Keloid fibroblasts and normal fibroblasts were separated from keloid tissues and normal tissues. Thenfostered in DMEM cell medium (Zeye Biotechnology, Shanghai, China), embodying 10% fetal bovine serum at 37°C, 5%CO2as described previously [23 (link)]. The fibroblasts were seeded in 6-well plates at a density of 2 × 105/well and incubated to 40 ~ 50% confluence. Logarithmic growth phase cells were harvested for research and synthesis of siRNA negative control for circPTPN12, siRNA for circPTPN12, siRNA negative control for SMAD7, siRNA for SMAD7, miR-21-5p mimics, miR-21-5p inhibitor, and miR negative control (RiboBio, Guangzhou, China). According to the vendor manual, Lipofectamine®2000 (Hengfei Biotechnology, Shanghai, China) was used for cell transfection as described previously [24 (link)]. Cells at passages 3–5 were used for this study. Cells were divided into the normal group (NC), silencing circPTPN12(si-circPTPN12), siRNA control group (si-NC), The grouping of miR-21-5p, and SMAD7 is the same as above.
Mir 21 5p mimic
Manufactured by RiboBio
Sourced in China
The MiR-21-5p mimic is a synthetic RNA molecule designed to mimic the function of the natural miR-21-5p microRNA. MicroRNAs are small, non-coding RNA molecules that play a crucial role in gene expression regulation.
Automatically generated - may contain errors
Lab products found in correlation
Mir 21 5p inhibitor, by RiboBio (7 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rabbit anti ve cadherin, by Cell Signaling Technology (1 mentions)
Rabbit anti gapdh 5174, by Cell Signaling Technology (1 mentions)
Mir 21 5p mimic, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Mimic control, by RiboBio (1 mentions)
Mimic nc, by RiboBio (1 mentions)
Pcdna3.1 vector, by GenePharma (1 mentions)
Lipofectamine 2000, by RiboBio (1 mentions)
Dual luciferase reporter assay kit, by Vazyme (1 mentions)
Lipofectamine ltx with plus reagent, by Thermo Fisher Scientific (1 mentions)
U6 small nucleolar rna, by RiboBio (1 mentions)
12 protocols using mir 21 5p mimic
1
Keloid Fibroblast Regulation by circPTPN12 and miR-21-5p
2
Overexpression of miR124 and miR21-5p in MSCs
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MSCs were resuspended and seeded at 5 × 104cells/ml into six-well culture plates and incubated at 37°C before transfection. MSCs were divided into four groups. according to instructions provided by the manufacturer, when the cell density get to 80%, 50 nM synthetic miR124 mimics (RiboBio, Guangzhou, China) were transfected by Lipofectamine 2000 (Invitrogen, Carlsbad, CA) into MSCs to overexpress miR124 in MSCs and this group is defined as miR124-MSCs group. In the same way, miR21-5p mimics (RiboBio, Guangzhou, China) and miRNA mimics negative control (RiboBio, Guangzhou, China) were respectively transfected into MSCs and they were respectively defined as miR21-5p-MSCs group and mimics control-MSCs group (MC-MSCs). The miR124 mimics and miR21-5p mimics were co-transfected into MSCs that was named as the miR124+21-5p-MSCs group. In addition, MSCs treated with L-DMEM only was also used as a control group that was defined as normal control-MSCs group (NC-MSCs). After incubation for 48 h at 37°C with 5% CO2 in a humidified incubator, the cells were collected for the further experiments. All the miRNA mimics used in the study are listed in Table 1 and the experiments were performed in triplicates and repeated in three independent experiments.
3
Western Blot and Immunofluorescence Staining Protocol
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Rabbit anti-SOX7 (BA3297-2, diluted at 1:1000 for WB (western blotting assay)) was obtained from Boster (Wuhan, China), rabbit anti-VE-cadherin (#2500, diluted at 1:1000 for WB and 1:200 for IF (immunofluorescence staining) and rabbit anti-GAPDH (#5174S, diluted at 1: 1000 for WB) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). miR-21-5p mimics (miR10000076), miR-21-5p inhibitor (miR20000076) were purchased from RiboBio Co., Ltd., China.
miR-21-5p mimics are chemically synthesized miRNA which is easy to control transient transfection into the cell, while miR-21-5p inhibitor is single-stranded synthetic inhibitor having complementary sequences to target miRNA.
PM2.5 were sampled from Beijing and analyzed as our previous research [7 (link)].
miR-21-5p mimics are chemically synthesized miRNA which is easy to control transient transfection into the cell, while miR-21-5p inhibitor is single-stranded synthetic inhibitor having complementary sequences to target miRNA.
PM2.5 were sampled from Beijing and analyzed as our previous research [7 (link)].
4
Overexpression and silencing of miR-21-5p
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PmirGLO-SMAD7 (HedgehogBio, Shanghai, China) was constructed. The miR-21-5p mimic, miR-21-5p inhibitor ,and negative control were designed and synthesized by RiboBio Co, Ltd. (Guangzhou, China). Lipofectamine 3000 reagent was used to transfect them into MHCC97H and HepG2 cells in a 24-well plate.
5
miR-21-5p Mimic Transfection Protocol
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The miR-21-5p mimic and its negative control (NC) were synthesized by RiboBio (Guangzhou, China). Wild-type cells (5.0×105 cells/well) were grown in 6-well plates in 2 ml culture medium. When cell confluence reached 50%–60%, the miR-21-5p mimic and its negative control were treated with Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. The cells were transfected in Opti-MEM (Gibco, USA) for 8 h, and then the medium was changed to normal culture medium. After 48 h, the cells were harvested for western blot and qRT-PCR. All groups were plated in 6-well culture plates at the same time and were harvested 48 h later.
6
Transfection of miR-21-5p Mimic in H9c2 Cells
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miR-21-5p mimic (5′-UAGCUUAUCAGACUGAUGUUGA-3′), mimic control (MC; 5′-UUCUCCGAACGUGUCACGUUU-3′), SKP2 overexpression plasmid, and the empty plasmids (which were used as the negative control) (NC.) were commercially ordered from RIBOBIO (Guangzhou, China).
Prior to cell transfection, H9c2 cells (3.0 × 105) that were cultured in DMEM were added to a six-well plate and maintained until the confluence reached 80%. Then, 2.0 μg of mimic or plasmids was transfected into the cells using 3 μl of lipofectamine 3000 transfection reagent (L3000015, Invitrogen, Waltham, MA, United States) and incubated for 48 h.
Prior to cell transfection, H9c2 cells (3.0 × 105) that were cultured in DMEM were added to a six-well plate and maintained until the confluence reached 80%. Then, 2.0 μg of mimic or plasmids was transfected into the cells using 3 μl of lipofectamine 3000 transfection reagent (L3000015, Invitrogen, Waltham, MA, United States) and incubated for 48 h.
7
Regulation of miR-21-5p and HIF-1α in HUVECs
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HUVECs were plated in a 6-well plate and transfected with 30 nM of miR-21-5p mimic NC, miR-21-5p mimic, miR-21-5p inhibitor (RiboBio, Guangzhou, China), 50 nM of si-NC or si-VHL, si-NC or si-HIF-1α, and si-NC or si-PDH-E1α (RiboBio) when cells reached 50 to 60% confluence. The sequences of the small interfering RNAs (siRNAs), miR-21-5p mimic, and inhibitors used in this study are shown in Table S1 . siRNA Lipofectamine 3000 (Thermofisher Scientific, Agawam, MA, USA) was used for cell transfection experiments according to the manufacturer’s instructions. HUVECs were treated with 200 µg/ml of N-SCs-Exos or H-SCs-Exos.
8
CircYAP1 Overexpression and Silencing
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The overexpressing circYAP1 plasmids and siRNA of circYAP1 were colligated by Sangon. MiR‐21‐5p mimic or NC mimic was purchased from RiboBio. Cell transfection was performed by utilizing Lipofectamine 2000 Reagent (Invitrogen). After 48 hours, cells were collected with the highest transfection efficiency.
9
Investigating MEG3 and miR-21-5p Interactions
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The pcDNA3.1-MEG3 plasmid (termed MEG3) was constructed by sub-cloning MEG3 sequence into pcDNA 3.1 vector (Shanghai GenePharma Co., Ltd.). The cells were transfected with MEG3 and and pcDNA 3.1 empty vector (vector) to a final concentration of 100 nM using Lipofectamine® 2000 (cat. no. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. miR-21-5p inhibitor (5′-UAGCUUAUCAGACUGAUGUUGA-3′), miR-21-5p mimic (5′-UAGCUUAUCAGACUGAUGUUGA-3′) and their negative controls (NC, 5′-CCCAGAATGTTGACAGCTGCCTCTT-3′) were synthesized by Guangzhou RiboBio Co., Ltd. The cells were transfected with miR-21-5p mimic or inhibitor to a final concentration of 50 nM using Lipofectamine 2000 according to the manufacturer's protocols. Subsequent experiments were performed 24 h after transfection.
In order to assess the effects of miR-21-5p on MEG3-mediated cellular responses, cells were transfected with MEG3, miR-21-5p mimic, miR-21-5p inhibitor, MEG3+miR-21-5p mimic and their negative controls using Lipofectamine 2000, respectively.
In order to assess the effects of miR-21-5p on MEG3-mediated cellular responses, cells were transfected with MEG3, miR-21-5p mimic, miR-21-5p inhibitor, MEG3+miR-21-5p mimic and their negative controls using Lipofectamine 2000, respectively.
10
Modulating miR-21-5p and SPRY2 in Angiogenesis
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MiR-21-5p mimic, miR-21-5p inhibitor and their negative controls were purchased from RiboBio (Guangzhou, China). Briefly, the cells were cultured in 6-well culture plates and transfected with MiR-21-5p mimic, miR-21-5p inhibitor or their negative controls using Lipofectamine 3000. After 48 h of incubation, subsequent experiments were performed.
To assess whether SPRY2 knockdown can achieve similar effects on angiogenesis and fibroblast function as miR-21-5p, SPRY2 siRNA obtained from RiboBio (Guangzhou, China) was used to knock down the expression of SPRY2 in HUVECs and HSFs. Briefly, cells were transfected with si-SPRY2 or negative control siRNA (si-NC) using Lipofectamine 3000 according to the manufacturer’s instructions. Forty-eight hours later, the knockdown efficiency was verified by qRT-PCR. The sequences of si-SPRY2 were as follows: forward: 5ʹ-AUAAACAAGGCAAAAAGAGGG-3ʹ and reverse: 5ʹ-CUCUUUUUGCCUUGUUUAUGG-3ʹ. Overexpression of SPRY2 was achieved by transfecting SPRY2 cDNA (Wei Zheng, Shandong, China) and using Lipofectamine 3000.
To assess whether SPRY2 knockdown can achieve similar effects on angiogenesis and fibroblast function as miR-21-5p, SPRY2 siRNA obtained from RiboBio (Guangzhou, China) was used to knock down the expression of SPRY2 in HUVECs and HSFs. Briefly, cells were transfected with si-SPRY2 or negative control siRNA (si-NC) using Lipofectamine 3000 according to the manufacturer’s instructions. Forty-eight hours later, the knockdown efficiency was verified by qRT-PCR. The sequences of si-SPRY2 were as follows: forward: 5ʹ-AUAAACAAGGCAAAAAGAGGG-3ʹ and reverse: 5ʹ-CUCUUUUUGCCUUGUUUAUGG-3ʹ. Overexpression of SPRY2 was achieved by transfecting SPRY2 cDNA (Wei Zheng, Shandong, China) and using Lipofectamine 3000.
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