Journal: Molecular Medicine Reports

Article Title: MicroRNA-199a-5p regulates FOXC2 to control human vascular smooth muscle cell phenotypic switch

doi: 10.3892/mmr.2021.12266

Figure Lengend Snippet: Expression levels of miR-199a-5p are decreased in human varicose vein tissues. (A) RT-qPCR revealed that the expression levels of VSMC differentiation biomarkers were decreased in varicose vein tissues (n=10). *P<0.05 vs. great saphenous vein tissues. (B) Western blot analysis revealed that the protein expression levels of VSMC differentiation biomarkers were decreased and the expression levels of FOXC2 were increased in varicose vein tissues (n=3). *P<0.05 vs. great saphenous vein tissues. (C) miR-199a-5p was downregulated and FOXC2 was upregulated in varicose vein tissues, as determined by RT-qPCR. (n=10). *P<0.05 vs. great saphenous vein tissues. miR-199a-5p, microRNA-199a-5p; FOXC2, forkhead box C2; MYH11, myosin heavy chain 11; RT-qPCR, Reverse transcription-quantitative PCR; SM22α, smooth muscle 22α; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell.

Article Snippet: Subsequently, membranes were incubated overnight at 4°C with the following primary antibodies: FOXC2 (1:500; cat. no. ab245510; Abcam), smooth muscle 22α (SM22α; 1:500; cat. no. 10493-1-AP; ProteinTech Group, Inc.), smooth muscle actin (SMA; 1:800; cat. no. 55135-1-AP; ProteinTech Group, Inc.), MYH11 (1:1,000; cat. no. 21404-1-AP; ProteinTech Group, Inc.), calponin (1:1,000; cat. no. bs-0095R; BIOSS), proliferating cell nuclear antigen (PCNA; 1:3,000; cat. no. ab92552; Abcam) and β-actin (1:5,000; cat. no. bs-0061R; BIOSS), followed by incubation with secondary antibodies (1:5,000; cat. no. bs-0296G-HRP; BIOSS) at room temperature for 2.5 h. Protein bands were visualized with an ECL western blotting kit (Cell Signaling Technology, Inc.) using cSeries Imager (Azure Biosystems, Inc.).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: MicroRNA-199a-5p regulates FOXC2 to control human vascular smooth muscle cell phenotypic switch

doi: 10.3892/mmr.2021.12266

Figure Lengend Snippet: miR-199a-5p regulates the expression levels of VSMC biomarkers. (A) Reverse transcription-quantitative PCR confirmed that miR-199a-5p was overexpressed and knocked down post-transfection with the miR-199a-5p mimics and inhibitor, respectively (n=3). (B) Overexpression or knockdown of miR-199a-5p promoted or inhibited the expression of VSMC differentiation biomarkers, respectively. *P<0.05 vs. control (n=10). (C) Western blot analysis of VSMC differentiation biomarkers. *P<0.05 vs. control (n=3). miR-199a-5p, microRNA-199a-5p; MYH11, myosin heavy chain 11; SM22α, smooth muscle 22α; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell.

Article Snippet: Subsequently, membranes were incubated overnight at 4°C with the following primary antibodies: FOXC2 (1:500; cat. no. ab245510; Abcam), smooth muscle 22α (SM22α; 1:500; cat. no. 10493-1-AP; ProteinTech Group, Inc.), smooth muscle actin (SMA; 1:800; cat. no. 55135-1-AP; ProteinTech Group, Inc.), MYH11 (1:1,000; cat. no. 21404-1-AP; ProteinTech Group, Inc.), calponin (1:1,000; cat. no. bs-0095R; BIOSS), proliferating cell nuclear antigen (PCNA; 1:3,000; cat. no. ab92552; Abcam) and β-actin (1:5,000; cat. no. bs-0061R; BIOSS), followed by incubation with secondary antibodies (1:5,000; cat. no. bs-0296G-HRP; BIOSS) at room temperature for 2.5 h. Protein bands were visualized with an ECL western blotting kit (Cell Signaling Technology, Inc.) using cSeries Imager (Azure Biosystems, Inc.).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Over Expression, Knockdown, Control, Western Blot

Journal: Molecular Medicine Reports

Article Title: MicroRNA-199a-5p regulates FOXC2 to control human vascular smooth muscle cell phenotypic switch

doi: 10.3892/mmr.2021.12266

Figure Lengend Snippet: FOXC2 rescue experiments. (A) RT-qPCR confirmed that FOXC2 was overexpressed and knocked down post-transfection with pcDNA3.1-FOXC2 vector or FOXC2 siRNA, respectively. *P<0.05 vs. control (n=3). (B) CCK-8 confirmed that VSMC proliferation was enhanced after transfection with miR-199a-5p mimics + FOXC2 vector compared with miR-199a-5p mimics alone. *P<0.05 (n=10). (C) Transwell migration assays revealed that FOXC2 enhanced VSMC migration. Magnification, ×40. *P<0.05 (n=10). (D) Western blot analysis revealed that the expression levels of VSMC differentiation biomarkers were decreased in cells transfected with miR-199a-5p mimics + FOXC2 vector compared with those in cells transfected with miR-199a-5p mimics only. *P<0.05 (n=3). (E) RT-qPCR revealed that FOXC3 decreased the expression levels of VSMC differentiation biomarkers compared with those in cells transfected with miR-199a-5p mimics only. *P<0.05 (n=10). (F) RT-qPCR was used to detect the expression levels of phenotypic transition biomarkers. *P<0.05 vs. control (n=10). (G) CCK-8 confirmed that proliferation of VSMCs was reduced in response to FOXC2 silencing, but increased in response to FOXC2 overexpression. *P<0.05 vs. control (n=3). (H) Wound healing assay revealed that migration of VSMCs was reduced post-transfection with the FOXC2 siRNA, but increased following the overexpression of FOXC2 compared with the control group. *P<0.05 vs. control (n=3). FOXC2, forkhead box C2; miR-199a-5p, microRNA-199a-5p; MYH11, myosin heavy chain 11; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; SM22α, smooth muscle 22α; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell; CCK-8, Cell Counting Kit-8.

Article Snippet: Subsequently, membranes were incubated overnight at 4°C with the following primary antibodies: FOXC2 (1:500; cat. no. ab245510; Abcam), smooth muscle 22α (SM22α; 1:500; cat. no. 10493-1-AP; ProteinTech Group, Inc.), smooth muscle actin (SMA; 1:800; cat. no. 55135-1-AP; ProteinTech Group, Inc.), MYH11 (1:1,000; cat. no. 21404-1-AP; ProteinTech Group, Inc.), calponin (1:1,000; cat. no. bs-0095R; BIOSS), proliferating cell nuclear antigen (PCNA; 1:3,000; cat. no. ab92552; Abcam) and β-actin (1:5,000; cat. no. bs-0061R; BIOSS), followed by incubation with secondary antibodies (1:5,000; cat. no. bs-0296G-HRP; BIOSS) at room temperature for 2.5 h. Protein bands were visualized with an ECL western blotting kit (Cell Signaling Technology, Inc.) using cSeries Imager (Azure Biosystems, Inc.).

Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Control, CCK-8 Assay, Migration, Western Blot, Expressing, Over Expression, Wound Healing Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA, Cell Counting

Journal: Journal of Human Genetics

Article Title: Compound heterozygous variants in MYH11 underlie autosomal recessive megacystis-microcolon-intestinal hypoperistalsis syndrome in a Chinese family

doi: 10.1038/s10038-019-0651-z

Figure Lengend Snippet: Compound heterozygous variants in MYH11 in a family with MMIHS. a Prenatal ultrasonography image at 13 weeks of gestation for the index fetus demonstrated a distended bladder (2.56 cm × 2.32 cm). b Prenatal ultrasonography image at 17 weeks shows a progressive distention of the bladder (9.5 cm × 7.16 cm) in the index fetus. c Sanger sequencing validates the exome sequencing variant of c.2051 G > A (p.R684H) in MYH11 (NM_001040114). d Sanger sequencing validates the exome sequencing variant of c.3540_3541delinsTT (p.(E1180D, Q1181Ter)) in MYH11 (NM_001040114). e Protein expression of MYH11 in the control (Ctrl) and proband umbilical cord tissues. Arrows point to the band location for protein MYH11

Article Snippet: Anti-MYH11 antibody (1:50, Monoclonal rabbit IgG, BM5659, Boster Biological Technology, China) was used as a primary antibody.

Techniques: Sequencing, Variant Assay, Expressing, Control

Journal: Journal of Human Genetics

Article Title: Compound heterozygous variants in MYH11 underlie autosomal recessive megacystis-microcolon-intestinal hypoperistalsis syndrome in a Chinese family

doi: 10.1038/s10038-019-0651-z

Figure Lengend Snippet: Summary of clinical and molecular findings of four genes involved in autosomal recessive MMIHS

Article Snippet: Anti-MYH11 antibody (1:50, Monoclonal rabbit IgG, BM5659, Boster Biological Technology, China) was used as a primary antibody.

Techniques: Sequencing