The scinderin gene (SCIN) is the direct target of miR3085-3p in chondrocytes

MiR-3085-3p was shown to play a crucial role in cartilage biology, with potential impacts in osteoarthritis (OA). Insight into this miRNA function could be of practical importance for future miRNA-based therapy, however, little is known regarding the biological roles of this miRNA. The physiologic function of an individual miRNA is dictated through its mRNA targets, and as SCIN (scinderin, also known as adseverin) was reported to be involved in chondrocyte differentiation, maturation, and phenotype maintenance, this study aimed to prove SCIN is a direct target of miRNA-3085-3p. Bioinformatics algorithms were utilized for predicting their interacting sites. Gain- and loss-of-function experiments with miRNA-3085-3p were performed and SCIN expression was measured by real-time RT-PCR. SCIN 3?UTR regions harboring either the miR-3085-3p seed site or its mutant version were cloned into pmirGLO downstream of a reporter firefly luciferase encoding gene. The effect of miR-3085-3p on this region was determined by the luciferase assay. Four binding sites of miR-3085-3p in SCIN 3?UTR were identified. SCIN expression level was found to be inversely correlated with the level of miRNA-3085-3p. MiR3085-3p directly binds to its binding sites in SCIN 3? UTR. These data suggest that SCIN is the direct target of miR-3085-3p in chondrocyte cells.

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Effect of miR-301a/PTEN pathway on the proliferation and apoptosis of cervical cancer

Innate Immunity ◽

10.1177/1753425919840702 ◽

2019 ◽

Vol 25 (4) ◽

pp. 217-223 ◽

Cited By ~ 3

Author(s):

Li-na Peng ◽

Wen-tian Shi ◽

Huan-rong Feng ◽

Chuan-yu Wei ◽

Qi-nan Yin

Keyword(s):

Cervical Cancer ◽

Cervical Carcinoma ◽

Hela Cells ◽

Pten Expression ◽

Cell Counting ◽

Luciferase Assay ◽

Loss Of Function ◽

Reverse Pattern ◽

Significant Difference ◽

Direct Target

The aim of this study was to evaluate the effect of the miR-301a/PTEN pathway in cervical cancer. miR-301a and PTEN expression were measured by quantitative real-time PCR (qRT-PCR) in tissues samples and HeLa cells. PTEN protein level was determined by Western blotting. Dual reporter luciferase assay was performed to validate PTEN as a direct target of miR-301a. The gain- and loss-of function assay was performed by miR-301a overexpression and silencing. Cell proliferation was monitored by cell counting Kit-8 (CCK-8). Cell apoptosis was quantitated by flow cytometry. SPSS was used to analyze the significant difference in the treatments. miR-301a demonstrated a significantly higher expression in cervical carcinoma tissues compared with the paired non-carcinoma tissues ( n = 12), while PTEN expression was found to be significantly lower in cervical carcinoma tissues than their paired non-carcinoma tissues ( n = 12). In addition, PTEN was identified as the direct target of miR-301a. Moreover, overexpression of miR-301a significantly promoted HeLa cells proliferation and anti-apoptosis which had a reverse pattern after PTEN overexpression. Our results confirm PTEN as a direct target of miR-301a in HeLa cells and suggest that miR-301a/PTEN pathway contributes to the development and progression of cervical cancer.

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ACOX1, regulated by C/EBPα and miR-25-3p, promotes bovine preadipocyte adipogenesis

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0250 ◽

2021 ◽

Author(s):

Feng Zhang ◽

Qi Xiong ◽

Hu Tao ◽

Yang Liu ◽

Nian Zhang ◽

...

Keyword(s):

Fatty Acid ◽

Binding Sites ◽

Site Directed Mutagenesis ◽

Luciferase Assay ◽

Directed Mutagenesis ◽

Loss Of Function ◽

Mobility Shift ◽

Promoter Deletion Analysis ◽

Factor C ◽

Peroxisomal Fatty Acid

Acyl-Coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme in peroxisomal fatty acid β-oxidation of fatty acids. Previous studies have reported that ACOX1 was correlated with the meat quality of livestock, while the role of ACOX1 in intramuscular adipogenesis of beef cattle and its transcriptional and post-transcriptional regulatory mechanisms remain unclear. In the present study, gain-of-function and loss-of-function assays demonstrated that ACOX1 positively regulated the adipogenesis of bovine intramuscular preadipocytes. The C/EBPα-binding sites in the bovine ACOX1 promoter region at -1142 to -1129 bp, -831 to -826 bp, and -303 to -298 bp were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) further showed that these three regions are C/EBPα-binding sites, both in vitro and in vivo, indicating that C/EBPα directly interacts with the bovine ACOX1 promoter and inhibits its transcription. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the ACOX1 3’untranslated region (3’UTR). Taken together, our findings suggest that ACOX1, regulated by transcription factor C/EBPα and miR-25-3p, promotes adipogenesis of bovine intramuscular preadipocytes via regulating peroxisomal fatty acid β-oxidation.

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microRNA-186 in extracellular vesicles from bone marrow mesenchymal stem cells alleviates idiopathic pulmonary fibrosis via interaction with SOX4 and DKK1

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02083-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jing Zhou ◽

Yang Lin ◽

Xiuhua Kang ◽

Zhicheng Liu ◽

Wei Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Idiopathic Pulmonary Fibrosis ◽

Pulmonary Fibrosis ◽

Extracellular Vesicles ◽

Luciferase Assay ◽

Therapeutic Effects ◽

Loss Of Function ◽

Fibroblast Activation ◽

Promising Therapeutic Approach

Abstract Background Previous reports have identified that human bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) with their cargo microRNAs (miRNAs) are a promising therapeutic approach for the treatment of idiopathic pulmonary fibrosis (IPF). Therefore, we explored whether delivery of microRNA-186 (miR-186), a downregulated miRNA in IPF, by BMSC EVs could interfere with the progression of IPF in a murine model. Methods In a co-culture system, we assessed whether BMSC-EVs modulated the activation of fibroblasts. We established a mouse model of PF to evaluate the in vivo therapeutic effects of BMSC-EVs and determined miR-186 expression in BMSC-EVs by polymerase chain reaction. Using a loss-of-function approach, we examined how miR-186 delivered by BMSC-EVs affected fibroblasts. The putative relationship between miR-186 and SRY-related HMG box transcription factor 4 (SOX4) was tested using luciferase assay. Next, we investigated whether EV-miR-186 affected fibroblast activation and PF by targeting SOX4 and its downstream gene, Dickkopf-1 (DKK1). Results BMSC-EVs suppressed lung fibroblast activation and delayed IPF progression in mice. miR-186 was downregulated in IPF but enriched in the BMSC-EVs. miR-186 delivered by BMSC-EVs could suppress fibroblast activation. Furthermore, miR-186 reduced the expression of SOX4, a target gene of miR-186, and hence suppressed the expression of DKK1. Finally, EV-delivered miR-186 impaired fibroblast activation and alleviated PF via downregulation of SOX4 and DKK1. Conclusion In conclusion, miR-186 delivered by BMSC-EVs suppressed SOX4 and DKK1 expression, thereby blocking fibroblast activation and ameliorating IPF, thus presenting a novel therapeutic target for IPF.

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Decreased level of miR-1301 promotes colorectal cancer progression via activation of STAT3 pathway

Biological Chemistry ◽

10.1515/hsz-2020-0301 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Fangfang Yang ◽

Hua Wang ◽

Bianbian Yan ◽

Tong Li ◽

Lulu Min ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Migration ◽

Cancer Progression ◽

Luciferase Assay ◽

Migration And Invasion ◽

Loss Of Function ◽

Aberrant Expression ◽

Cell Migration And Invasion ◽

Lovo Cells ◽

Colorectal Cancer Progression

Abstract The molecular pathogenesis of colorectal cancer (CRC) has been widely investigated in recent years. Accumulating evidence has indicated that microRNA (miRNA) dysregulation participates in the processes of driving CRC initiation and progression. Aberrant expression of miR-1301 has been found in various tumor types. However, its role in CRC remains to be elucidated. In the present study, we identified miR-1301 was enriched in normal colorectal tissues and significantly down-regulated in CRC. Decreased level of miR-1301 strongly correlated with aggressive pathological characteristics, including advanced stage and metastasis. Bioinformatics and dual luciferase assay demonstrated that STAT3 is a direct target of miR-1301. Gain and loss-of-function assays showed that miR-1301 had no effect on cell proliferation. Overexpression of miR-1301 suppressed cell migration and invasion capacity of pSTA3-positive LoVo cells, but not pSTAT3-negative SW480 cells, while inhibition of miR-1301 consistently promoted cell migration and invasion in both cell lines. Additionally, miR-1301 inhibition restored the suppressed migration and invasion of STAT3- knockdown LoVo cells. MiR-1301 functioned as a tumor suppressor to modulate the IL6/STAT3 signaling pathway. In summary, this study highlights the significant role of miR- 1301/STAT3 axis in CRC metastasis.

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Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057112465184 ◽

2012 ◽

Vol 18 (4) ◽

pp. 453-461 ◽

Cited By ~ 17

Author(s):

Ellen Siebring-van Olst ◽

Christie Vermeulen ◽

Renee X. de Menezes ◽

Michael Howell ◽

Egbert F. Smit ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Firefly Luciferase ◽

Luciferase Assay ◽

Luciferase Reporter ◽

Chemical Components ◽

Simple Liquid ◽

Luciferase Reporter Construct ◽

Reagent Cost ◽

Firefly Luciferase Gene

The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

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Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS

10.1101/221978 ◽

2017 ◽

Cited By ~ 1

Author(s):

Maksym Bobrovskyy ◽

Jane K. Frandsen ◽

Jichuan Zhang ◽

Anustup Poddar ◽

Muhammad S. Azam ◽

...

Keyword(s):

Small Rna ◽

Binding Sites ◽

Sugar Transport ◽

Enteric Bacteria ◽

Sugar Uptake ◽

Rna Chaperone ◽

Target Mrnas ◽

Mrna Targets ◽

Molecular Features ◽

Response To Stress

ABSTRACTThe mechanisms by which small RNA (sRNA) regulators select and prioritize target mRNAs remain poorly understood, but serve to promote efficient responses to environmental cues and stresses. We sought to uncover mechanisms that establish regulatory hierarchy for a model sRNA, SgrS, found in enteric bacteria and produced under conditions of metabolic stress when sugar transport and metabolism are unbalanced. SgrS post-transcriptionally controls a nine-gene regulon to restore growth and homeostasis under stress conditions. An in vivo reporter system was used to quantify SgrS-dependent regulation of target genes and established that SgrS exhibits a clear preference for certain targets, and regulates those targets efficiently even at low SgrS levels. Higher SgrS concentrations are required to regulate other targets. The position of targets in the regulatory hierarchy is not well-correlated with the predicted thermodynamic stability of SgrS-mRNA interactions or the SgrS-mRNA binding affinity as measured in vitro. Detailed analyses of SgrS interaction with asd mRNA demonstrate that SgrS binds cooperatively to two sites and remodels asd mRNA secondary structure. SgrS binding at both sites increases the efficiency of asd mRNA regulation compared to mutants that have only a single SgrS binding site. Our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features. The sRNA-mRNA interaction, including the number and position of sRNA binding sites on the mRNA and cofactors like the RNA chaperone Hfq, seem to tune the efficiency of regulation of specific mRNA targets.IMPORTANCETo survive, bacteria must respond rapidly to stress and simultaneously maintain metabolic homeostasis. The small RNA (sRNA) SgrS mediates the response to stress arising from imbalanced sugar transport and metabolism. To coordinate the stress response, SgrS regulates genes involved in sugar uptake and metabolism. Intrinsic properties of sRNAs such as SgrS allow them to regulate extensive networks of genes. To date, sRNA regulation of targets has largely been studied in the context of “one sRNA-one target”, and little is known about coordination of multi-gene regulons and sRNA regulatory network structure. Here, we explore the molecular basis for regulatory hierarchy in sRNA regulons. Our results reveal a complex interplay of factors that influence the outcome of sRNA regulation. The number and location of sRNA binding sites on mRNA targets and the participation of an RNA chaperone dictate prioritized regulation of targets to promote an efficient response to stress.

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Normal adenylate ribonucleotide content in mouse embryos homozygous for the t12 mutation

Development ◽

10.1242/dev.78.1.43 ◽

1983 ◽

Vol 78 (1) ◽

pp. 43-51

Author(s):

Horst Spielmann ◽

Robert P. Erickson

Keyword(s):

In Vitro Culture ◽

Normal Values ◽

Firefly Luciferase ◽

Blastocyst Stage ◽

Mouse Embryos ◽

Luciferase Assay ◽

Preimplantation Mouse Embryos ◽

Preimplantation Mouse

The recently improved firefly luciferase assay was used to determine ATP, ADP or AMP in single preimplantation mouse embryos from crosses yielding lethal t12/t12 embryos. Normal values of the three adenylate ribonucleotides were found in freshly collected 2-cell and 4-cell embryos and during in vitro culture to the blastocyst stage. A decrease in adenylate ribonucleotide content was seen in putative t12/t12 embryos only when they were degenerating.

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Kernicterus in Rats with an Inherited Deficiency of Glucuronyal Transferase

PEDIATRICS ◽

10.1542/peds.24.1.73 ◽

1959 ◽

Vol 24 (1) ◽

pp. 73-73

Keyword(s):

Binding Sites ◽

Renal Excretion ◽

Practical Importance ◽

Human Beings ◽

Human Infants ◽

Toxic Agent ◽

History Of ◽

The Central Nervous System ◽

The Brain ◽

Comprehensive Study

Aside from the great theoretic interest, the experiments described in this paper have considerable practical importance in providing a means of studying the efficacy of treatments designed to prevent kernicterus. A comprehensive study of a strain of rats (the Gunn strain) which have a hereditary deficiency of the enzyme required to conjugate bilirubin, and thus develope jaundice due to increased concentration of unconjugated bilirubin in the blood and tissues. The rats developed kernicterus which was apparently identical with that seen in human beings and is the only example of kernicterus in animals that fulfills rigid criteria outlined by the authors. Extensive data on the natural history of the bilirubinemia and development of kernicterus in the rats, as determined by chemical and pathologic techniques, are provided. Bilirubin itself is incriminated as the toxic agent producing the characteristic changes in the brain in kernicterus. Sulfonamides were found to augment the toxic effects of bilirubin, apparently because of competition between bilirubin and sulfonamides for binding sites on serum albumin. Neither infection nor hypoxia appeared to aggravate the effects of bilirubin. Administration of sodium glucuronate to jaundiced rats was followed by a decrease in bilirubin in the serum which at times exceeded 50%. This was not accompanied by any postponement of the onset of signs of damage to the central nervous system and did not prevent development of kernicterus. It appeared that the decrease in bilirubin in the serum may have resulted from an increased transferral to tissues rather than elimination through renal excretion. On the basis of the knowledge of this strain of rats, it should be possible to explore the usefulness of proposed therapeutic regimens in the experimental animal without jeopardizing the course of human infants who might be successfully treated with exchange transfusion pending the discovery of a more satisfactory therapy.

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A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription

Nucleic Acids Research ◽

10.1093/nar/gkz1053 ◽

2019 ◽

Cited By ~ 2

Author(s):

Yuanyuan Wang ◽

Yalan Sheng ◽

Yongqiang Liu ◽

Wenxin Zhang ◽

Ting Cheng ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Single Molecule ◽

Molecular Mechanisms ◽

Epigenetic Mark ◽

Loss Of Function ◽

H3k4 Methylation ◽

Adenine Methylation ◽

Direct Target ◽

Rna Polymerase Ii Transcription ◽

Basal Fungi

Abstract Rediscovered as a potential eukaryotic epigenetic mark, DNA N6-adenine methylation (6mA) varies across species in abundance and its relationships with transcription. Here we characterize AMT1—representing a distinct MT-A70 family methyltransferase—in the ciliate Tetrahymena thermophila. AMT1 loss-of-function leads to severe defects in growth and development. Single Molecule, Real-Time (SMRT) sequencing reveals that AMT1 is required for the bulk of 6mA and all symmetric methylation at the ApT dinucleotides. The detection of hemi-methylated ApT sites suggests a semi-conservative mechanism for maintaining symmetric methylation. AMT1 affects expression of many genes; in particular, RAB46, encoding a Rab family GTPase involved in contractile vacuole function, is likely a direct target. The distribution of 6mA resembles H3K4 methylation and H2A.Z, two conserved epigenetic marks associated with RNA polymerase II transcription. Furthermore, strong 6mA and nucleosome positioning in wild-type cells is attenuated in ΔAMT1 cells. Our results support that AMT1-catalyzed 6mA is an integral part of the transcription-associated epigenetic landscape. AMT1 homologues are generally found in protists and basal fungi featuring ApT hyper-methylation associated with transcription, which are missing in animals, plants, and true fungi. This dichotomy of 6mA functions and the underlying molecular mechanisms may have implications in eukaryotic diversification.

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AHK3-Mediated Cytokinin Signaling Is Required for the Delayed Leaf Senescence Induced by SSPP

International Journal of Molecular Sciences ◽

10.3390/ijms20082043 ◽

2019 ◽

Vol 20 (8) ◽

pp. 2043

Author(s):

Yanan Wang ◽

Xiyu Zhang ◽

Yanjiao Cui ◽

Lei Li ◽

Dan Wang ◽

...

Keyword(s):

Leaf Senescence ◽

Protein Phosphatase ◽

Developmental Process ◽

Loss Of Function ◽

Cytokinin Signaling ◽

Phenotypic Analysis ◽

Exogenous Cytokinin ◽

Cytokinin Treatment ◽

Encoding Gene ◽

Multiple Signaling

Leaf senescence is a highly-programmed developmental process regulated by an array of multiple signaling pathways. Our group previously reported that overexpression of the protein phosphatase-encoding gene SSPP led to delayed leaf senescence and significantly enhanced cytokinin responses. However, it is still unclear how the delayed leaf senescence phenotype is associated with the enhanced cytokinin responses. In this study, we introduced a cytokinin receptor AHK3 knockout into the 35S:SSPP background. The phenotypic analysis of double mutant revealed that AHK3 loss-of-function reversed the delayed leaf senescence induced by SSPP. Moreover, we found the hypersensitivity of 35S:SSPP to exogenous cytokinin treatment disappeared due to the introduction of AHK3 knockout. Collectively, our results demonstrated that AHK3-mediated cytokinin signaling is required for the delayed leaf senescence caused by SSPP overexpression and the detailed mechanism remains to be further elucidated.

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