MBD1 knockdown inhibits proliferation, migration and angiogenesis of human umbilical vein endothelial cells in vitro

doi:10.12122/j.issn.1673-4254.2026.07.12

Abstract

Abstract:

Objective To investigate the effects of methyl-CpG binding domain protein 1 (MBD1) on proliferation, migration, and angiogenesis in human umbilical vein endothelial cells (HUVECs). Method Cultured HUVECs were infected with a lentivirus carrying sh-MBD1 or a scramble shRNA (sh-NC), and the successfully infected cells were screened using puromycin. RT-qPCR and Western blotting were used to assess MBD1 gene silencing efficiency. The changes in proliferation, cell cycle, and migration of the infected cells were assessed using CCK-8 and colony formation assays, flow cytometry, scratch assay, and Transwell assay. The impact of MBD1 knockdown on angiogenic capacity of HUVECs was evaluated using angiogenesis assay. Immunofluorescence staining was used to investigate the expression of vascular endothelial growth factor A (VEGFA) in the infected HUVECs. Result Compared with the sh-NC cells, HUVECs infected with the lentivirus carrying sh-MBD1 exhibited significantly reduced proliferation capacity and migration ability. The total length of vessels formed in vitro, closed vascular loops, and the number of branching points were all lower in sh-MBD1 cells than in sh-NC cells. VEGFA expression was significantly reduced in sh-MBD1 cells compared to the sh-NC cells. Conclusion MBD1 promotes proliferation, migration, and angiogenesis of HUVECs possibly by regulating the expression of VEGFA.

Key words: angiogenesis, methyl-CpG-binding domain protein 1, human umbilical vein endothelial cells, vascular endothelial growth factor A, cell proliferation, cell migration

Yutong DAN, Tingfu ZHANG, Hongmei LIU, Peng ZHANG. MBD1 knockdown inhibits proliferation, migration and angiogenesis of human umbilical vein endothelial cells in vitro[J]. Journal of Southern Medical University, 2026, 46(7): 1585-1592.

Figures/Tables 6

Fig.1 Construction and validation of human umbilical vein endothelial cells (HUVECs) with MBD1 knockdown. A: Fluorescence microscopy of HUVECs 48 h after lentivirus infection (Original magnification: ×50). B: qPCR detection of MBD1 mRNA expression levels in both groups (n=3). C: Western blotting for assessing interference efficiency and statistics of MBD1 protein expression levels (n=4). **P<0.01, ***P<0.001 vs sh-NC group.

Fig.2 Effect of MBD1 knockdown on migration capacity of HUVECs. A: Observation and statistical analysis of scratch healing rates in HUVECs at 12 h (n=15). B: HUVECs scratch healing rate and statistics at 12 h (n=14). C: Transwell assay for assessing migration capacity of the infected cells (n=7). Scale bar=100 μm. **P<0.01, ****P<0.0001 vs sh-NC group.

Fig.3 Effect of MBD1 knockdown on proliferation of HUVECs. A: Results of clonogenic assay of the infected HUVECs (n=6). B: CCK-8 assay for assessing cell viability of the infected cells (n=8). C: Proliferation curves of the infected cells based on CCK-8 assays (n=3). *P<0.01, ***P<0.001, ****P<0.0001 vs sh-NC group.

Fig.4 Effect of MBD1 knockdown on cell cycle of HUVECs. A: Distribution and proportion of cells in G0/G1 phase, S phase, and G2/M phase in the two groups. B: Statistical analysis of the proliferation index in the two groups (n=7). ****P<0.0001 vs sh-NC group.

Fig.5 Effect of MBD1 knockdown on angiogenic capacity of HUVECs. A: Schematic diagram of tubule formation experimental workflow. B: Photographs of the cells at 2, 4, and 6 h post-tubing induction. C: Photographs of both cell groups after 24 h of tubule formation observed under a fluorescence microscope. D: Statistical analysis of total tubule length, meshes, and branching points at 6 h in both groups (n=6). Scale bar=100 μm. **P<0.01 vs sh-NC group.

Fig.6 Immunofluorescence staining for detection of VEGFA Expression in HUVECs. A: VEGFA fluorescence expression (Scale bar=100 μm). B: Statistical analysis of VEGFA fluorescence intensity in the two cell groups. ****P<0.0001 vs sh-NC group.

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