T helper 17 cell (Th17) and regulatory T cell (Treg) and the Th17/Treg ratio.@*METHODS@#A total of 79 children with ITP (ITP group) and 40 healthy children (control group) were selected as subjects. According to the treatment response, the 79 children with ITP were divided into three groups: complete response (n=40), partial response (n=18), and non-response (n=21). Quantitative real-time PCR was used to measure the expression level of miR-106b-5p. Flow cytometry was used to measure the frequencies of Th17 and Treg, and the Th17/Treg ratio was calculated. The correlation of the expression level of plasma miR-106b-5p with the frequencies of Th17 and Treg and the Th17/Treg ratio was analyzed.@*RESULTS@#Compared with the control group, the ITP group had significantly higher levels of miR-106b-5p, Th17, and Th17/Treg ratio (P<0.05) and a significantly lower level of Treg (P<0.05). After treatment, the ITP group had significant reductions in the levels of miR-106b-5p, Th17, and Th17/Treg ratio (P<0.05) and a significant increase in the level of Treg (P<0.05). Compared with the partial response and non-response groups, the complete response group had significantly lower levels of miR-106b-5p, Th17, and Th17/Treg ratio (P<0.05) and a significantly higher level of Treg (P<0.05). The correlation analysis showed that in the children with ITP, the expression level of plasma miR-106b-5p was positively correlated with the Th17 level and the Th17/Treg ratio (r=0.730 and 0.816 respectively; P<0.001) and was negatively correlated with the Treg level (r=-0.774, P<0.001).@*CONCLUSIONS@#A higher expression level of miR-106b-5p and Th17/Treg imbalance may be observed in children with ITP. The measurement of miR-106b-5p, Th17, Treg, and Th17/Treg ratio during treatment may be useful to the evaluation of treatment outcome in children with ITP.