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A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity In Vivo.

ABSTRACT: MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity in vivo by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.

SUBMITTER: Phiwpan K

PROVIDER: S-EPMC4452368 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity In Vivo.

Phiwpan Krung K Guo Jie J Zhang Wei W Hu Tanyu T Boruah Bhargavi M BM Zhang Jianhua J Zhou Xuyu X

PloS one 20150601 6

MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity in vivo by inserting miR-155-5p target sequence downstream of a reporter transg ...[more]

PMID: 26030404

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Project description:The pathophysiology of most neurodegenerative diseases includes aberrant accumulation of protein aggregates. Recent evidence highlights the role of protein degradation pathways in neurodegeneration. Concurrently, genetic tools have been generated to enable zebrafish, Danio rerio, to be used as an animal model to study neurodegenerative processes. In addition to optical clarity and fast ex utero development, the zebrafish brain is relatively small and has conserved structures with its mammalian counterparts. To take advantage of this model organism and to aid further studies on autophagy and neurodegeneration, we created a stable transgenic zebrafish line that expresses eGFP-Map1lc3b specifically in post-mitotic neurons under the elavl3 promoter. This line is useful for indirectly monitoring autophagic activity in neurons in vivo and screening for macroautophagy/autophagy-modulating compounds. We determined the applicability of this transgenic line by modulating and quantifying the number of autophagosomes via treatment with a known autophagy inducer (rapamycin) and inhibitors (3-methyladenine, protease inhibitors). Additionally, we proposed an in vivo method for quantifying rates of autophagosome accumulation, which can be used to infer occurrence of autophagic flux. Last, we tested two FDA-approved drugs currently undergoing clinical studies for Parkinson disease, isradipine and nilotinib, and found that isradipine did not modulate autophagy, whereas nilotinib induced both autophagosome number and autophagic flux. It is hoped that others will find this line useful as an in vivo vertebrate model to find or validate autophagy modulators that might be used to halt the progression of neurodegenerative diseases. Abbreviations: 3MA: 3-methyladenine; BafA: bafilomycin A1; dd: dorsal diencephalon; dpf: days post fertilization; e: eye; eGFP: enhanced green fluorescent protein; Elavl3: ELAV like neuron-specific RNA binding protein 3; FDA: Food and Drug Administration; hb: habenula; hpt, hours post treatment; Map1lc3b: microtubule-associated protein 1 light chain 3 beta; nt: neural tube; ot, optic tectum; P/E: pepstatin A and E64d; PD: Parkinson disease; PMTs: photomultiplier tubes; PTU: 1-phenyl-2-thiourea; Ta: annealing temperature; Tel, telencephalon.

Project description:Objective:To study the role of LncRNA PTTG3P in colorectal cancer (CRC) and the underlying mechanism. Patients and Methods:The expression level of LncRNA PTTG3P was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in CRC cell lines and tumor tissues from 43 CRC patients, and the correlations between LncRNA PTTG3P expression and the clinicopathological indicators and prognosis of CRC patients were analyzed. Then, a PTTG3P knockdown model in CRC cell lines HCT-8 and HCT-116 was constructed. Finally, the relationship between LncRNA PTTG3P and microRNA-155-5p was explored through luciferase reporter experiments and recovery experiments. Results:qRT-PCR results showed that LncRNA PTTG3P was markedly up-regulated in CRC tumor tissues than that in adjacent tissues. Meanwhile, patients with high LncRNA PTTG3P expression had higher rates of lymph node metastasis and distant metastasis. In addition, cell functional experiments suggested that knocking down PTTG3P markedly reduced the migration abilities of CRC cells. Subsequently, bioinformatics analysis and luciferase reporter gene experiments suggested that LncRNA PTTG3P could directly bind to microRNA-155-5P. Analysis of CRC tissue samples showed that microRNA-155-5P expression was markedly reduced in CRC and was negatively correlated with LncRNA PTTG3P. Finally, the recovery experiments also suggested that there was a mutual regulation between LncRNA PTTG3P and microRNA-155-5P, and silencing microRNA-155-5P can reverse the inhibitory effect of knocking down PTTG3P on the malignant progression of CRC. Conclusion:In summary, LncRNA PTTG3P level was markedly increased in CRC, and was highly correlated to the incidence of lymph node metastasis and distant metastasis in CRC patients. In addition, LncRNA PTTG3P might promote the ability of CRC to invade and migrate by downregulating microRNA-155-5P. Therefore, dissecting the aberrant regulation of LncRNA PTTG3P/microRNA-155-5P may be valuable for early screening, guidance treatment, and recurrence detection of CRC.

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A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity In Vivo.

Publications

A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity In Vivo.

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