CircRNA Galntl6 sponges miR-335 to ameliorate stress-induced hypertension through upregulating Lig3 in rostral ventrolateral medulla

Figures

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  Fig. 1. CircRNA Galntl6 expression was downregulated in RVLM of SIH rats. (A) Heatmap shows the top five differentially expressed circRNAs identified by RNA sequencing between SIH and control rats. (B) RT-qPCR was used to measure the relative expressions of three indicated circRNAs listed in (A). (C) CircRNA Galntl6 expression was detected by RT-qPCR in 11 tissues of rats. (D) Expressions of circRNA Galntl6 and Galntl6 mRNA in RVLM tissues treated with or without RNase R were examined by RT-qPCR. (E) RT-PCR and Sanger sequencing were used to verify the existence of circRNA Galntl6. (F) FISH was performed to evaluate the subcellular localization of circRNA Galntl6 in B104 cells. Data were expressed as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student's t-test (B and D) and one-way ANOVA, followed by post-hoc Bonferroni test (C). n = 6 rats per group (B–D). *p < 0.05, **p < 0.01 versus control group. ΔΔΔp < 0.001 versus heart group. ###p < 0.001 versus mock group.

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  Fig. 2. Overexpression of circRNA Galntl6 in RVLM improved SIH. (A) Radio telemetry was used to measure the MAP and HR in rats after circRNA Galntl6 overexpression. (B and C) Effects of circRNA Galntl6 overexpression on the sympathetic outflow were unveiled using RSNA recording test and plasma NE ELISA. (D) RVLM neuronal excitability was detected by immunostaining with c-Fos. Data were expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by post-hoc Bonferroni test (A–D). n = 6 rats per group (A–D). *p < 0.05, ***p < 0.001, and ns means nonsignificant versus SIH group.

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  Fig. 3. CircRNA Galntl6 restricted RVLM oxidative stress in SIH rats. (A–D) After microinjection of pLV-circRNA Galntl6 plasmid in the RVLM of SIH rats, the level of 8-OH-dG and the activities of SOD and CAT were determined by commercial kits, and ROS production was detected by DHE staining. Data were expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by post-hoc Bonferroni test (A–D). n = 6 rats per group (A–D). *p < 0.05, ***p < 0.001, and ns means nonsignificant versus SIH group.

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  Fig. 4. CircRNA Galntl6 bound to miR-335 and decreased its expression. (A) Predicted binding sites between circRNA Galntl6 and miR-335. (B) RNA pulldown assay showed that biotin-labeled miR-335 interacted with circRNA Galntl6. (C) Dual-luciferase reporter assay was used to confirm the targeted relationship between circRNA Galntl6 and miR-335. (D and E) RT-qPCR was used to measure the effect of circRNA Galntl6 overexpression or knockdown on miR‐335 expression in B104 cells. (F) Relative expression of miR-335 was detected by RT-qPCR in RVLM administrated with pLV-circRNA Galntl6 plasmid. Data were expressed as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student's t-test (B–E) and one-way ANOVA, followed by post-hoc Bonferroni test (F). n = 6 of independent cell culture preparations (B–E). n = 6 rats per group (F). ##p < 0.01, ###p < 0.001, and ns means nonsignificant versus agomir NC group. ΔΔp < 0.01 versus pLV-NC group. &&p < 0.01 versus si-NC group. **p < 0.01, and ns means nonsignificant versus SIH group.

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  Fig. 5. CircRNA Galntl6 repressed oxidative stress in RVLM of SIH rats by binding to miR-335. (A–C) Effects of circRNA Galntl6 overexpression and miR-335 agomir on the level of 8-OH-dG and activities of SOD and CAT in RVLM were measured using commercial kits. (D) Influences of circRNA Galntl6 upregulation and miR-335 agomir on ROS production in RVLM were detected by DHE staining. Data were expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by post-hoc Bonferroni test (A–D). n = 6 rats per group (A–D). *p < 0.05, **p < 0.01, ***p < 0.001, and ns means nonsignificant versus SIH group.

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  Fig. 6. Lig3 was a downstream mRNA target of miR-335. (A) Schematic graph of the potential binding sites for miR-335 within Lig3 3′ UTR. (B) The luciferase activities of Lig3 luciferase reporters (WT or MUT) were tested in B104 cells co-expressing miR-335 agomir or agomir NC. (C and D) RT-qPCR and Western blot analyses showed that the expression of Lig3 decreased or increased in B104 cells when miR-335 was overexpressed or downregulated, respectively. (E) Following miR-335 knockdown in RVLM of SIH rats, Lig3 expression was measured using RT-qPCR and Western blot assays. Data were expressed as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student's t-test (B–D) and one-way ANOVA, followed by post-hoc Bonferroni test (E). n = 3–6 of independent cell culture preparations (B–D). n = 3–6 rats per group (E). ##p < 0.01, ###p < 0.001, and ns means nonsignificant versus agomir NC group. Δp < 0.05, and ΔΔp < 0.01 versus antagomir NC group. **p < 0.01, and ***p < 0.001 versus SIH group.

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  Fig. 7. MiR-335 caused oxidative stress in RVLM of SIH rats via targeting Lig3. (A–C) Effects of miR-335 downregulation on the level of 8-OH-dG and activities of SOD and CAT in RVLM of SIH rats were reversed by Lig3 depletion. (D) Lig3 knockdown weakened the suppressive influence of miR-335 silencing on ROS production in RVLM of SIH rats. Data were expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by post-hoc Bonferroni test (A–D). n = 6 rats per group (A–D). *p < 0.05, **p < 0.01, ***p < 0.001, and ns means nonsignificant versus SIH group.

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  Fig. 8. CircRNA Galntl6 regulated Lig3 expression via targeting miR-335. (A and B) Effects of circRNA Galntl6 overexpression and miR-335 agomir on the expression of Lig3 were revealed using RT-qPCR and Western blot analyses in B104 cells. (C and D) RT-qPCR and Western blot assays were used to illustrate the effects of circRNA Galntl6 knockdown and miR-335 antagomir on Lig3 expression in B104 cells. (E and F) After microinjection of pLV-circRNA Galntl6 plasmid and miR-335 agomir into RVLM of SIH rats, the expression of Lig3 was measured by RT-qPCR and Western blot analyses. Data were expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA, followed by post-hoc Bonferroni test (A–F). n = 6 of independent cell culture preparations (A and C). n = 3 of independent cell culture preparations (B and D). n = 6 rats per group (E). n = 3 rats per group (F). #p < 0.05, ##p < 0.001, and ns means nonsignificant versus pLV-NC group. Δp< 0.05, ΔΔΔp< 0.001, and ns means nonsignificant versus si-NC group. *p < 0.05, ***p < 0.001, and ns means nonsignificant versus SIH + pLV-NC group.