Journal: Molecular Medicine Reports

Article Title: Rs41291957 polymorphism in the promoter region of microRNA-143 serves as a prognostic biomarker for patients with intracranial hemorrhage

doi: 10.3892/mmr.2021.11928

Figure Lengend Snippet: TLR2 is a direct target gene of miR-143. (A) Computational analysis of the regulatory relationship between miR-143 and TLR2 mRNA. (B) Luciferase assay of THP-1 cells co-transfected with wild-type or mutant TLR2 mRNA, and miR-143 or miRNA controls. (C) Luciferase assay of HPASMC cells co-transfected with wild-type or mutant TLR2 mRNA, and miR-143 or miRNA controls. (D) Relative expression of miR-143 in THP-1 cells transfected with miR-143 mimics. (E) Relative expression of miR-143 in HPASMC cells transfected with miR-143 mimics. n=3. *P<0.05 vs. negative control group. TLR2, Toll-like receptor 2; miR-143, microRNA-143; miR-cont, microRNA-control; 3′-UTR, 3′-untranslated region; WT, wild-type; MUT, mutant; NC, negative control.

Article Snippet: THP-1 (human monocytic cells) and human pulmonary artery smooth muscle cells (HPASMC) obtained from American Type Culture Collection were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Luciferase, Transfection, Mutagenesis, Expressing, Negative Control, Control

Journal: Molecular Medicine Reports

Article Title: Rs41291957 polymorphism in the promoter region of microRNA-143 serves as a prognostic biomarker for patients with intracranial hemorrhage

doi: 10.3892/mmr.2021.11928

Figure Lengend Snippet: IL-16 mRNA is not a target of miR-143. (A) Computational analysis of the regulatory relationship between miR-143 and IL-16 mRNA. (B) Luciferase assay of THP-1 cells co-transfected with wild-type or mutant IL-16 mRNA, and miR-143 or miRNA controls. (C) Luciferase assay of HPASMC cells co-transfected with wild-type or mutant IL-16 mRNA, and miR-143 or miRNA controls. IL-16, interleukin-16; miR-143, microRNA-143; 3′-UTR, 3′-untranslated region; WT, wild-type; MUT, mutant.

Article Snippet: THP-1 (human monocytic cells) and human pulmonary artery smooth muscle cells (HPASMC) obtained from American Type Culture Collection were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Luciferase, Transfection, Mutagenesis

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-212-5p, an anti-proliferative miRNA, attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents

doi: 10.1016/j.omtn.2022.06.008

Figure Lengend Snippet: Expression of miR-212-5p is induced in PASMCs and the lung of PH subjects Expression of miR-212-5p was measured by qRT-PCR analysis in the following samples. (A) PASMCs of human PH patients (n = 4) or normal donors (control, n = 9). (B) PASMCs isolated from control mice (N, exposed to room air, n = 8) or mice with hypoxia-induced PH (H, 10% O 2 , 3 weeks, n = 7). (C) Lungs of control mice (N, exposed to room air, n = 5 for each time point) or mice exposed to hypoxia for 1, 2, or 3 weeks (H, 10% O 2 , n = 5 for each time point). (D) Lungs of control rats (CTRL, given DMSO and stayed in room air, n = 6) or rats with Sugen/hypoxia-induced severe PH (SuHx, n = 5). Data are presented as mean ± SEM. ∗ or ∗∗, versus CTRL or N. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: MiR-212-5p is an “anti-proliferative” miRNA in PASMC (A) Normal human PASMCs (hPASMCs, Lonza) were transfected with miR-212-5p or negative control miR inhibitor (Anti-miR-212 versus Anti-Neg).

Techniques: Expressing, Quantitative RT-PCR, Control, Isolation

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-212-5p, an anti-proliferative miRNA, attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents

doi: 10.1016/j.omtn.2022.06.008

Figure Lengend Snippet: MiR-212-5p is an “anti-proliferative” miRNA in PASMC (A) Normal human PASMCs (hPASMCs, Lonza) were transfected with miR-212-5p or negative control miR inhibitor (Anti-miR-212 versus Anti-Neg). BrdU incorporation assay was performed. Proliferation level of PASMCs transfected with negative control miR inhibitor (Anti-Neg) was set as 1 and served as controls. Proliferation level of PASMCs transfected with miR-212-5p inhibitor (Anti-miR-212) was compared with that of control cells. Inhibition of miR-212-5p induced PASMC proliferation in vitro in room air. (B) Normal hPASMC (Lonza) were transfected with miR-212-5p or negative control miR mimic and then exposed to room air (N) or hypoxia (H, 1% O 2 ) for 24 h. BrdU incorporation assay was performed to determine the relative proliferation level of these cells. Proliferation level of PASMCs transfected with negative control miR mimic (Neg mimic) and exposed to room air was set as 1 and served as controls. Proliferation levels of PASMCs in other groups were compared with that of control cells. miR-212-5p mimic significantly suppressed PASMC proliferation in both room air and hypoxia. (C) Normal hPASMCs (Lonza) were infected with adenoviral particles that express miR-212-5p (Ad-miR212, 100 PFUs/cell) or control adenoviral particles (Ad-Ctrl, 100 PFUs/cell) on day 1. Then cells were collected for cell counting on day 3 and 5, respectively. Overexpression of miR-212-5p significantly suppressed cell growth in room air. (D) Human PASMCs of PH patient (PH hPASMCs) were transfected with miR-212-5p or negative control miR mimic. BrdU incorporation assay was performed and relative cell proliferation level was compared, as described above. miR-212-5p mimic also significantly suppressed proliferation of PH hPASMCs. Data are presented as mean ± SEM. ∗ or ∗∗ versus room air Anti-Neg, Neg mimic control, or Ad-Ctrl, respectively; ## versus hypoxic Neg mimic control. ∗p < 0.05, ∗∗ or ##p < 0.01.

Article Snippet: MiR-212-5p is an “anti-proliferative” miRNA in PASMC (A) Normal human PASMCs (hPASMCs, Lonza) were transfected with miR-212-5p or negative control miR inhibitor (Anti-miR-212 versus Anti-Neg).

Techniques: Transfection, Negative Control, BrdU Incorporation Assay, Control, Inhibition, In Vitro, Infection, Cell Counting, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-212-5p, an anti-proliferative miRNA, attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents

doi: 10.1016/j.omtn.2022.06.008

Figure Lengend Snippet: SMC-specific knockout of miR-212 significantly exacerbates hypoxia-induced PH in mice (A) The strategy to generate smooth muscle cell (SMC)-specific miR-212 knockout mice and mouse genotyping (B). (C) The expression levels of miR-212-5p in freshly isolated mouse PASMCs (mPASMCs) from sm-212 −/ − mice (n = 4) and their 212-fl/fl littermates (n = 4). sm-212 −/− and their 212-fl/fl littermates were exposed to room air or hypoxia (10% O 2 ) for 3 weeks and then PH indices were measured. SMC-specific knockout of miR-212 significantly exacerbated hypoxia-induced RVSP elevation (D), RV hypertrophy (E), and pulmonary vessel wall thickening (F–G). V: vessels. Data are presented as mean ± SEM. ∗, ∗∗, or ∗∗∗ versus fl/fl control in room air; ## or ### versus fl/fl control in hypoxia; ∗p < 0.05, ∗∗ or ##p < 0.01, ###p < 0.001.

Article Snippet: MiR-212-5p is an “anti-proliferative” miRNA in PASMC (A) Normal human PASMCs (hPASMCs, Lonza) were transfected with miR-212-5p or negative control miR inhibitor (Anti-miR-212 versus Anti-Neg).

Techniques: Knock-Out, Expressing, Isolation, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-212-5p, an anti-proliferative miRNA, attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents

doi: 10.1016/j.omtn.2022.06.008

Figure Lengend Snippet: MiR-212-5p is an “anti-proliferative” miRNA in PASMC (A) Normal human PASMCs (hPASMCs, Lonza) were transfected with miR-212-5p or negative control miR inhibitor (Anti-miR-212 versus Anti-Neg). BrdU incorporation assay was performed. Proliferation level of PASMCs transfected with negative control miR inhibitor (Anti-Neg) was set as 1 and served as controls. Proliferation level of PASMCs transfected with miR-212-5p inhibitor (Anti-miR-212) was compared with that of control cells. Inhibition of miR-212-5p induced PASMC proliferation in vitro in room air. (B) Normal hPASMC (Lonza) were transfected with miR-212-5p or negative control miR mimic and then exposed to room air (N) or hypoxia (H, 1% O 2 ) for 24 h. BrdU incorporation assay was performed to determine the relative proliferation level of these cells. Proliferation level of PASMCs transfected with negative control miR mimic (Neg mimic) and exposed to room air was set as 1 and served as controls. Proliferation levels of PASMCs in other groups were compared with that of control cells. miR-212-5p mimic significantly suppressed PASMC proliferation in both room air and hypoxia. (C) Normal hPASMCs (Lonza) were infected with adenoviral particles that express miR-212-5p (Ad-miR212, 100 PFUs/cell) or control adenoviral particles (Ad-Ctrl, 100 PFUs/cell) on day 1. Then cells were collected for cell counting on day 3 and 5, respectively. Overexpression of miR-212-5p significantly suppressed cell growth in room air. (D) Human PASMCs of PH patient (PH hPASMCs) were transfected with miR-212-5p or negative control miR mimic. BrdU incorporation assay was performed and relative cell proliferation level was compared, as described above. miR-212-5p mimic also significantly suppressed proliferation of PH hPASMCs. Data are presented as mean ± SEM. ∗ or ∗∗ versus room air Anti-Neg, Neg mimic control, or Ad-Ctrl, respectively; ## versus hypoxic Neg mimic control. ∗p < 0.05, ∗∗ or ##p < 0.01.

Article Snippet: Inhibition of miR-212-5p induced PASMC proliferation in vitro in room air. (B) Normal hPASMC (Lonza) were transfected with miR-212-5p or negative control miR mimic and then exposed to room air (N) or hypoxia (H, 1% O 2 ) for 24 h. BrdU incorporation assay was performed to determine the relative proliferation level of these cells.

Techniques: Transfection, Negative Control, BrdU Incorporation Assay, Control, Inhibition, In Vitro, Infection, Cell Counting, Over Expression

Journal: Cell Proliferation

Article Title: Super‐Enhancer Target Gene CBP/p300‐Interacting Transactivator With Glu/Asp‐Rich C‐Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling

doi: 10.1111/cpr.13817

Figure Lengend Snippet: CITED2 can affect the proliferation of idiopathic PAH patient cells. (A) The overexpression efficiency of the CITED2 was detected in iPAH patient cells. (B) Expression changes of CITED2 in iPAH patient cells. (C) The CCK8 experiment showed that overexpression of CITED2 could affect the growth and viability of cells. (D, E) After overexpression of CITED2, the levels of cell cycle‐related proteins decreased in patients. (F) Overexpression of CITED2 in hypoxia‐treated hPASMCs reduced Ki67 levels in the cells. (G) Flow cytometry results showed that overexpression of CITED2 reduced the proportion of cells in the G2/M phase and S phase and affected cell cycle progression. (H) After overexpression of CITED2, the expression of PCNA in patient cells decreased. (I, J) Overexpression of CITED2 in hypoxia‐treated hPASMCs also affected the expression of cell cycle‐related proteins. (Bar = mean ± S.E.M, * p < 0.05; ** p < 0.01).

Article Snippet: Normal hPASMCs were purchased from Procell Life Science & Technology Co. Ltd.

Techniques: Over Expression, Expressing, Flow Cytometry