Journal: Molecular Medicine Reports

Article Title: MicroRNA-125a-mediated regulation of the mevalonate signaling pathway contributes to high glucose-induced proliferation and migration of vascular smooth muscle cells

doi: 10.3892/mmr.2020.11077

Figure Lengend Snippet: miR-125a expression is inversely related to HG-induced VSMC proliferation and migration. (A) VSMCs were incubated with different concentrations of glucose (5.6, 11.1, 22.2 and 44.5 mM) for 48 h. Cell proliferation was measured using the Cell Counting Kit-8 assay. (B) A wound healing assay was performed to investigate the (C) migration of HG treated-VSMCs (magnification, ×20). Cell proliferation was inhibited using mitomycin C before wound scratching. (D) Expression of miR-125a in HG-treated VSMCs was analyzed by reverse transcription-quantitative PCR. Data are presented as the mean ± SD. n=3. *P<0.05, **P<0.01; ns, not significant; miR, microRNA; HG, high glucose; VSMCs, vascular smooth muscle cells.

Article Snippet: For further assessment of miRNA expression, miRNAs from the VSMCs in the aortic media were extracted using mirVana miRNA isolation kit (Thermo Fisher Scientific, Inc.). cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio, Inc.) at 42°C for 1 h according to the manufacturer's instructions with specific rno-miR-125a RT primers (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGAC-3′; Guangzhou RiboBio Co., Ltd.). qPCR was performed using the SYBR Premix Ex Taq kit (Takara Bio, Inc.) under the following conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and at 60°C for 30 sec in an ABI Prism 7900 system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Migration, Incubation, Cell Counting, Wound Healing Assay, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: MicroRNA-125a-mediated regulation of the mevalonate signaling pathway contributes to high glucose-induced proliferation and migration of vascular smooth muscle cells

doi: 10.3892/mmr.2020.11077

Figure Lengend Snippet: Transfection with miR-125a abrogates HG-induced VSMC proliferation and migration. (A) Cell Counting Kit-8 assay results demonstrated that transfection with a miR-125a mimic reduced HG-induced VSMC proliferation. (B) A wound healing assay (C) showed that miR-125a transfection reduced HG-induced VSMC migration. Magnification, ×20. Cell proliferation was inhibited by using mitomycin C before wound scratching. Data are presented as the mean ± SD. n=3. *P<0.05, **P<0.01; ns, not significant; miR, microRNA; H.G, high glucose; N.G, normal glucose; VSMC, vascular smooth muscle cell; NC, negative control.

Article Snippet: For further assessment of miRNA expression, miRNAs from the VSMCs in the aortic media were extracted using mirVana miRNA isolation kit (Thermo Fisher Scientific, Inc.). cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio, Inc.) at 42°C for 1 h according to the manufacturer's instructions with specific rno-miR-125a RT primers (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGAC-3′; Guangzhou RiboBio Co., Ltd.). qPCR was performed using the SYBR Premix Ex Taq kit (Takara Bio, Inc.) under the following conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and at 60°C for 30 sec in an ABI Prism 7900 system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Techniques: Transfection, Migration, Cell Counting, Wound Healing Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: MicroRNA-125a-mediated regulation of the mevalonate signaling pathway contributes to high glucose-induced proliferation and migration of vascular smooth muscle cells

doi: 10.3892/mmr.2020.11077

Figure Lengend Snippet: Validation of HMGCR as a direct target of miR-125a. (A) Target site of miR-125a in the HMGCR 3′-UTR. (B) Dual-luciferase activity of the WT and Mut HMGCR 3′-UTR reporter in the presence of miR-125a or miR-NC. (C) Western blot analysis of HMGCR expression in VSMCs transfected with a miR-125a mimic, miR-125a inhibitor or miR-NC. Data are presented as the mean ± SD. n=3. **P<0.01; ns, not significant; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; miR, microRNA; WT, wild-type; Mut, mutant; 3′-UTR, 3′-untranslated region; NC, negative control; VSMC, vascular smooth muscle cell; UT, untreated VSMCs.

Article Snippet: For further assessment of miRNA expression, miRNAs from the VSMCs in the aortic media were extracted using mirVana miRNA isolation kit (Thermo Fisher Scientific, Inc.). cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio, Inc.) at 42°C for 1 h according to the manufacturer's instructions with specific rno-miR-125a RT primers (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGAC-3′; Guangzhou RiboBio Co., Ltd.). qPCR was performed using the SYBR Premix Ex Taq kit (Takara Bio, Inc.) under the following conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and at 60°C for 30 sec in an ABI Prism 7900 system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Techniques: Luciferase, Activity Assay, Western Blot, Expressing, Transfection, Mutagenesis, Negative Control

Journal: Molecular Medicine Reports

Article Title: MicroRNA-125a-mediated regulation of the mevalonate signaling pathway contributes to high glucose-induced proliferation and migration of vascular smooth muscle cells

doi: 10.3892/mmr.2020.11077

Figure Lengend Snippet: miR-125a regulates the expression levels of key enzymes in the mevalonate signaling pathway. (A) Protein expression levels of HMGCR, FDPS, SQS and GGTase-I were measured by western blotting. miR-125a mimic transfection reversed HG-induced dysregulation of these proteins in VSMCs. (B) miR-125a inhibitor transfection induced the activation of the mevalonate signaling pathway in VSMCs cultured under normal conditions (5.6 nM glucose). Data are presented as the mean ± SD. n=3. *P<0.05, **P<0.01. ns, not significant; miR, microRNA; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; FDPS, farnesyl diphosphate synthase; SQS, squalene synthase; GGTase-I, geranylgeranyltransferase type I; H.G, high glucose; N.G, normal glucose; VSMC, vascular smooth muscle cell; NC, negative control.

Article Snippet: For further assessment of miRNA expression, miRNAs from the VSMCs in the aortic media were extracted using mirVana miRNA isolation kit (Thermo Fisher Scientific, Inc.). cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio, Inc.) at 42°C for 1 h according to the manufacturer's instructions with specific rno-miR-125a RT primers (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGAC-3′; Guangzhou RiboBio Co., Ltd.). qPCR was performed using the SYBR Premix Ex Taq kit (Takara Bio, Inc.) under the following conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and at 60°C for 30 sec in an ABI Prism 7900 system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Western Blot, Transfection, Activation Assay, Cell Culture, Negative Control

Journal: Molecular Medicine Reports

Article Title: MicroRNA-125a-mediated regulation of the mevalonate signaling pathway contributes to high glucose-induced proliferation and migration of vascular smooth muscle cells

doi: 10.3892/mmr.2020.11077

Figure Lengend Snippet: High glucose-induced atherosclerosis is associated with miR-125a-mediated dysregulation of the mevalonate signaling pathway. (A) Media thickness and MCSA of the thoracic aorta from Sprague Dawley rats at time 0, and 5, 10 and 20 weeks after streptozotocin injection. (B) miR-125a expression in the aortic media. (C) Protein expression levels of HMGCR, FDPS, SQS and GGTase-I in the aortic media were measured by western blotting. Data are presented as the mean ± SD. n=6. *P<0.05, **P<0.01. ns, not significant; MCSA, media cross-sectional area; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; FDPS, farnesyl diphosphate synthase; SQS, squalene synthase; GGTase-I, geranylgeranyltransferase type I; miR, microRNA.

Article Snippet: For further assessment of miRNA expression, miRNAs from the VSMCs in the aortic media were extracted using mirVana miRNA isolation kit (Thermo Fisher Scientific, Inc.). cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio, Inc.) at 42°C for 1 h according to the manufacturer's instructions with specific rno-miR-125a RT primers (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGAC-3′; Guangzhou RiboBio Co., Ltd.). qPCR was performed using the SYBR Premix Ex Taq kit (Takara Bio, Inc.) under the following conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and at 60°C for 30 sec in an ABI Prism 7900 system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Techniques: Injection, Expressing, Western Blot

Journal: Journal of Virology

Article Title: ERK Is a Critical Regulator of JC Polyomavirus Infection

doi: 10.1128/JVI.01529-17

Figure Lengend Snippet: JCPyV induces multiphasic ERK activation upon infection. (A) SVG-A cells were infected with JCPyV (MOI = 1 FFU/cell) for the specified duration and were analyzed via Western blotting using antibodies specific for phosphorylated ERK (pERK) and total ERK (protein control). The percentage of pERK was determined using phosphorylated ERK and total ERK band intensities, determined by using the equation [pERK/(pERK + total ERK)] × 100. The percentage of pERK for JCPyV-infected samples was normalized to the value for mock-infected samples at each time point (100%; dashed line). Percentages of phosphorylated ERK for each time point were plotted via box-and-whisker plot and are representative of results from three independent experiments. (B) SVG-A cells were either pretreated (PT) for 1 h prior to JCPyV infection with U0126 (10 μM) or DMSO (1:1,000) or incubated in complete medium. Cells were then infected with JCPyV (MOI = 0.1 FFU/cell) at 4°C for 1 h. Following infection, cells were washed with medium and then incubated in media with inhibitors for the indicated times. At the specified time point, medium was removed, cells were washed, complete medium was added, and cells were incubated at 37°C for 72 h. Cells were fixed and stained by indirect immunofluorescence using a VP1-specific antibody, and infectivity was scored by quantitating nuclear VP1 expression. Data are representative of the average number of infected cells per visual field for five 20× fields of view for triplicate samples. Data are representative of results from three independent experiments. Error bars indicate SDs. Student's t test was used to determine statistical significance. *, P < 0.05.

Article Snippet: Antibodies used for pERK Western blot analysis were a rabbit-specific phosphorylated ERK antibody (Cell Signaling Technology; no. 9101) and mouse-specific total ERK antibody (Cell Signaling Technology; no. 4696) and corresponding secondary anti-mouse 680 antibody (LICOR) and goat anti-rabbit 800 antibody (LICOR).

Techniques: Activation Assay, Infection, Western Blot, Whisker Assay, Incubation, Staining, Immunofluorescence, Expressing