miR-152-3p aggravates vascular endothelial cell dysfunction by targeting DEAD-box helicase 6 (DDX6) under hypoxia

Figures & data

Table 1. Sequences of primers used for RT-qPCR

Gene	Sequence (5ʹ→3ʹ)
miR-152-3p forward	TCAGTGCATGACAGAACTTGG
miR-152-3p reverse	CTCTACAGCTATATTGCCAGCCA
DDX6 forward	TTGCTAGCCAAGAAGATTTCTC
DDX6 reverse	ACGATTTCGATGTTCCTGC
ATP2A2 forward	GTATGGCAGGAAAGAAATGCT
ATP2A2 reverse	CTGTCGATACACTTTGCCC
QKI forward	TTTCTGTGGACGCCTAGAG
QKI reverse	TTCCGTACTCTGCTAATTTCTG
SLC25A44 forward	TTCTATGCAGGTTGAGGGC
SLC25A44 reverse	AGATGATTCTGGCCGAGAG
GADD45A forward	AACGACATCAACATCCTGC
GADD45A reverse	AATGTGGATTCGTCACCAG
GAPDH forward	CCTCCTGTTCGACAGTCAG
GAPDH reverse	CATACGACTGCAAAGACCC
U6 forward	CTTTGGCAGCACATATACCA
U6 reverse	CTCATTCAGAGGCCATGCT

Figure 1. Inhibition of miR-152-3p mitigates hypoxia-induced angiogenesis in HUVECs. (a) miR-152-3p expression in HUVECs under hypoxia was examined by RT-qPCR in thrice. (b) The efficiency of miR-152-3p downregulation was measured by RT-qPCR in triplicate. (c) CCK-8 assays were conducted to detect the viability of HUVECs transfected with miR-152-3p inhibitor under hypoxia. each experiment was repeated three times. (d) Tube formation assays were applied to determine effects of mir-152-3p inhibitor on angiogenesis. the experiment was conducted in triplicate. (e) Western blot analyses were conducted three times to examine levels of angiogenesis-associated proteins (VEGFA and ANGII). **p < 0.01, ***p < 0.001

Figure 2. Inhibition of miR-152-3p alleviates hypoxia-induced endothelial permeability of HUVECs. (a) Western blot analysis was employed to examine levels of tight junction proteins (ZO-1 and occludin) in HUVECs under hypoxia. Each experiment was performed in triplicate. (b) Endothelial permeability of indicated cells was detected utilizing endothelial cell permeability in vitro assays. the assay was performed in thrice. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 3. DDX6 is targeted by miR-152-3p. (a) Expression levels of candidate mRNAs in HUVECs transfected with miR-152-3p inhibitor or NC inhibitor were examined utilizing RT-qPCR in thrice. (b) Western blot analyses were conducted three times to measure effects of miR-152-3p inhibitor on the protein level of DDX6 in cells. (c) The possible binding site between miR-152-3p and DDX6 was predicted from the starBase, and luciferase reporter assays were performed in thrice to explore the interaction between miR-152-3p and DDX6. (d) DDX6 expression in HUVECs under hypoxia was examined utilizing RT-qPCR analysis in triplicate. **p < 0.01, ***p < 0.001

Figure 4. miR-152-3p promotes tube formation of HUVECs by targeting DDX6. (a) The knockdown efficiency of DDX6 in HUVECs under hypoxia was detected by RT-qPCR in thrice. (b) CCK-8 assays were carried out three times to probe effects of miR-152-3p inhibition and DDX6 silencing on cell viability. (c) The angiogenesis in cells with different transfection was measured by tube formation assays. Each experiment was repeated in triplicate. (d) Western blot analyses were employed to probe protein levels of angiogenesis-associated factors in cells with transfection of miR-152-3p inhibitor and sh-DDX6. Each analysis was performed three times. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 5. miR-152-3p facilitates endothelial permeability of HUVECs by downregulating DDX6 expression. (a) Protein levels of ZO-1 and occludin in cells with transfection of NC inhibitor, miR-152-3p inhibitor or miR-152-3p inhibitor + sh-DDX6 were examined by western blot analyses. each analysis was performed in thrice. (b) The rescue effect of DDX6 on the decrease in cell permeability induced by miR-152-3p inhibition was probed utilizing endothelial cell permeability in vitro assays. Each experiment was performed in thrice. *p < 0.05, ***p < 0.001