Knockdown of circ_0003204 alleviates oxidative low-density lipoprotein-induced human umbilical vein endothelial cells injury: Circulating RNAs could explain atherosclerosis disease progression

Figures

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  Figure 1. Effects of ox-LDL on HUVECs injury and circ_0003204 expression. (a) HUVECs were treated with 50, 100, and 200 μg/mL ox-LDL for 24 h, and the viability of HUVECs was measured by CCK8 assay. (b) HUVECs were treated with 100 μg/mL ox-LDL for 12, 24, and 48 h, respectively. CCK8 assay was used to detect the viability of HUVECs. (c–k) HUVECs were treated with 100 μg/mL ox-LDL for 24 h. (c) The apoptosis of HUVECs was assessed by flow cytometry. (d) The activity of caspase-3 was determined using Caspase-3 Activity Assay Kit. (e–g) The contents of IL-6, IL-1β, and TNF-α were tested via the IL-6, IL-1β, and TNF-α ELISA Assay Kits, respectively. (h–j) The expression of ROS and MDA and the activity of LDH were measured using the ROS, MDA, and LDH Assay Kits, respectively. (k) The expression of circ_0003204 was determined by qPCR. *P < 0.05.

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  Figure 2. Effects of circ_0003204 silencing on ox-LDL-induced HUVECs injury. HUVECs were transfected with si-circ_0003204 or si-NC, followed by treatment with ox-LDL. (a) The qPCR was used to detect circ_0003204 expression. (b) CCK8 assay was used to determine the viability of HUVECs. (c) Flow cytometry was performed to test the apoptosis of HUVECs. (d) The activity of caspase-3 was measured using Caspase-3 Activity Assay Kit. (e–g) IL-6, IL-1β, and TNF-α ELISA Assay Kits were used to assess the contents of IL-6, IL-1β, and TNF-α in ox-LDL-induced HUVECs. (h–j) The expression of ROS and MDA and the activity of LDH were evaluated using the ROS, MDA, and LDH Assay Kits, respectively. *P < 0.05.

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  Figure 3. circ_0003204 served as a sponge of miR-330-5p. (a) The fragments of circ_0003204 containing the binding sites or mutant binding sites of miR-330-5p were exhibited. (b and c) Dual-luciferase reporter assay was performed to determine the luciferase activity of circ_0003204 WT or MUT vector in ox-LDL-induced HUVECs. (d) RNA pull-down assay was used to verify the interaction between circ_0003204 and miR-330-5p. The relative expression of circ_0003204 in Bio-miR-330-5p probe or Bio-miR-330-5p MUT probe was detected using qPCR. (e) The miR-330-5p expression in ox-LDL-induced HUVECs or normal HUVECs was assessed by qPCR. (f) HUVECs were transfected with circ_0003204 overexpression plasmid or si-circ_0003204 or their negative controls (pcDNA or si-NC), followed by treatment with ox-LDL. The qPCR was used to test the expression of miR-330-5p. *P < 0.05.

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  Figure 4. Effects of miR-330-5p inhibitor on ox-LDL-induced HUVECs injury. HUVECs were transfected with si-NC, si-circ_0003204, si-circ_0003204 + in-miR-NC, or si-circ_0003204 + in-miR-330-5p, followed by treatment with ox-LDL. (a) The expression of miR-330-5p was measured using qPCR. (b) CCK8 assay was performed to test the viability of HUVECs. (c) The apoptosis of HUVECs was determined via flow cytometry. (d) Caspase-3 Activity Assay Kit was used to assess the activity of caspase-3. (e–g) The contents of IL-6, IL-1β, and TNF-α were detected using the IL-6, IL-1β, and TNF-α ELISA Assay Kits, respectively. (h–j) The ROS, MDA, and LDH Assay Kits were used to measure the expression of ROS and MDA and the activity of LDH. *P < 0.05.

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  Figure 5. TLR4 was a target of miR-330-5p. (a) The sequences of TLR4 3′UTR contained the binding sites or mutant binding sites of miR-330-5p were presented. (b and c) The luciferase activity of TLR4 3′UTR WT or MUT vector in ox-LDL-induced HUVECs was measured using dual-luciferase reporter assay. (d) The interaction between TLR4 and miR-330-5p was confirmed via RNA pull-down assay, and the relative expression of TLR4 in Bio-miR-330-5p probe or Bio-miR-330-5p MUT probe was determined using qPCR. (e) The protein level of TLR4 in ox-LDL-induced HUVECs or normal HUVECs was detected by WB analysis. (f) The protein level of TLR4 was tested using WB analysis to evaluate the effect of miR-330-5p mimic on TLR4 expression. (g) HUVECs were transfected with si-NC, si-circ_0003204, si-circ_0003204 + in-miR-NC, or si-circ_0003204 + in-miR-330-5p, followed by treatment with ox-LDL. WB analysis was performed to assess the expression of TLR4. *P < 0.05.

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  Figure 6. Effects of miR-330-5p mimic and TLR4 overexpression on ox-LDL-induced HUVECs injury. HUVECs were transfected with miR-NC, miR-330-5p, miR-330-5p + pcDNA, or miR-330-5p + TLR4, followed by treatment with ox-LDL. (a) The protein level of TLR4 was determined using WB analysis. (b) The viability of HUVECs was detected via CCK8 assay. (c) The apoptosis of HUVECs was measured using flow cytometry. (d) Caspase-3 Activity Assay Kit was used to determine the activity of caspase-3. (e–g) The IL-6, IL-1β, and TNF-α ELISA Assay Kits were used to detect the contents of IL-6, IL-1β, and TNF-α, respectively. (h–j) The expression of ROS and MDA and the activity of LDH were assessed using the ROS, MDA, and LDH Assay Kits, respectively. *P < 0.05.

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  Figure 7. The circ_0003204/miR-330-5p/TLR4 axis regulated the activity of the NF-κB signaling pathway. (a) HUVECs were transfected with si-NC, si-circ_0003204, si-circ_0003204 + in-miR-NC, or si-circ_0003204 + in-miR-330-5p, followed by treatment with ox-LDL. The relative expression of p-p65/p65 and p-IκBα/IκBα was measured using WB analysis. (b) HUVECs were transfected with miR-NC, miR-330-5p, miR-330-5p + pcDNA, or miR-330-5p + TLR4, followed by treatment with ox-LDL. WB analysis was used to determine the relative expression of p-p65/p65 and p-IκBα/IκBα. *P < 0.05.

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  Figure 8. The schematic diagram of the main findings of this research. In ox-LDL-induced HUVECs, circ_0003204 inhibited the viability and promoted the apoptosis, oxidative stress, and inflammatory response of cells via mediating the activity of NF-κB signaling pathway by regulating the miR-330-5p/TLR4 axis.