Knockdown of KCNQ1OT1 attenuates cardiac hypertrophy through modulation of the miR-2054/AKT3 axis

Figures

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  Figure 1. LncRNA KCNQ1OT1 is up-regulated in hypertrophic cardiomyocytes. (A) Immunofluorescence results for the effect of Ang-II on cell surface areas. (B) Changes in the expression of LINC00850, LINCMD1, MALAT1, and KCNQ1OT1 in cardiomyocytes under Ang-II. (C) Detection of the expression of KCNQ1OT1 in TAC and sham mice. (D) The mRNA expression of hypertrophy markers by RT-PCR. (E) Transfection efficiency of shKCNQ1OT1 by RT-PCR. *, P<0.05. TAC, transverse aortic constriction.

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  Figure 2. Knockdown of KCNQ1OT1 inhibits cardiomyocytes’ hypertrophy induced by Ang-II. (A) Immunofluorescence results for the effect of KCNQ1OT1 knockdown on cell surface areas. Scale bar: 200 microns. (B,C) mRNA or protein expression of ANF, BNP, and β-MHC by shKCNQ1OT1. *, P<0.05.

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  Figure 3. Directional interaction between KCNQ1OT1 and miR-2054, miR-2054 and AKT3. (A) The complementary sequence between KCNQ1OT1 and miR-2054 was obtained from the online program starbase v2.0 (http://starbase.sysu.edu.cn/). (B) Overexpression of KCNQ1OT1 and miR-2054 in cardiomyocytes transfected by recombinant adenovirus. (C) Perform a luciferase reporter gene test to detect luciferase activity at the binding site on KCNQ1OT1 when miR-2054 is overexpressed. (D) RNA pull-down test showed that lncRNA KCNQ1OT1 can be enriched by biotin-labeled miR-2054, but not by biotin-labeled miR-2054 mutant. (E) Spearman correlation analysis was used to analyze the correlation between KCNQ1OT1 level and miR-2054 level in the TAC group. (F) The complementary sequence between miR-2054 and AKT3 was obtained from Targetscan Human 7.2 (http://www.targetscan.org/). (G) Luciferase reporter assay is conducted to detect the luciferase activity of the binding site on AKT3. (H) The correlation between the levels of miR-2054 and AKT3 in the TAC group was analyzed by Spearman correlation analysis. *, P<0.05. TAC, transverse aortic constriction.

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  Figure 4. LncRNA KCNQ1OT1 acts as CeRNA by competing with miR-2054. (A,B) Immunofluorescence for cell surface areas of Ang-II-induced cardiomyocytes under shKCNQ1OT1 and sh-miR-2054. (C) mRNA expression of ANF, BNP and β-MHC under shKCNQ1OT1 and sh-MIR-2054. (D) ANF, BNP, and β-MHC protein levels under shKCNQ1OT1 and sh-miR-2054. *, P<0.05.

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  Figure 5. miR-2054 attenuates Ang-II-induced cardiac hypertrophy by targeting AKT3. (A,B) Expression of AKT3 under shKCNQ1OT1 and sh-miR-2054. (C) and (D) Cell surface area under Adv-miR-2054 and Adv-AKT3. (E) mRNA expression of ANF, BNP and β-MHC under Adv-miR-2054 and Adv-AKT3. (F) ANF, BNP, and β-MHC protein expression were under Adv-miR-2054 and Adv-AKT3. *, P<0.05.