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Objective To explore the effect of endoplasmic reticulum stress activating transcription factor 6(ATF6)on the expression of reproduction related gene heat shock protein family A member 1 like(HSPA1L)and preliminari-ly clarify its regulatory molecular mechanism.Methods The ATF6 over-expression plasmid was transfected into HEK-293T cells and the over-expression efficiency was verified by RT-qPCR and Western blot.The transcriptome sequen-cing information of testis tissue of male ATF6 knockout mice was used to screen five reproduction related genes down-stream of ATF6.The dual luciferase reporter assay selected the downstream genes with high promoter activity and de-tected the effect of over-expression of ATF6 on the promoter activity of downstream genes.The possible binding sites of ATF6 and downstream gene promoters were predicted by gene-regulation.RT-qPCR and Western blot were used to detect the effect of over-expression of ATF6 on downstream gene expression in HEK-293T cells.Whether ATF6 binds to downstream gene promoters was determined by electrophoretic mobility shift assay(EMSA).Results The expres-sion of ATF6 mRNA(P＜0.001)and protein(P＜0.001 and P＜0.05)in HEK-293T cells was significantly increased after transfection.HSPA1L(P＜0.001 and P＜0.05),a reproductive related gene downstream of ATF6 was screened by transcriptome sequencing and dual luciferase reporter assay.ATF6 promoted the truncated promoter activity of HSPA1L(P＜0.001).After over-expression of ATF6,the expression of HSPA1L was significantly increased(P＜0.001).The differences were statistically significant.ATF6 protein could bind to the aagtcgtcac DNA sequence of HSPA1L promoter.Conclusions ATF6,a key molecule of endoplasmic reticulum stress,regulates the expression of reproduction related gene HSPA1L by binding to the promoter of HSPA1L.This result will lay a foundation for further research on the prevention or treatment of endoplasmic reticulum stress(ERS)related male infertility.
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The human genome encrypts around 20,000 protein coding genes, constituting around 1% of the total human genome sequence. The rest of it initially labeled as a “junk DNA” is transcribed to a distinct class of non-coding RNAs (ncRNAs) which do not code for any protein in the cell and their presence was quite intriguing to the researchers. The recent studies, however, have surprisingly revealed the vital roles of these ncRNAs in regulating an array of diverse cellular and biological processes in different organs including brain. The dysfunction of these regulatory ncRNAs in human brain causes certain neurological disorders and brain tumors which earlier have been widely linked to various risk factors such as oxidative stress, genetic mutations, aberrant protein degradation and dysfunctional neural network. This review provides an overview of different types of ncRNAs, their regulatory roles in brain functions and neurological disorders along with their prospects to be used as potential biomarkers and therapeutics.
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The many-banded krait, Bungarus multicinctus, has been recorded as the animal resource of JinQianBaiHuaShe in the Chinese Pharmacopoeia. Characterization of its venoms classified chief phyla of modern animal neurotoxins. However, the evolutionary origin and diversification of its neurotoxins as well as biosynthesis of its active compounds remain largely unknown due to the lack of its high-quality genome. Here, we present the 1.58 Gbp genome of B. multicinctus assembled into 18 chromosomes with contig/scaffold N50 of 7.53 Mbp/149.8 Mbp. Major bungarotoxin-coding genes were clustered within genome by family and found to be associated with ancient local duplications. The truncation of glycosylphosphatidylinositol anchor in the 3'-terminal of a LY6E paralog released modern three-finger toxins (3FTxs) from membrane tethering before the Colubroidea divergence. Subsequent expansion and mutations diversified and recruited these 3FTxs. After the cobra/krait divergence, the modern unit-B of β-bungarotoxin emerged with an extra cysteine residue. A subsequent point substitution in unit-A enabled the β-bungarotoxin covalent linkage. The B. multicinctus gene expression, chromatin topological organization, and histone modification characteristics were featured by transcriptome, proteome, chromatin conformation capture sequencing, and ChIP-seq. The results highlighted that venom production was under a sophisticated regulation. Our findings provide new insights into snake neurotoxin research, meanwhile will facilitate antivenom development, toxin-driven drug discovery and the quality control of JinQianBaiHuaShe.
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Myocardial fibrosis is a common pathological feature in various advanced cardiovascular diseases, and progressive fibrosis is the pathological basis for the development and progression of many cardiac arrhythmias and heart failure. There are no effective reversal drugs for myocardial fibrosis, which is related to the lack of understanding of the molecular mechanisms. Noncoding RNAs are a class of RNAs that do not function as coding proteins, and have been found to be intimately involved in the life cycle of cardiomyocyte differentiation, transcription and apoptosis, and are important regulators of cardiovascular diseases. An increasing number of studies have shown that noncoding RNAs regulate the proliferation and transformation of cardiac fibroblasts through related signaling pathways and can be used as potential biomarkers and novel therapeutic targets for cardiac fibrosis. This article reviews the relationship between noncoding RNAs and cardiac fibrosis.
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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.
Subject(s)
MicroRNAs/genetics , Droughts , Stress, Physiological/genetics , Crops, Agricultural/genetics , Crop ProductionABSTRACT
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.
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.
Subject(s)
Edible Grain , MicroRNAs/analysis , MicroRNAs/genetics , DroughtsABSTRACT
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.
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@#Diabetic retinopathy(DR)is one of the chronic microvascular complications of diabetes mellitus in the eye, leading to an increasing incidence of visual quality loss and even blindness in diabetic patients. However, so far, the mechanism of pathological changes leading to DR is very complex and not completely elucidated, and clinical treatment is mostly based on different stages of DR with symptomatic treatment, such as retinal laser photocoagulation, anti-vascular endothelial factor treatment, vitreous excision and membrane peeling surgery, <i>etc</i>. There is still a lack of safe and effective treatment methods, and it is therefore necessary to find new targets for diagnosis and treatment. With the development of molecular biology analysis technology, circular RNA(circRNA)has become one of the important research hotspots at present. It is reported that under the stimulation of various factors, the regulated expression of genes-circRNA regulates the development of DR by regulating the functions of retinal microvascular pericytes, retinal microvascular endothelial cells, and retinal pigment epithelial cells, which are involved in the pathological processes such as retinal basement membrane thickening, vascular leakage, apoptosis, inflammatory response and neovascularization. Therefore, this paper focuses on the research progress of cyclic RNA, competitive endogenous RNA and microRNA in the molecular mechanism of DR pathological alterations and therapeutic prospects, in order to provide some reference for clinical and basic research.
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@#Metastasis-associated lung adenocarcinoma transcript 1(MALAT1)is one of the first identified LncRNA associated with human diseases. Unlike most members of the LncRNA family, MALAT1 is found in almost all human tissues and expressed at a relatively high level. At present, MALAT1 is known to play a vital role in the pathophysiological process of many diseases such as tumors, cardiovascular diseases, and nervous system diseases. In recent years, studies have found that MALAT1 may be involved in many ocular diseases(such as diabetic retinopathy, cataracts, glaucoma, retinoblastoma, neonatal retinopathy, <i>etc</i>.)play an important role in the pathological development process, and it is expected to become an effective target for the diagnosis and treatment of eye diseases. This article summarizes the research progress of eye diseases in which MALAT1 has participated in recent years.
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Bronchial asthma is a common heterogeneous chronic inflammatory disorder of airways characterized by airway hyperreactivity， mucus hypersecretion， and airflow obstruction. The incidence of asthma has been on the rise worldwide， and about 45.7 million adults in China suffer from asthma. Asthma is considered a costly disease， resulting in a significant economic and social burden. microRNAs （miRNAs） are long noncoding RNAs that regulate gene expression. They play a role in asthma through their activity in multiple immune and non-immune cell subsets. They impact multiple facets of critical cell function including cell survival， proliferation， and differentiation， which in turn induce the occurrence of airway spasm， mucus hypersecretion， and asthma symptoms. Traditional Chinese medicine has a long history in the treatment of asthma. Over the past a few decades， a growing number of herbs have proven effective in treating asthma in clinical trials or asthma inflammation in animal models. Chinese medicine has the features of multiple components and multiple targets. Evidence suggests that Chinese medicine and components of Chinese medicine can regulate immune homeostasis， improve airway inflammation and airway remodeling by modulating microRNA expression for asthma treatment. However， there is a lack of systematic and detailed reviews on the regulation of asthma-related microRNA expression by Chinese medicine. The article aims to review the correlation between microRNAs and asthma in animal experiments and clinical trials in recent years， as well as the mechanism of microRNA regulation by Chinese medicine in the treatment of asthma， with the intention of providing a reference for basic research and clinical application.
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This study analyzed RNA expression of genes for three serum tumor markers, alpha fetoprotein (AFP), human chorionic gonadotropin (hCG), and lactate dehydrogenase (LDH), in patients with testicular germ cell tumors (TGCT) type 2. The gene AFP encodes AFP, the gene for chorionic gonadotropin beta polypeptide 5 (CGB5) encodes a major part of the specific beta subunit of hCG, and the genes for LDH subunit A (LDHA), LDH subunit B (LDHB), and LDH subunit C (LDHC) encode three different subunits of LDH. LDHB encodes the LDHB subunit present as a tetramer in LDH isoenzyme 1 (LDH-1). We examined three datasets with 203 samples of normal testis tissue (NT) and TGCT type 2. Yolk sac tumor (YST) expressed RNA of AFP fourteen thousand times higher than seminoma (SE), embryonal carcinoma (EC), and teratoma (TER) combined (P = 0.00015). In the second microarray, choriocarcinoma (CC) expressed RNA of CGB5 ten times higher than other histologic types of TGCT combined. EC expressed RNA of LDHB twice higher than SE, YST and TER combined (P = 0.000041). EC expressed RNA of LDHB higher than that YST expressed RNA of AFP and that CC expressed RNA of CGB5. In conclusion, TGCT type 2 expressed RNA of LDHB markedly higher than the RNA of 23 other candidate genes for TGCT type 2.
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In eukaryote cells,transcription from genome DNA is a key process of gene expression.The transcription products contain not only messenger RNAs that code proteins,but also various types of non-coding RNAs.During transcription,some of the gene loci produce more than one kind of RNA molecule,including coding RNAs and more often non-coding RNAs.These gene loci that generate several kinds of RNA molecules are named supergenes.According to the transcription pattern,supergenes are divided into three types,known as types Ⅰ,Ⅱ and Ⅲ.In this review,we summarize the transcription pattern of each type of supergene,and exposit the role of these genes in cells.
Subject(s)
Gene Expression , RNA, MessengerABSTRACT
Ensuring agricultural food security is a major concern for the future world, and being the second mostconsumed crop, rice yield needs an urgent upliftment. Grain yield is a pleiotropic trait that employs a plethoraof genes functioning in complex signalling cascades. The yield related genes are controlled by variousregulatory factors including the microRNAs (miRNAs), the small 20–22 nucleotide (nt) non-coding RNAs,which have emerged as the master ribo-regulators of eukaryotic genes. Plant miRNAs can bind to highlycomplementary sequences in the target messenger RNAs (mRNAs) and negatively regulate gene expression tocoordinate the various biological processes involved in plant development. In rice, an ideal plant architecture(IPA) has been regarded as the key to attain high yield and several miRNAs have been deciphered to playimportant roles in orchestrating vital regulatory procedures for achieving optimum plant morphological yieldrelated traits like less unproductive tillers, more panicle branches and heavier grains. In this review, we presentand discuss the various genetic engineering strategies undertaken to manipulate the miRNA-mRNA expressionlevels in order to achieve improved grain output by modulation of rice plant architecture and recent advancesmade in this regard
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BRAF is a serine/threonine kinase that harbors activating mutations in ∼7% of human malignancies and ∼60% of melanomas. Despite initial clinical responses to BRAF inhibitors, patients frequently develop drug resistance. To identify candidate therapeutic targets for BRAF inhibitor resistant melanoma, we conduct CRISPR screens in melanoma cells harboring an activating BRAF mutation that had also acquired resistance to BRAF inhibitors. To investigate the mechanisms and pathways enabling resistance to BRAF inhibitors in melanomas, we integrate expression, ATAC-seq, and CRISPR screen data. We identify the JUN family transcription factors and the ETS family transcription factor ETV5 as key regulators of CDK6, which together enable resistance to BRAF inhibitors in melanoma cells. Our findings reveal genes contributing to resistance to a selective BRAF inhibitor PLX4720, providing new insights into gene regulation in BRAF inhibitor resistant melanoma cells.
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Objective To investigate the effect of Huangkui extract powder (HK) on the expression of nephrin and podocin proteins in mouse podocytes induced by high glucose,which is involved in the treatment of diabetic nephropathy (DN).Methods Cultured mouse podocytes (MPC5) were incubated in high glucose and HK at 5.6 mmol/L NG,5.6 mmol/L NG + 0.45 g/L HK,25 mmol/L HG,25 mmol/L HG + 0.45 g/L HK,respectively.The 5.6 mmol/L NG group was used as normal control.After 24 hours of intervention,we detected podocyte apoptosis by Annexin-V FITC/PI double staining,measured the mRNA and protein expression of nephrin and podocin by qRT-PCR and Western blot.Results Compared with the control group (5.6 mmol/L,NG),the apoptosis rate of podocytes in the high glucose concentration group (25 mmol/L,HG) was significantly higher [(20.39 ± 0.03) ％ vs.(17.70 ± 0.91) ％,t =2.947,P ＜ 0.05)].The apoptosis rate of podocytes in the 25 mmol/L HG + 0.45 g/L HK group was significantly lower than that in the 25 mmol/L HG group [(11.96 ± 1.11) ％ vs.(20.39 ± 0.03) ％,t =7.586,P ＜ 0.01].The results of qRT-PCR and Western blot showed that the expression of nephrin and podocin was significantly inhibited by high glucose concentration compared with the control group[(0.489 ±0.040) vs.(0.721 ±0.022),t =4.992,P ＜0.01;(0.387 ±0.014) vs.(0.778 ±0.036),t =10.050,P ＜0.01],and the expression of podocin and nephrin was increased by appropriate concentration of H K [(0.603 ± 0.013) vs.(0.489 ± 0.040),t =2.653,P＜0.05;(0.640±0.024) vs.(0.387 ±0.014),t=8.946,P＜0.01].Conclusion Podocyte apoptosis can be induced by prolonged high glucose treatment,but a certain concentration of HK can inhibit podocyte death induced by high glucose.The possible mechanism is that HK may inhibit the apoptosis of podocytes by regulating the expression of podocin and nephrin in podocytes at high glucose concentration,thus plays a protective role on podocytes.
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@#Long non-coding RNAs(LncRNAs)refer to RNAs that are longer than 200 nucleotides and cannot encode a protein. With the application of next-generation sequencing technology, whole-genome analysis shows that LncRNAs can regulate gene expression in immune response, epigenetic, gene transcription and post-transcriptional levels, thereby participating in the maintenance of cell proliferation and apoptosis, tissue homeostasis and other physiological processes. LncRNAs have been shown to involve in initiation and development of a variety of human diseases in recent years. This article reviews the research progress of LncRNAs in common diseases of ophthalmology, in order to early diagnosis and treatment of related ophthalmic diseases.
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@#Myopia is one of the most common eye diseases in the world. MicroRNAs(miRNAs)are small, non-protein-coded single-stranded RNAs with a length of about 20-25 nucleotides,which are widely involved in the process of post-transcriptional regulation of many physiological and pathological processes of organisms. It has attracted extensive attention for the regulation role of miRNAs in the process of induction and development of myopia in recent years. This article reviews the regulatory role and related signal pathways of miRNAs in the development of myopia. It will provide highlights for the understanding of molecular mechanism of myopia and benefit for potential molecular diagnosis of myopia in the future.
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@#Tooth agenesis is a common tooth number deficiency that occurs in the tooth-forming process or earlier period of tooth germ development and has a serious impact on the maxillofacial development, aesthetics and masticatory function of patients. According to the presence or absence of systemic symptoms, tooth agenesis can be divided into syndromic tooth agenesis and nonsyndromic tooth agenesis. In recent years, the discovery of new related genes, new mutation sites and related molecular mechanisms has become a major direction of gene research. This article will review the current research progress of the signaling pathways related to nonsyndromic tooth agenesis, such as the WNT/beta-catenin pathway, TGF-β/BMP pathway, PAX9, MSX1, and the EDA/EDAR/NF-κb pathway, and their molecular mechanisms. The interaction between Pax9 activating the Wnt/β-catenin and TGF-β/BMP pathways, MSX1 activating the TGF-β/BMP pathway, and Wnt activating the EDA/EDAR/NF-κb pathway was also found, which provides a new theoretical basis for the prevention and treatment of tooth agenesis. The molecular mechanism of nonsyndromic tooth agenesis is rarely studied; thus, the exploration of its mechanism will become one of the main research directions in the future.
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Infertility or subfertility in bovine males may be related to spermatic microRNAs (miRNAs), whose function seems to be associated with the regulation of gene expression, degradation orstorage of messenger RNAs (mRNAs) for later translation into early embryonic development. Thus, the purpose of this study was to identify differentially expressed miRNAs in semen samples from bulls (Bos taurus) with low and high efficiency in the in vitro embryo production (IVEP) and to evaluate if they can be used as markers of semen efficiency for IVEPs. In order to identify miRNA markers of semen efficiency in thein vitro embryo production, eight semen samples from each animal, one bull with high and two bulls with low efficiency in IVEPs were used to perform the RNAseq technique for miRNAs. Initially the samples were washed with PBS to remove the extender semen and subsequently were submitted to RNA extraction protocols performed according to procedures described by mirVana™ miRNA Isolation Kit. Then, the amplification of the miRNAs was carried out, not to mention the preparation of the library (Ion Total RNA-Seq Kit v2), the PCR emulsion reaction, enrichment, as well as the injection of the sample on the chip by the Ion Chef equipment. The sequencing was done on Ion Proton equipment. The comparison between the samples was established using two methodologies for searching for targets to increase the robustness of the analytical procedure: the miRanda program using as cutoff minimum free energy of the hybridization -20 kcal/Mol, 100% of identity between nucleotides 2 and 8 of the miRNA, and the RNAhybrid program, using as cutoff minimum free energy of hybridization -20 kcal/mol. In sum, 1306 miRNAs were identified in the samples. The bta-miR-380-5p, bta-miR-155, bta-miR-30c and bta-miR-34a genes were identified by the Bioinformatics as being strongly differentially expressed between the groups, indicating that these genes may present themselves as possible efficiency markers. However, it has become clear that there is no single miRNA that marks different types and causes of fertility problems.
A infertilidade ou subfertilidade em machos bovinos pode estar relacionada a microRNAs espermáticos (miRNAs), cuja função parece estar associada à regulação da expressão gênica, degradação ou armazenamento de RNAs mensageiros (mRNAs), para posterior tradução no desenvolvimento embrionário inicial. Assim, o objetivo deste estudo foi identificar miRNAs diferencialmente expressos em amostras de sêmen de touros (Bos taurus) com baixa e alta eficiência na produção in vitro de embriões (PIVE) e avaliar se eles podem ser utilizados como marcadores de eficiência do sêmen em PIVEs. Para identificar miRNA marcadores da eficiência de sêmen em PIVE, oito amostras de sêmen de cada animal, sendo um touro com alto e dois touros com baixa eficiência, foram utilizados para realizar a técnica de RNAseq para miRNAs. Inicialmente as amostras foram lavadas com PBS para remover o diluente do sêmen e, posteriormente, foram submetidas a protocolos de extração de RNA realizados de acordo com os procedimentos descritos pelo Kit de isolamento de miRNA mirVana ™. Em seguida, foi realizada a amplificação dos miRNAs, a preparação da biblioteca (Ion RNA-Seq Kit v2), a reação de emulsão de PCR, enriquecimento e a injeção das amostras no chip apropriado utilizando o equipamento Ion. Chef. O sequenciamento foi realizado no equipamento Ion Proton. A comparação entre as amostras foi estabelecida utilizando duas metodologias de busca de alvos para aumentar a robustez do procedimento analítico: o programa miRanda utilizando como valor de corte a energia mínima livre de hibridização -20 kcal / Mol e 100% de identidade entre os nucleotídeos 2 e 8 do miRNA, e o programa RNAhybrid, utilizando como valor de corte a energia mínima livre de hibridização -20 kcal / mol. Em suma, 1306 miRNAs foram identificados nas amostras. Os genes bta-miR-380-5p, bta-miR-155, bta-miR-30c e bta-miR-34a foram identificados pela bioinformática como sendo fortemente diferencialmente expressos entre os grupos, indicando que esses genes podem se apresentar como possíveis marcadores de eficiência. No entanto, ficou claro que não existe um único miRNA que marque diferentes tipos e causas de problemas de fertilidade.
Subject(s)
RNA , Cattle , Embryo Research , InfertilityABSTRACT
Bacillus subtilis can be widely used as an important microorganism for metabolic engineering and recombinant proteins expression in industrial biotechnology and synthetic biology. However, it is difficult to make accurate regulation of exogenous gene by biological tools in B. subtilis, which limits the application of B. subtilis in synthetic biology. The purpose of this study is to develop regulatory tools for precise control of gene expression by using non-coding RNAs, by which the activation of heterologous gene could be achieved without the auxiliary protein factors. We constructed the synthetic riboswitch E and aptazyme AZ using the theophylline aptamer. Six different native promoters from B. subtilis were functionally adapted with the E and AZ to fabricate an array of novel regulatory elements activated by theophylline. Then, we determined the performance of these elements using green fluorescence protein as reporter, and then further verified using red fluorescence protein and pullulanase as cargo proteins. Results showed that the same kind of RNA elements with different promoters showed different levels of efficiency. Promoter PsigW and E combination (sigWE) had the highest induction rate in B. subtilis. Compared with the control group, it can produce the induction rate of 16.8. Promoter PrpoB and AZ combination (rpoBAZ) showed the highest induction rate of 6.2. SigWE mediated mCherry induction rate was 9.2, and P43E mediated pullulanase induction rate was 32.8, in which enzyme activity reached 81 U/mL. This study confirmed that GFP, mCherry and pullulan can all be regulated by riboswitch and aptazyme, but there were differences between different combinations of promoters with RNA regulators.