Effects of miR-338-3p/TCF4 on choroidal microvascular endothelial cell proliferation and apoptosis

Jiancheng LI; Zhanyun BU; Junhui PAN

ABSTRACT

Objective:

To investigate whether microRNA-338-3p (miR-338-3p) affects the proliferation and apoptosis of choroidal microvascular endothelial cells by regulating the expression of T cell factor 4 (TCF4).

Methods:

Human choroidal microvascular endothelial cells were cultured in vitro, and they were divided into vascular endothelial growth factor (VEGF) group and Normal control group.The Cultured cells in the VEGF group were divided into four subgroups, and were transfected with miR-NC, miR-338-3p mimics, miR-338-3p mimics + pcDNA, miR-338-3p mimics + pcDNA-TCF4 before VEGF treatment, respectively.Quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-338-3p and TCF4.MTT assay was used to detect cell proliferation.Flow cytometry was used to detect the apoptosis rate.The double luciferase report experiment verified the targeting relationship of miR-338-3p and TCF4.Western blot was used to detect the expression of Ki-67, PCNA, bax, and bcl-2.

Results:

After VEGF treatment, the expression level of miR-338-3p was significantly reduced, the expression level of TCF4 mRNA was significantly increased, the cell viability was significantly increased, and the apoptosis rate was significantly decreased, the levels of Ki-67, PCNA, bcl-2 protein were increased significantly, and the level of bax protein was decreased significantly (all at P<0.05). After miR-338-3p overexpression, the cell viability was significantly reduced, the apoptosis rate was significantly increased, and the levels of Ki-67, PCNA, and bcl-2 proteins were significantly reduced, the level of bax protein was increased significantly (all at P<0.05). Double luciferase reporting experiments confirmed that miR-338-3p targeted TCF4.Compared with the VEGF + miR-338-3p + pcDNA group, the cell viability of the VEGF + miR-338-3p + pcDNA-TCF4 group was significantly increased ([56.48±13.20]% vs. [96.24±16.24]%), and the apoptosis rate was significantly reduced ([30.59±3.57]% vs.[12.36±1.29]%), the levels of Ki-67, PCNA and bcl-2 protein were significantly increased (0.41±0.11 vs. 0.96±0.19; 0.44±0.10 vs. 0.97±0.20; 0.55±0.12 vs. 0.98±0.15), and the levels of bax protein were significantly decreased (0.87±0.13 vs. 0.42±0.11) ( t=5.700, 14.408, 7.516, 7.111, 6.715, 7.927; all at P<0.01).

Conclusions:

Overexpression of miR-338-3p can negatively regulate TCF4 expression, thereby inhibite choroidal microvascular endothelial cell proliferation and induce apoptosis.