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. 2024 Feb 29;25(5):2816.
doi: 10.3390/ijms25052816.

Transcriptomic Analyses and Experimental Validation Identified Immune-Related lncRNA-mRNA Pair MIR210HG- BPIFC Regulating the Progression of Hypertrophic Cardiomyopathy

Yuan Zhang  1   2 , Jiuxiao Zhao  1 , Qiao Jin  1 , Lenan Zhuang  1   2
Affiliations

Transcriptomic Analyses and Experimental Validation Identified Immune-Related lncRNA-mRNA Pair MIR210HG- BPIFC Regulating the Progression of Hypertrophic Cardiomyopathy

Yuan Zhang et al. Int J Mol Sci. .

Abstract

Hypertrophic cardiomyopathy (HCM) is a disease in which the myocardium of the heart becomes asymmetrically thickened, malformed, disordered, and loses its normal structure and function. Recent studies have demonstrated the significant involvement of inflammatory responses in HCM. However, the precise role of immune-related long non-coding RNAs (lncRNAs) in the pathogenesis of HCM remains unclear. In this study, we performed a comprehensive analysis of immune-related lncRNAs in HCM. First, transcriptomic RNA-Seq data from both HCM patients and healthy individuals (GSE180313) were reanalyzed thoroughly. Key HCM-related modules were identified using weighted gene co-expression network analysis (WGCNA). A screening for immune-related lncRNAs was conducted within the key modules using immune-related mRNA co-expression analysis. Based on lncRNA-mRNA pairs that exhibit shared regulatory microRNAs (miRNAs), we constructed a competing endogenous RNA (ceRNA) network, comprising 9 lncRNAs and 17 mRNAs that were significantly correlated. Among the 26 lncRNA-mRNA pairs, only the MIR210HG-BPIFC pair was verified by another HCM dataset (GSE130036) and the isoprenaline (ISO)-induced HCM cell model. Furthermore, knockdown of MIR210HG increased the regulatory miRNAs and decreased the mRNA expression of BPIFC correspondingly in AC16 cells. Additionally, the analysis of immune cell infiltration indicated that the MIR210HG-BPIFC pair was potentially involved in the infiltration of naïve CD4+ T cells and CD8+ T cells. Together, our findings indicate that the decreased expression of the lncRNA-mRNA pair MIR210HG-BPIFC was significantly correlated with the pathogenesis of the disease and may be involved in the immune cell infiltration in the mechanism of HCM.

Keywords: bioinformatics analysis; hypertrophic cardiomyopathy; immune cell infiltration; non-coding RNAs.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Heatmap and volcano plot illustrating the DEGs identified in the GSE180313 dataset: (A) Heatmap showing all DEGs. (B) Volcano plot. HCM: hypertrophic cardiomyopathy; CTRL: disease-free heart; DEGs: differentially expressed genes.
Figure 2
Figure 2
WGCNA for GSE180313: (A) Analysis of the scale-free index and mean connectivity was carried out for threshold powers ranging from 1 to 20. We chose a β value of 5 as the soft threshold; this choice led to a scale-free R2 value of 0.85. (B) Cluster dendrogram of genes in the WGCNA network, with assigned module colors. (C) Analysis of module–clinical trait associations. Each column represents a clinical trait, while each row represents a module. Each grid cell contains the correlation (left) and p-value (right). The table is color-coded based on the correlation, as shown in the color legend. The correlation coefficients range from −1 to 1, and the association is positively related to the absolute value. (D) Heatmap of gene expression profiles in the yellow module. (E) Heatmap of gene expression profiles in the turquoise module.
Figure 3
Figure 3
Correlation test of lncRNA–mRNA pairs with disease characteristics in the GSE180313 and GSE130036 datasets: (A) The network of immune-related ceRNAs. The red ovals are lncRNAs, the gray triangles are miRNAs, and the blue cubes are mRNAs. (B) Expression levels of lncRNAs included in the ceRNA network among the HCM and CTRL samples in the GSE180313 training dataset. (C) Expression levels of mRNAs included in the ceRNA network among the HCM and CTRL samples in the GSE180313 training dataset. (D) The lncRNAs included in the ceRNA network among the HCM and CTRL samples’ expression levels in the GSE130036 validation dataset. (E) The mRNAs included in the ceRNA network among the HCM and CTRL samples’ expression levels in the GSE130036 validation dataset. * p-Value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001; ns: no significance.
Figure 4
Figure 4
Validation of the immune-related ceRNA network: (A–C) Validation of correlations of immune-related lncRNA–mRNA pairs in the GSE180313 training dataset and the GSE130036 validation dataset. The pink and green bars showed the expression levels of the indicated genes and the blue lines indicated the linear regression line. (D) The ceRNA network confirmed by the GSE180313 training dataset and the GSE130036 validation dataset. The red ovals are lncRNAs, the gray triangles are miRNAs, and the blue cubes are mRNAs.
Figure 5
Figure 5
Validation of the immune-related lncRNA–mRNA pairs in vitro: (A) Actin-tracker staining results of the control and ISO groups. (B) The mRNA expression levels of Nppa and Nppb in the control and ISO groups. (C,D) The expression levels of lncRNA–mRNA pairs in the control and ISO groups. (E) The expression levels of MIR210HG-regulated immune-related miRNAs in the control and ISO groups. (F) The expression levels of the MIR210HG–BPIFC pair in the siRNA-NC, siRNA-1, and siRNA-2 groups. (G) The expression levels of MIR210HG-regulated immune-related miRNA in the siRNA-NC, siRNA-1, and siRNA-2 groups. (H) The mRNA expression levels of Nppa and Nppb in the siRNA-NC, siRNA-1, and siRNA-2 groups. (I) Actin-tracker staining results of the siRNA-NC, siRNA-1, and siRNA-2 groups. * p-Value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001, ns: no significance. Control: normal cultured AC16 cells; ISO: ISO-induced AC16 cells; siRNA-NC: NC-FAM-induced AC16 cells; MIR210HG-siRNA-1: one of the siRNAs targeting MIR210HG-induced AC16 cells; MIR210HG-siRNA-2: the other siRNA targeting MIR210HG-induced AC16 cells. Scale bar = 50 µm.
Figure 6
Figure 6
Analysis of immune cell infiltration: (A) The 22 immune cells’ proportions in HCM and CTRL samples. (B) The immune cells’ differential expression analysis in the CTRL and HCM groups. (C) Pearson’s correlation analysis of BPIFC and the infiltrating immune cells; statistically significant immune cells are marked in red, with p-values < 0.05. (D) Pearson’s correlation analysis of MIR210HG and the infiltrating immune cells; statistically significant immune cells are marked in red, with p-values < 0.05. * p-Value < 0.05, ns: no significance.
Figure 7
Figure 7
The lncRNA MIR210HG regulates the mRNA BPIFC via the miRNAs miR-216b, miR-24, miR-34c, etc. Comparing with the normal heart, the decreased expression of lncRNA MIR210HG prompts more binding of the mediating miRNAs (miR-145, miR-216b, miR-24, miR-34c, etc) with the mRNA of BPIFC and induces the degradation of BPIFC, associated with the hypertrophy of cardiomyocytes in HCM. The arrows indicated the decreased expression.
Figure 8
Figure 8
Study flowchart.
See this image and copyright information in PMC

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