Mir 21 5p mimic

Manufactured by GenePharma

Sourced in China

MiR-21-5p mimic is a synthetic RNA molecule designed to mimic the function of the naturally occurring microRNA, miR-21-5p. MicroRNAs are small non-coding RNA molecules that play a role in the regulation of gene expression. The MiR-21-5p mimic can be used in research applications to study the biological functions and effects of miR-21-5p.

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12 protocols using mir 21 5p mimic

Exosomal miR-21-5p Regulates Melanogenesis

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To explore the roles of exosomes on melanogenesis in PIG1 cells, PIG1 cells were treated with 100 μg/mL exosomes derived from the healthy volunteers (Normal-exo) or 100 μg/mL exosomes derived from the vitiligo patients (Patients-exo) for 24 h.
The miR-21-5p mimic, miR-21-5p inhibitor and corresponding negative control were synthesized from GenePharma (Shanghai, China). The sequences for cell transfection were listed in Table 1. For overexpression of SATB1 (SATB1 OE), the cDNA of SATB1 was cloned into pcDNA3.1 vector. pcDNA3.1 vector was considered as the overexpression of negative control (NC OE). When cells reached 70% confluence, PIG1 cells were transfected with oligonucleotide or plasmid using the Lipofectamine RNAiMAX reagent or Lipofectamine 2000 (Invitrogen, USA).

Zhang C., Guo W., Wang S., Di Y, & Wu D. (2022). Peripheral Blood of Vitiligo Patients-Derived Exosomal MiR-21-5p Inhibits Melanocytes Melanogenesis via Targeting SATB1. Iranian Journal of Public Health, 51(12), 2706-2716.

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Overexpression of miR-21-5p in THP-1 cells

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miR-21-5p mimic (final concentration, 5 nM) was synthesized by GenePharma, and was incubated in THP-1 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for 6 h to over-express the gene expression level of miR-21-5p using the HiPerFect transfection reagent (QIAGEN, Hilden, Germany). The efficiency of the transfection was detected by quantitative RT-PCR.

Chen Y.C., Hsu P.Y., Su M.C., Chin C.H., Liou C.W., Wang T.Y., Lin Y.Y., Lee C.P., Lin M.C, & Hsiao C.C. (2020). miR-21-5p Under-Expression in Patients with Obstructive Sleep Apnea Modulates Intermittent Hypoxia with Re-Oxygenation-Induced-Cell Apoptosis and Cytotoxicity by Targeting Pro-Inflammatory TNF-α-TLR4 Signaling. International Journal of Molecular Sciences, 21(3), 999.

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Transfection of miR-21-5p and CDKN2C in Melanoma

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The miR‐21‐5p mimic, inhibitor and their negative controls (NCs) were purchased from GenePharma Co. Ltd. (Shanghai, China). Small interference RNA for CDKN2C (si‐CDKN2C) and its NC (si‐NC) were also synthesized by GenePharma Co. Ltd. The CDKN2C overexpression plasmid was constructed by inserting CDKN2C cDNA into a pcDNA3.1 vector by GenePharma Co. Ltd. For transfection, A375 or M14 cells were seeded at a density of 2 × 10⁵ cells per well in a six‐well culture dish, and transfection was performed when 80% confluence was achieved. A total of two 8‐µL (500 ng·µL⁻¹) plasmids (pcDNA3.1‐CDKN2C, si‐CDKN2C) or mimics (miR‐21‐5p mimic, inhibitor and NC) and 8 µL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were suspended in 100 µL Opti‐MEM (Gibco), and the final concentration of CDKN2C plasmids or mimics used was 1 mg·mL⁻¹. Then, the mixture was added into the cell culture and incubated for 48 h.

Yang Z., Liao B., Xiang X, & Ke S. (2020). miR‐21‐5p promotes cell proliferation and G1/S transition in melanoma by targeting CDKN2C. FEBS Open Bio, 10(5), 752-760.

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MiR-21 Mimic and PDCD4 Silencing

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The miR-21‑5p mimic, si-PDCD4 and the corresponding NC sequences were purchased from GenePharma (Guangzhou, China). Briefly, CC cells were seeded in a six-well plate. When the cells were 70–80% confluent, the miR-21 inhibitor, miR-21 inhibitor NC, miR-21 mimic, miR-21 mimic NC, or si-PDCD4 at a final concentration of 20 nM was transfected into HeLa or SiHa cells using Lipofectamine™ 3000 (Invitrogen/Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. For the rescue experiment, the miR-21 mimic or NC was transfected into cell lines stably transfected with GAS5. Functional experiments were performed after transfection for 48 h.

Fang X., Zhong G., Wang Y., Lin Z., Lin R, & Yao T. (2020). Low GAS5 expression may predict poor survival and cisplatin resistance in cervical cancer. Cell Death & Disease, 11(7), 531.

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Exploring miR-21-5p's Role in Neuronal Autophagy

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To study the role of miR-21-5p, miR-21-5p mimic and negative control (NC) mimic (Gene Pharma, Shanghai, China) were transfected into HT-22 neurons as previously described (Table 1) [25 (link),39 (link)]. Briefly, the miR-21-5p mimic or NC mimic was diluted to an accurate concentration of 20 mM. After that, 5 mL miR-21-5p mimic or NC mimic were mixed with 5 mL Lipofectamine-3000 (Thermo Fisher Scientific) in 500 mL serum-free DMEM/F12 and incubated for 20 minutes at room temperature. This transfection solution was washed twice with PBS and then added to the culture plates. After 48 hours of transfection, the neurons were prepared for the further experiment.
To further clarify the effect of miR-21-5p on autophagy through targeting Rab11a, a recombinant vector that overexpresses Rab11a was used (GenePharma, Shanghai, China), and for specific gene knockdown on Rab11a mRNA, the vectors of siRNA targeting Rab11a (si-Rab11a) or scramble siRNA Control (si-Control) was used (GenePharma, Shanghai, China) (Table 1). They were transfected into HT22 neurons with Lipofectamine-3000 as described above.

Li D., Huang S., Zhu J., Hu T., Han Z., Zhang S., Zhao J., Chen F, & Lei P. (2019). Exosomes from MiR-21-5p-Increased Neurons Play a Role in Neuroprotection by Suppressing Rab11a-Mediated Neuronal Autophagy In Vitro After Traumatic Brain Injury. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 25, 1871-1885.

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Regulation of MAPK10 by miR-21-5p

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miR-21-5p mimic and negative control (NC) were synthesized by Shanghai GenePharma Co., Ltd. pcDNA3-MAPK10 and pcDNA3 were purchased from Addgene (cat. no. 13758). A549 (1×10³/well) and H1299 (1×10³/well) cells were seeded in 96-well plates, cultured for 24 h and transfected with 50 nM miR-21-5p mimic, 50 nM miR-NC mimic, 2 µg pcDNA3 or 2 µg pcDNA3-MAPK10 using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 h, A549 and H1299 cells were collected for subsequent experiments.

Wu X., Li X, & Xu G. (2020). Propofol suppresses the progression of non-small cell lung cancer via downregulation of the miR-21-5p/MAPK10 axis. Oncology Reports, 44(2), 487-498.

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Targeting TLR4 and ALKBH5 in PMVEC Cells

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Speci c small interfering RNAs (siRNAs) targeting TLR4 (si-TLR4) and ALKBH5 (si-ALKBH5), siRNAnegative control (si-NC), miR-21-5p mimic, NC mimic, miR-21-5p inhibitor, and NC inhibitor were purchased from Shanghai GenePharma Co., Ltd. To overexpress TLR4 or ALKBH5, the sequences of TLR4 or ALKBH5 were inserted into a pcDNA3.1 plasmid to obtain the TLR4 or ALKBH5 overexpression plasmid oe-TLR4 or oe-ALKBH5, and an empty pcDNA3.1 plasmid was used as the negative control (oe-NC). Plasmid DNA, siRNA, miR-mimic or miR-inhibitor was transfected into PMVEC cells (1×10 5 ), which were subcultured at a density of 80%, with Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scienti c, Inc.) at 37˚C. After transfection for 48 h, the transfection e ciency was detected via RT-qPCR and western blot, and then subsequent experiments were performed.

Liu Z., Li R., Wang Y., Niu X., He X., He C., Li Y., & Ai L. (2021). ALKBH5 Regulates m6A Methylation-modified Pre-miR-21 to Promote Lung Injury in Obstructive Sleep Apnea Rat Model.

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Lung Cancer Cell Line Manipulation

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All lung cancer cell lines H1650, H1581, H460, H1299, and A549, and normal human bronchial epithelial cells HBE, were grown in 10% fetal bovine serum (FBS)‐supplemented RPMI1‐1640 complete medium. Cells were typically grown in an incubator at 37°C and 5% CO₂. circ‐SAR1A overexpression vector (ov‐SAR1A), negative control (ov‐NC), TXNIP lentiviral expression vector (sh‐TXNIP), negative control sh‐NC, miR‐21‐5p mimics, miR‐21‐5p inhibitor, and negative control (miR‐NC) were purchased from GenePharma. These plasmids were transfected individually or combined into A549 cells for subsequent experiments.

Zhao Y., Wan Y, & He T. (2022). Circ_SAR1A regulates the malignant behavior of lung cancer cells via the miR‐21‐5p/TXNIP axis. Journal of Clinical Laboratory Analysis, 36(5), e24366.

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Regulation of NSCLC by HNRNPA2B1

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HNRNPA2B1 overexpression plasmids, lentivirus-mediated HNRNPA2B1 shRNA (sh-HNRNPA2B1, 5ʹ-CCGTAAGCTCTTTATTGGTGGCTTA-3ʹ), si-MEG3, miR-21-5p mimics and inhibitor were purchased from GenePharma (Shanghai, China). The sh-NC, vector and miR-NC were used as the control groups. NSCLC cell lines were planted in 6-well plates 24 h prior to sh-HNRNPA2B1, si-MEG3, miR-21-5p mimics or inhibitor transfection with 50–60% confluence, and then mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacture instructions.

Li K., Gong Q., Xiang X.D., Guo G., Liu J., Zhao L., Li J., Chen N., Li H., Zhang L.J., Zhou C.Y., Wang Z.Y, & Zhuang L. (2023). HNRNPA2B1-mediated m6A modification of lncRNA MEG3 facilitates tumorigenesis and metastasis of non-small cell lung cancer by regulating miR-21-5p/PTEN axis. Journal of Translational Medicine, 21, 382.

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Dual Luciferase Reporter Assay for miR-21 Targeting

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Plasmid pmirGLO was purchased from Promega Corporation (Madison, WI, USA). BHT101
and Hth83 cell lines were seeded into 24-well plates. pmirGLO-BISPR-wt,
pmirGLO-BISPR-mut, pmirGLO-Bcl-2 wt, pmirGLO-Bcl-2-mut, miR-21-5p mimics, and
miR-21-5p inhibitor (GenePharma, Shanghai, China) were constructed and
co-transfected into BHT101 and Hth83 cell lines using Lipofectamine 2000 (Life
Technologies, Carlsbad, CA, USA) for 48 h. Subsequently, dual luciferase
reporter assay system kit (Promega Corporation) was applied to examine the
luciferase activity.

Zhang H., Cai Y., Zheng L., Zhang Z., Lin X, & Jiang N. (2018). LncRNA BISPR promotes the progression of thyroid papillary carcinoma by regulating miR-21-5p. International Journal of Immunopathology and Pharmacology, 32, 2058738418772652.

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