Mir 29a

Manufactured by Thermo Fisher Scientific

Sourced in United States

MiR-29a is a microRNA (miRNA) that functions as a regulator of gene expression. It is involved in various biological processes and is associated with several diseases. The core function of MiR-29a is to modulate the expression of target genes by binding to complementary sequences in their mRNA transcripts, leading to translational repression or mRNA degradation.

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9 protocols using mir 29a

Engineered Synoviocyte Responses to LPS

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Synoviocytes (type B, primary cells) were purchased from Sigma-Aldrich (Cat. 408OA-05A). These cells are derived from an adult OA patient. Synoviocytes were cultivated in human synoviocyte media (Cell applications). Cell culture conditions were 5% CO 2 , 37°C and 95% humidity. In cases of LPS treatment, synoviocytes were treated with LPS at dosages of 0, 2, 4, 6, 8 and 10 µg/ml for 48h prior to the subsequent analysis.
Overexpression of circCTNNA1 and miR-29a was reached in synoviocytes by transfecting pcDNA3.1-circCTNNA1 vector (Invitrogen) or miR-29a (Invitrogen) using Lipofectamine 2000 (Invitrogen). NC experiments were transfections of NC miRNA or empty pcDNA3.1 vector. Untransfected cells were used as control (C) cells. Expression vectors and miRNAs were rst mixed with transfection reagent to form transfection mixtures, followed by incubation with cells for 6h. After that, cells were washed with fresh medium. Prior to the subsequent assays, cells were cultured for 48h.

Liu W., Yang H., Feng X., Song J, & Zhong W. (2022). Circular RNA circCTNNA1 is downregulated in osteoarthritis and sponges miR-29a to suppress LPS-induced apoptosis of synoviocytes. Immunopharmacology and immunotoxicology, 44(1).

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Engineered Synoviocyte Responses to LPS

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Liu W., Yang H., Feng X., Song J., & Zhong W. (2021). Circular RNA circCTNNA1 is downregulated in osteoarthritis and sponges miR-29a to suppress LPS-induced apoptosis of synoviocytes.

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Intercellular miR-29a Transfer via Exosomes

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MIN 6 cells were transfected with fluorescently labelled miR‐29a (Invitrogen). Next, exosomes were isolated from the MIN 6 cell culture medium as previously described. The MVs were incubated with cultured hepatocytes for 6 h in RPMI 1640 medium supplemented with 10% FBS. The hepatocytes were washed, fixed and then observed using a confocal microscope (FV1000; Olympus, Tokyo). The excitation wavelengths were 405 nm for DAPI and 532 nm for fluorescently labelled miR‐29a. The image size was 1024×1024 pixels.

Li J., Zhang Y., Ye Y., Li D., Liu Y., Lee E., Zhang M., Dai X., Zhang X., Wang S., Zhang J., Jia W., Zen K., Vidal‐Puig A., Jiang X, & Zhang C. (2021). Pancreatic β cells control glucose homeostasis via the secretion of exosomal miR‐29 family. Journal of Extracellular Vesicles, 10(3), e12055.

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Affinity Pulldown of miR-29a and KLF4

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Biotin-labeled miR-29a or KLF4 probe and the NC probe were synthesized by RiboBio (Guangzhou, China). 1 × 10⁶ AD-VSMCs cells were harvested and lysed. Probe-coated beads were generated by incubation with miR-29a or KLF4 probe and C-1 magnetic beads (Life Technologies, USA). Cells lysate with miR-29a or KLF4 probe or NC probe were cultured for 24 hours 4°C. At last, the pull down complex was eluted and subjected to RT-qPCR analysis.

Xu Z., Zhong K., Guo G., Xu C., Song Z., Wang D, & Pan J. (2021). circ_TGFBR2 Inhibits Vascular Smooth Muscle Cells Phenotypic Switch and Suppresses Aortic Dissection Progression by Sponging miR-29a. Journal of Inflammation Research, 14, 5877-5890.

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Quantification of Exosomal miRNAs in Rheumatoid Arthritis

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Studies were approved by the Institutional Review Board of the University of Illinois at Chicago, and all donors gave informed written consent. RA patients were diagnosed according to the American College of Rheumatology 1987 revised criteria (23 (link)).
RNA was extracted from 1 ml of RA SF, osteoarthritis (OA) SF, RA plasma, and normal plasma. The RNA (10 ng) was reverse transcribed, and levels of miRNA were determined by real-time reverse transcription–polymerase chain reaction (RT-PCR) using a microRNA assay for hsa-let-7b according to the manufacturer’s instructions (Life Technologies). The copy number of exosomal miR-let-7b was calculated via a standard curve using the synthetic miR-let-7b (Gen-Script) and plotting the C_t values against the copy number, as described previously (24 (link)).
Mononuclear cells were isolated by Histopaque gradient centrifugation. Monocytes and T cells were isolated from normal and RA peripheral blood (PB) and/or SF using a negative selection kit (StemCell Technologies) (25 (link),26 (link)), and neutrophils were isolated from normal PB (27 (link)). The copy number of miR-let-7b and miR-29a (Life Technologies) in exosomes was determined in PB T cells, in vitro differentiated macrophages, and neutrophils, as well as in RA SF macrophages and RA ST fibroblasts.

Kim S.J., Chen Z., Essani A.B., Elshabrawy H.A., Volin M.V., Volkov S., Swedler W., Arami S., Sweiss N, & Shahrara S. (2016). Identification of a Novel Toll-like Receptor 7 Endogenous Ligand in Rheumatoid Arthritis Synovial Fluid That Can Provoke Arthritic Joint Inflammation. Arthritis & rheumatology (Hoboken, N.J.), 68(5), 1099-1110.

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Kidney microRNA Expression Analysis

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Experiments were performed on kidneys harvested on D2. We extracted miRs using an isolation kit (mirVana; Thermo Fisher Scientific), and we used total RNA enriched with miRs. For reverse-transcriptase reaction, 5 ng of RNA was used, employing the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). The miRs studied were miR-29a, miR-29b, miR-335 and miR-34a (Applied Biosystems; Thermo Fisher Scientific). RNU48, RNU44, U47 and U6 (Applied Biosystems; Thermo Fisher Scientific) were tested as possible housekeeping genes; we found U6 to be the best suited and used it in our analysis.
To perform a quantitative polymerase chain reaction (qPCR), we used TaqMan Universal PCR Master Mix II (Thermo Fisher Scientific). The data were analysed with DataAssist software (Applied Biosystems; Thermo Fisher Scientific) and relative gene expression calculated as 2^–ΔΔCt, where Ct is the threshold cycle.

Rodrigues C.E., Capcha J.M., de Bragança A.C., Sanches T.R., Gouveia P.Q., de Oliveira P.A., Malheiros D.M., Volpini R.A., Santinho M.A., Santana B.A., Calado R.D., Noronha I.D, & Andrade L. (2017). Human umbilical cord-derived mesenchymal stromal cells protect against premature renal senescence resulting from oxidative stress in rats with acute kidney injury. Stem Cell Research & Therapy, 8, 19.

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miRNA Profiling from Milk Samples

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Total RNA extraction and purification from milk, as well as specific miRNA analysis, were performed as previously described [19 (link)]. The small RNA-specific RT and PCR primers used were U6 snRNA (Assay ID: 001973), miR-26a (Assay ID: 000405), miR-27a (Assay ID: 000408), miR-29a (Assay ID: 002112), miR-103 (Assay ID: 000439), miR-200a (Assay ID: 000502), miR-200b (Assay ID: 001800), miR-221 (Assay ID: 000524), and miR-222 (Assay ID: 002276) (Applied Biosystems). The threshold cycle (Ct) was calculated by the instrument’s software (StepOne Software v2.2.2, Applied Biosystems) and miRNA expression was calculated as a percentage of the control group at day 5 of lactation using the 2^−ΔΔCt method [61 (link)]. Expression of U6 snRNA and β-actin were used as references. Finally, miRNA expression was calculated according to each reference and data are presented as the average of both values. The primers used for the β-actin gene are detailed in Table S1 and were supplied by Sigma.

Alonso-Bernáldez M., Asensio A., Palou-March A., Sánchez J., Palou A., Serra F, & Palou M. (2022). Breast Milk MicroRNAs Related to Leptin and Adiponectin Function Can Be Modulated by Maternal Diet and Influence Offspring Phenotype in Rats. International Journal of Molecular Sciences, 23(13), 7237.

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Quantification of miRNA and mRNA Expression

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PCR quantification was carried out essentially as previously described [9 (link), 15 (link)]. To quantify the expression level of miRNAs, we utilised stem-loop qRT-PCR for miR-29a (assay ID: 002112; Applied Biosystems, Foster City, CA, USA), miR-29b (assay ID: 000413), and miR-29c (assay ID: 000587) following the manufacturer’s protocol. TaqMan probes and primers were used for ITGB1 expression (assay ID: Hs00559595_m1; Applied Biosystems). GUSB (assay ID: Hs99999908_m1; Applied Biosystems) and RNU48 (assay ID: 001006; Applied Biosystems) were used as internal controls.

Koshizuka K., Kikkawa N., Hanazawa T., Yamada Y., Okato A., Arai T., Katada K., Okamoto Y, & Seki N. (2017). Inhibition of integrin β1-mediated oncogenic signalling by the antitumor microRNA-29 family in head and neck squamous cell carcinoma. Oncotarget, 9(3), 3663-3676.

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Quantification of Mature miRNA and mRNA Expression

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Mature miRNA expression was quantified using TaqMan microRNA assays (ABI, Life Technologies, Carlsbad, CA, USA). Total RNA (5 ng) was reverse-transcribed using miRNA specific primers and the TaqMan Reverse Transcription Kit (ABI, Life Technologies, Carlsbad, CA, USA). TaqMan miRNA assays were performed on a LC480 LightCycler (Roche, Basel, Switzerland), using the TaqMan Universal PCR Master Mix (ΑΒΙ, Life Technologies, Carlsbad, CA, USA), and analyzed with the LC480 analysis software. Values were normalized to the endogenous control sno202. For quantification of IFN-γ mRNA expression, 10 ng RNA was transcribed using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Expression of IFN-γ was analyzed using TaqMan gene expression assays and the data was normalized to GAPDH endogenous control. Relative fold changes of miRNA or mRNA expression were determined with the 2^−ΔΔCT method [18 (link)]. Mmu-miRNA and mRNA detection assays were obtained from Applied Biosystems: sno202 (assay ID: TM 001232); miR-125a-5p (assay ID: TM 002198); miR-155 (assay ID: TM 002571); miR-200c (assay ID: TM 002300); miR-29a (assay ID: TM002112); IFN-γ mRNA (assay ID: Mm 01168134_m1); and GAPDH (assay ID: Mm 99999915_g1).

Christaki E., Diza E., Giamarellos-Bourboulis E.J., Papadopoulou N., Pistiki A., Droggiti D.I., Georgitsi M., Machova A., Lambrelli D., Malisiovas N., Nikolaidis P, & Opal S.M. (2015). NK and NKT Cell Depletion Alters the Outcome of Experimental Pneumococcal Pneumonia: Relationship with Regulation of Interferon-γ Production. Journal of Immunology Research, 2015, 532717.

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