Polycystic Ovary Syndrome: Novel and Hub lncRNAs in the Insulin Resistance-Associated lncRNA–mRNA Network

Figures

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  Figure 1. Differentially expressed lncRNAs and mRNAs associated with insulin resistance in patients with polycystic ovary syndrome (PCOS). (A) Heatmap of differentially expressed transcripts in the muscle of patients with PCOS and insulin resistance, compared with levels in controls. The columns represent 10 patients with PCOS (Var1, blue columns) and 13 healthy women (Var1 yellow columns). Var2 represents the number of samples. The rows represent lncRNAs and their mRNA neighbors. The red and green rows are based on expression in different samples. Red rows indicate upregulation, whereas green rows indicate downregulation. The darker the color, the greater the difference. (B) Heatmap of significantly changed transcripts after pioglitazone treatment. The columns represent 10 obese women with PCOS before (Var1 yellow columns) and after pioglitazone treatment (Var1 blue columns). Var2 represents the number of samples. (C) Venn diagram of commonly identified lncRNAs and mRNAs in both dataset 1 (PCOS and control) and dataset 2 (before and after treatment); 181 and 1,322 similar lncRNAs and mRNAs were identified respectively and then mapped into the global triple network.

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  Figure 2. Insulin-resistance lncRNA–mRNA network (IRLMN). The blue nodes represent mRNA, and the red nodes represent lncRNAs. There were 47 lncRNA nodes, 852 mRNA nodes, and 5,705 edges in the network. The nodes with colorful labels are lncRNAs that are highly related to insulin resistance in our study. The size of the nodes represents the degrees of nodes in the network.

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  Figure 3. Module analysis of lncRNA and mRNA interactions. (A) Heatmap of insulin resistance-associated lncRNA–mRNA network (IRLMN). The bidirectional hierarchical cluster was performed with R software. The red ellipses represent the module (the same for the rectangles in the cluster tree). The color scale of blue to yellow indicates differences in correlations (from 0 to 1). The closer the color to yellow (to 1), the stronger the correlation. (B) Module network extracted from IRLMN, consisting of six lncRNAs (red triangles), three RNA-binding proteins (RBPs) (yellow rectangles), and 58 mRNAs (blue ellipses). (C) Gene ontology (GO) enrichment analysis of module; the x-axis is the −log10 of the p-value, and p < 0.05 was considered statistically significant. The y-axis shows the biological processes. (D) Subpathway enrichment analysis of the module; the x-axis is the −log10 of the p-value, and p < 0.05 was considered statistically significant. The y-axis shows the subpathways.

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  Figure 4. Weighted gene co-expression network analysis (WGCNA) of differentially expressed genes associated with insulin resistance in patients with polycystic ovary syndrome (PCOS). (A) Hierarchical clustering of 23 skeletal muscle samples (10 cases and 13 controls) from women with and without PCOS. The rows represent the types of samples. The red row represents cases, and the white row represents controls. (B) Hierarchical clustering of 13 samples (seven cases and six controls) after deleting the outlier samples. (C) Gene dendrogram obtained by average linkage hierarchical clustering. The color row underneath the dendrogram shows the module assignment determined by the Dynamic Tree Cut. (D) Module–PCOS relationship. The y-axis shows the modules. The first row of each module is the R2 value; and the closer R2 is to 1, the stronger the correlation is. The second row of each module is the p-value, and p < 0.01 was considered statistically significant. The blue module was most closely correlated with insulin resistance of PCOS (R2 = 0.98, p-value = 2e−09). (E) The gene ontology (GO) enrichment analysis of the module; the x-axis is −log10 of p-value, and p < 0.05 was considered statistically…

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  Figure 5. Elevated expression of lncRNA RP11-151A6.4 in the subcutaneous and omental adipose tissues of patients with insulin resistance. Expression level of RP11-151A6.4 in the subcutaneous and omental adipose tissues of people with and without insulin resistance. IR, insulin resistance; IS, insulin sensitive; SA, subcutaneous adipose tissue; OA, omental adipose tissue. *p < 0.05 and ns: not significant, p > 0.05, according to two-tailed Student’s t-test.

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  Figure 6. Elevated expression of lncRNA RP11-151A6.4 in the ovarian granulosa cells of patients with insulin resistance. The expression level was detected via qRT-PCR and normalized to β-actin levels. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. (A) The expression level of RP11-151A6.4 was increased in patients with PCOS compared with control women. (B) The expression level of RP11-151A6.4 was correlated with body mass index (BMI) in patients with PCOS. (C) The expression level of RP11-151A6.4 was elevated in PCOS patients with hyperinsulinemia (fasting insulin > 23 mIU/L). (D) Higher HOMA-IR values were associated with higher expression levels of RP11-151A6.4. Ranges of HOMA-IR values were as follows: tertile I (0.95 to 2.79), tertile II (2.79 to 4.49), and tertile III (4.49 to 20.73).