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

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: miR-637 and miR-661 suppress HPASMC proliferation and migration. HPASMCs were cultured with specific microRNAs (miRNAs) as indicated for 48 h under hypoxic culture conditions, followed by qRT‒PCR. ( B , C ) Cells from ( A ) were seeded into 96-well plates, followed by MTT ( B ) and EdU ( C ), the statistical analysis were performed. D Cells from ( A ) were subjected to a transwell assay, the statistical analysis were performed.The data are presented as the means ± SDs ( n = 5). The statistical test used was the paired t test. ***, p < 0.001 versus miR-NC.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Cell Culture, Transwell Assay

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: miR-637 and miR-661 inhibit the expression of TRIM29. ( A ) HPASMCs were transfected with miR-637 or miR-661, followed by RNA-seq analysis. Venn diagram of gene changes after the overexpression of two different microRNAs (FC: fold change). ( B , C ) HPASMCs were overexpressing miR-637 or miR-661, followed by RNA-seq analysis. The transcript volcano plot shows the differentially expressed genes as indicated. ( D ) HPASMC cells were transfected with indicated plasmids for 48 h, followed by luciferase assay. ( E ) HPASMCs were overexpressing miR-637 or miR-661 under hypoxic culture conditions, followed by RT‒PCR and WB. Original blots are presented in Supplementary Fig. 1.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Transfection, RNA Sequencing, Over Expression, Luciferase

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: TRIM29 attenuates the miR-637- and miR-661-induced inhibition of AKT/mTOR signalling. ( A ) Statistical analysis of the results of the GO enrichment assay was performed after the indicated miRNAs were overexpressed. B-C HPASMCs were cultured for different durations under hypoxic conditions, followed by WB ( B ) and qRT‒PCR ( C ). ( D ) qRT‒PCR was used to detect the expression level of TRIM29 in the serum of patients with pulmonary hypertension and healthy volunteers. ( E ) HPASMCs were overexpressing miR-637 or miR-661 under hypoxic culture conditions, followed by RT‒PCR and WB. ( F ) WB assays were performed after the inhibition of miR-637 and miR-661. ( G ) HPASMCs were transfected with Flag-TRIM29 for 48 h under hypoxic culture conditions, and the cells were subjected to WB analysis. ( H ) TRIM29 was knocked out in HAPSMCs under hypoxic culture conditions, and cell lysates were then prepared for WB analysis. ( I ) HPASMCs were transfected with Flag-TRIM29 or overexpressed with microRNA as indicated for 48 h under hypoxic culture conditions, followed by WB analysis. Original blots are presented in Supplementary Figs. 2–7.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Inhibition, Cell Culture, Expressing, Transfection

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: miR-637 and miR-661 attenuate TRIM29-induced HPASMC proliferation and migration. ( A ) HPASMCs were transfected with the indicated plasmids or infected with TRIM29 sgRNA for 48 h of hypoxia, followed by the MTT assay. ( B – E ) HPASMCs were transfected with Flag-TRIM29, infected with TRIM29 sgRNA lentivirus or overexpressed with microRNA as indicated for 48 h under hypoxic culture conditions, followed by the EdU assay ( B ), wound healing assay ( D ) and the statistical analysis were performed ( C , E ). The data are presented as the means ± SDs ( n = 5). The statistical test used was the paired t test. ***, p < 0.001 versus miR-NC.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Transfection, Infection, MTT Assay, EdU Assay, Wound Healing Assay

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