Gene Silencing of ANGPTL3 Induces PCSK9: Exploring the Biological Significance in the Hepatoma Huh7 Cell Line

Figures

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  Figure 1. RNA-seq analysis after gene silencing of ANGPTL3 and PCSK9. (A) schematic representation of the experimental protocol for RNA-seq analysis. (B) Principal component analysis (PCA) of samples selected for RNA-seq data analysis and differential expression genes exploration. (C) Venn diagram showing the distribution of differentially expressed transcripts across different conditions. In (panel B), colors and symbols identify the treatment groups as indicated in the legend; in (panel C), each colored circle represents the indicated siRNA condition.

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  Figure 2. Heat map and volcano plots of differentially expressed genes. (A,C,E) Heat map of upregulated and downregulated mRNA levels in response to ANGPTL3-siRNA (panel A), PCSK9-siRNA (panel C), and ANGPTL3/PCSK9-siRNA (panel E). Data are expressed as the log scale of normalized counts. The heat map represents read-count expression. Different genes are indicated on the right side in the corresponding line. On the top parts of the graphs, the different experimental conditions are indicated. (B,D,F) Volcano plot showing the most relevant differentially expressed genes. Red dots show the downregulated genes and green dots the upregulated genes after transfection with ANGPTL3-siRNA (panel B), PCSK9-siRNA (panel D), and ANGPTL3/PCSK9-siRNA (panel F).

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  Figure 3. Differential regulation of gene and protein expression of ANGPTL3 and PCSK9 after siRNA transfection. (A,B) ANGPTL3 and PCSK9 gene expression analysis in response to different siRNA in Huh7 cells. p values were calculated using ANOVA one-way Benjamini and Hochberg FDR method. (C,G) Western blot analysis for ANGPTL3 and PCSK9 after 48 h transfection with scramble-siRNA and ANGPTL3-siRNA (panel C) and scramble-siRNA, PCSK9-siRNA, and double siRNA-ANGPTL3/PCSK9 (panel G). GAPDH was used as a loading control and quantifications of protein expression are shown in panels (D–F,H,I). ApoB secretion was determined by ELISA assay. (J,K). Real-time quantitative PCR for ANGPTL3 (panel J) and PCSK9 (panel K) from total RNA extracted from Huh7 cells transfected with indicated siRNA. p values were calculated using one-way ANOVA method. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. scramble-siRNA. Data from Western blot and real-time quantitative PCR are presented as mean ± SD of three independent experiments.

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  Figure 4. Enrichment analysis by Gene Ontology Biological Processes (GO-BP) database in response to ANGPTL3-siRNA (panel A), PCSK9-siRNA (panel B), and ANGPTL3/PCSK9-siRNA (panel C).

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  Figure 5. α1-antitrypsin and LMAN1 were downregulated in response to ANGPTL3-siRNA in Huh7 cells. (A,B) Differential expression of SERPINA1 and LMAN1 determined by NGS after different siRNA transfection. (C) Protein analysis from Huh7 cells after transfection with scramble-siRNA and ANGPTL3-siRNA. GAPDH was used as loading control. (D,E) Quantification analysis of Western blot. p values were calculated using ANOVA one-way Benjamini and Hochberg FDR method (A,B) or Student’s t-test (D,E). ** p < 0.01, **** p < 0.0001 vs. scramble-siRNA. Western blot analysis is presented as mean ± SD of four independent experiments.

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  Figure 6. FV and FVIII were significantly upregulated in response to ANGPTL3-siRNA. (A,B) FV and FVIII mRNA were determined by RT-qPCR after 48 h of different siRNA transfection. p values were calculated using one-way ANOVA method. * p < 0.05; ** p < 0.01; vs. scramble-siRNA. (C–H) ELISA assay for FV (C–E) and FVIII (F–H) from conditioned media of Huh7 cells after 48 h of different siRNA transfection. FV and FVIII determinations were normalized for total protein content of cell lysates. p values were calculated using Student’s t-test. * p < 0.05; ** p < 0.01, *** p < 0.001 vs. scramble-siRNA.