Long non-coding RNA CASC7 is associated with the pathogenesis of heart failure via modulating the expression of miR-30c

Abstract

MiRNAs can be used as promising diagnostic biomarkers of heart failure, while lncRNAs act as competing endogenous RNAs of miRNAs. In this study, we collected peripheral blood monocytes from subjects with or without HF to explore the association between certain lncRNAs, miRNAs and HF. Heart failure patients with preserved or reduced ejection fraction were recruited for investigation. ROC analysis was carried out to evaluate the diagnostic values of certain miRNAs and lncRNAs in HF. Luciferase assays were used to study the regulatory relationship between above miRNAs and lncRNAs. LncRNA overexpression was used to explore the effect of certain miRNAs in H9C2 cells. Expression of miR-30c was significantly decreased in the plasma and peripheral blood monocytes of patients suffering from heart failure, especially in these with reduced ejection fraction. On the contrary, the expression of lncRNA-CASC7 was remarkably increased in the plasma and peripheral blood monocytes of patients suffering from heart failure. Both miR-30c and lncRNA-CASC7 expression showed a promising efficiency as diagnostic biomarkers of heart failure. Luciferase assays indicated that miR-30c played an inhibitory role in lncRNA-CASC7 and IL-11 mRNA expression. Moreover, the overexpression of lncRNA-CASC7 suppressed the expression of miR-30c while evidently increasing the expression of IL-11 mRNA and protein in H9C2 cells. This study clarified the relationship among miR-30c, lncRNA-CASC7 and IL-11 expression and the risk of heart failure and showed that lncRNA-CASC7 is potentially involved in the pathogenesis of HF via modulating the expression of miR-30c.

Keywords: CASC7; IL-11; cardiomyocyte; heart failure; lncRNA; miR-30c; miRNA.

FIGURE 1

miR‐30c was down‐regulated in the plasma of heart failure patients (*P value <0.05 vs. control group; HF: heart failure; pEF: preserved ejection fraction; rEF: reduced ejection fraction). A, Expression of miR‐30c was decreased in the plasma of HFpEF and HFrEF patients. B, Expression of miR‐146a was not significantly changed in the plasma of HFpEF, HFrEF and control groups. C, Expression of miR‐221 was not significantly changed in the plasma of HFpEF, HFrEF and control groups. D, Expression of miR‐328 was not significantly changed in the plasma of HFpEF, HFrEF and control groups. E, Expression of miR‐375 was not significantly changed in the plasma of HFpEF, HFrEF and control groups

FIGURE 2

miR‐30c was down‐regulated in the peripheral blood monocytes of heart failure patients (*P value <0.05 vs. control group; HF: heart failure; pEF: preserved ejection fraction; rEF: reduced ejection fraction). A, Expression of miR‐30c was decreased in the peripheral blood monocytes of HFpEF and HFrEF patients. B, Expression of miR‐146a was not significantly different in the peripheral blood monocytes of HFpEF, HFrEF and control groups. C, Expression of miR‐221 was not significantly changed in the peripheral blood monocytes of HFpEF, HFrEF and control groups. D, Expression of miR‐328 was not significantly changed in the peripheral blood monocytes of HFpEF, HFrEF and control groups. E, Expression of miR‐375 was not significantly changed in the peripheral blood monocytes of HFpEF, HFrEF and control groups

FIGURE 3

ROC analysis for the diagnostic value of multiple candidate miRNAs indicated that miR‐30c was a promising biomarker for heart failure diagnosis. A, ROC analysis for the diagnostic value of miR‐30c in heart failure. B, ROC analysis showed that miR‐146a was unable to differentiate heart failure from normal control. C, ROC analysis showed that miR‐221 was unable to differentiate heart failure from normal control. D, ROC analysis showed that miR‐328 was unable to differentiate heart failure from normal control. E, ROC analysis showed that miR‐375 was unable to differentiate heart failure from normal control

FIGURE 4

lncRNA‐CASC7 was up‐regulated in the plasma of heart failure patients (*P value <0.05 vs. control group; HF: heart failure; pEF: preserved ejection fraction; rEF: reduced ejection fraction). A, Expression of lncRNA‐CCAT1 was not significantly different in the plasma of HFpEF, HFrEF and control groups. B, Expression of lncRNA‐CASC7 was elevated in the plasma of HFpEF and HFrEF patients. C, Expression of lncRNA‐AK017368 was not significantly different in the plasma of HFpEF, HFrEF and control groups. D, Expression of IL‐11 mRNA was not significantly different in the plasma of HFpEF, HFrEF and control groups

FIGURE 5

lncRNA‐CASC7 was up‐regulated in the peripheral blood monocytes of heart failure patients (*P value <0.05 vs. control group; HF: heart failure; pEF: preserved ejection fraction; rEF: reduced ejection fraction). A, Expression of lncRNA‐CCAT1 was not significantly different in the peripheral blood monocytes of HFpEF, HFrEF and control groups. B, Expression of lncRNA‐CASC7 was elevated in the peripheral blood monocytes of HFpEF and HFrEF patients. C, Expression of lncRNA‐AK017368 was not significantly different in the peripheral blood monocytes of HFpEF, HFrEF and control groups. D, Expression of IL‐11 mRNA was not significantly different in the peripheral blood monocytes of HFpEF, HFrEF and control groups

FIGURE 6

Pearson's correlation analysis indicated a negative relationship between CASC7 and miR‐30. A, Pearson's correlation analysis of serum CASC7 and serum miR‐30 showed a negative relationship between CASC7 and miR‐30. B, Pearson's correlation analysis of cellular CASC7 and cellular miR‐30 in PBMCs showed a negative relationship between CASC7 and miR‐30

FIGURE 7

ROC analysis for the diagnostic value of multiple candidate lncRNAs indicated that lncRNA‐CASC7 was a promising biomarker for heart failure diagnosis. A, ROC analysis showed that lncRNA‐CCAT1 was unable to differentiate heart failure from normal control. B, ROC analysis of the diagnostic value of lncRNA‐CASC7 in heart failure. C, ROC analysis showed that lncRNA‐AK017368 was unable to differentiate heart failure from normal control

FIGURE 8

miR‐30c inhibited the luciferase activity of the lncRNA‐CCAT1 and IL‐11 3′UTR plasmid (*P value <0.05 vs. control mimics group; WT: wild type; MUT: mutant). A, Sequence analysis showed a target of miR‐30c in lncRNA‐CASC7 (Binding site: 235bp‐257bp). B, Luciferase activity of WT lncRNA‐CASC7 vector was inhibited by miR‐30c mimics in H9C2 cells. C, Sequence analysis showed a target of miR‐30c in lncRNA‐ CCAT1 (Binding site: 121bp‐145bp). D, Luciferase activity of WT and MT lncRNA‐CASC7 vectors was not affected by miR‐30c mimics in H9C2 cells. E, Sequence analysis showed a target of miR‐30c in lncRNA‐ AK017368 (Binding site: 235bp‐257bp). F, Luciferase activity of WT lncRNA‐CASC7 vector was not suppressed by miR‐30c mimics in H9C2 cells. G, Sequence analysis showed a target of miR‐30c in IL‐11 3′UTR (Binding site: 367bp‐390bp). H, Luciferase activity of WT IL‐11 3′UTR vector was obstructed by miR‐30c mimics in H9C2 cells

FIGURE 9

lncRNA‐CASC7 suppressed the expression of miR‐30c in H9C2 cells (*P value <0.05 vs. NC group; NC: negative control). A, The successful transfection of plasmids carrying lncRNA‐CASC7 led to the overexpression of lncRNA‐CASC7 in H9C2 cells. B, Overexpression of lncRNA‐CASC7 reduced the expression of miR‐30c in H9C2 cells. C, Expression of miR‐30c was decreased in H9C2 cells transfected with lncRNA‐ CCAT1. D, Expression of miR‐30c was not affected in H9C2 cells transfected with lncRNA‐ AK017368. E, Overexpression of lncRNA‐CASC7 increased the expression of IL‐11 mRNA in H9C2 cells. F, Overexpression of lncRNA‐CASC7 increased the expression of IL‐11 in H9C2 cells