Indoxyl Sulfate Elevated Lnc-SLC15A1-1 Upregulating CXCL10/CXCL8 Expression in High-Glucose Endothelial Cells by Sponging MicroRNAs

Figures

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  Figure 1. Upregulation of lnc-SLC15A1-1 in the high-glucose-cultured HUVECs after indoxyl sulfate treatment. (A) MTT assay. Proliferation of HUVECs was examined in 30 mM D-glucose (HG) or HG with 0.1 mM indoxyl sulfate (HG + IS) for 4 days. (B) Principal component analysis (PCA) of samples. RNA sequencing was performed in HG + IS and HG groups (n = 3). Log-transformed fragments per kilobase of transcript per million mapped reads were used to perform PCA. (C) Volcano plot of differentially expressed lncRNAs. Selection criteria: fold change ≥1.5 or ≤−1.5 with p-value < 0.05. (D) Heatmap and hierarchical cluster analysis of 208 differentially expressed lncRNAs. Each column represents one sample, and each row represents one lncRNA. Red: upregulated lncRNAs; green: downregulated lncRNAs. (E) Relative expression levels of lnc-SLC15A1-1 by quantitative RT-PCR. (* p < 0.05, ** p < 0.01).

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  Figure 2. General characterization of lnc-SLC15A1-1. (A) Schematic representation of lnc-SLC15A1-1. (B) Phylogenetic tree of lnc-SLC15A1-1 across species. (C) The predicted secondary structure of lnc-SLC15A1-1. The minimum free energy structure was colored by base-pairing probabilities using the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi, accessed on 4 May 2020).

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  Figure 3. Identification of genes regulated by lnc-SLC15A1-1 in HUVECs by microarray analysis. (A) Relative expression levels of lnc-SLC15A1-1 by quantitative RT-PCR. HUVECs were transfected with 1.5 μg of lnc-SLC15A1-1 expression plasmids (pcDNA3.1(+)-lnc-SLC15A1-1). The lnc-SLC15A1-1 RNA expression levels were normalized to 18S rRNA (*** p < 0.001). (B) Principal component analysis. HUVECs were transfected with empty vector (EV; red spots) or lnc-SLC15A1-1 expression plasmids (Lnc; blue spots). The expression levels were analyzed by Affymetrix Clariom™ S Assay (n = 3). The expression levels of total mRNAs were used for plotting PCA. (C) Volcano plot of differentially expressed genes (DEGs) in HUVECs over-expressing lnc-SLC15A1-1. DEGs with fold change ≥1.5 or ≤−1.5 and p-value < 0.05 are labeled as red or green spots, respectively. (D) Heatmap with hierarchical clustering. Red color indicates upregulated genes; green color indicates downregulated genes.

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  Figure 4. Ingenuity Pathway Analysis of differentially expressed genes regulated by lnc-SLC15A1-1. (A) Canonical pathways of differentially expressed genes regulated by lnc-SLC15A1-1. Vertical orange line indicates the default threshold. The gray bars indicate no activity pattern. (B) Representative network of downstream genes regulated by lnc-SLC15A1-1. Red and blue stars indicate CXCL10 and CXCL8, respectively. Green color indicates downregulation, and red color indicate upregulation. Solid lines represent direct interaction (yellow), and dashed lines represent indirect interaction. Various shapes representing the functional class of the gene product. (C) Quantitative RT-PCR analyses of top 10 differentially expressed genes identified by microarray. Loading control: GAPDH. Experiments were repeated in triplicate, and the results are the mean ± SDs. (* p < 0.05).

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  Figure 5. Upregulation of IP-10 and IL-8 in HUVECs overexpressing lnc-SLC15A-1. (A,B) Total lysates from HUVECs transfected with 1.5 μg of lnc-SLC15A1-1 expression plasmid or empty vector (pcDNA3.1(+)) were analyzed by Western blotting. Loading control: β-actin. (C,D) Quantitative results of Western blotting. Analysis and quantification of Western blotting results of IP-10 and IL-8. Data are the means ± SDs (n = 3; * p < 0.05, ** p < 0.01). (E,F) Enzyme-linked immunosorbent assay (ELISA) of IP-10 and IL-8 in conditioned medium. Culture medium of HUVECs with/without overexpression of lnc-SLC15A1-1 was collected, followed by ELISA application. All data shown are the means ± SDs (* p < 0.05).

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  Figure 6. Lnc-SLC15A-1 distribution in HUVECs. Cytoplasmic and nuclear RNA were fractionated in HUVECs overexpressing lnc-SLC15A1-1 under HG and HG + IS treatment. Relative amounts of RNA were normalized to the total amount of RNA and detected by quantitative RT-PCR. Nuclear markers: BCAR4 snRNA. Cytoplasmic marker: GAPDH. Data shown are the means ± SDs (n = 8).

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  Figure 7. Lnc-SLC15A1-1 functions as a miRNA sponge. (A) Venn diagram of miRNAs predicted to interact with lnc-SLC15A1-1, CXCL10, and CXCL8. MiRNAs were predicted by miRDB (http://mirdb.org/index.html, accessed on 17 December 2020). (B) Relative expression levels of three predicted miRNAs. The expression levels of miRNAs in HUVECs overexpressing lnc-SLC15A1-1 or empty vector (pcDNA3.1(+)) were detected by quantitative RT-PCR and normalized to U6. Data are the means ± SDs (* p < 0.05, ** p < 0.01, and *** p < 0.001). (C) RNA immunoprecipitation (RIP) analysis of lnc-SLC15A1-1 using antibody against Argonaute-2 (AGO2) in HUVECs. The RIP enrichment of the AGO2-associated lncRNA was measured by quantitative RT-PCR and normalized to U6. The relative fold enrichment was calculated as compared to the IgG group. Data are the means ± SDs (n = 5 and ** p < 0.01, *** p < 0.001).