Figure 1.
PC4 acts as an RBP that stabilizes CCND1 mRNA. (A) Schematic representation showing the screening process used to identify PC4 as an essential RBP that promotes HCC. In the initial step, a comprehensive analysis of differential expression and survival data was conducted using the Liver Cancer Institute (LCI), TCGA-Liver Hepatocellular Carcinoma (LIHC), and Stanford datasets, as described by Dang et al. (2017). This analysis led to the identification of 148 RBPs that were clinically relevant in HCC. Among these RBPs, the top 20 candidates with the highest clinical relevance were selected. Notably, four of these candidates had previously unknown roles in HCC, making them particularly interesting for further investigation. To evaluate the potential impact of these four RBP candidates on cell proliferation, an siRNA screening was performed. Huh7 cells were treated with siRNAs targeting each candidate, and their effects on cell growth were assessed at day 1 and day 20. (B) Dot plot of gene ontology (GO) enrichment showing significant GO terms for differentially expressed genes after PC4 knockdown (left) and RNAs that are bound with and regulated by PC4 (right) in Huh7 cells. Color indicates P value and dot size denotes the number of genes enriched. Dots are not shown for terms with no statistically significant (P < 0.05) enrichment. (C) Venn diagrams showing the overlapping sets of data from three different categories related to PC4-bound RNA transcripts with a significant alternation upon PC4 knockdown. The first category represents differential expression transcripts identified by RNA-seq upon PC4 knockdown in the three cell lines, with the number of genes indicated. The second category represents PC4-bound RNA transcripts identified by RIP-seq in two independent experiments. The third category represents the intersection of both datasets. In the Venn diagrams, five PC4-upregulated RNA targets that are related to the cell cycle are shown in red, while five PC4-downregulated RNAs involved in the cell cycle are represented in blue. Furthermore, among the five RNAs shown in red, they belong to G1/S genes, whereas the other five RNAs in blue are categorized as G2/M genes. (D) Heatmap representing the mRNA half-life of five indicated genes in Huh7 cells with knockdown of NC or PC4, following treatment with actinomycin D (ActD) at different timepoints (h). The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. (E) Distribution of PC4-binding peaks across CCND1 mRNA from RIP-seq and eCLIP-seq, which were performed by anti-PC4 antibody in Huh7 cells. Data are generated from n = 2 biological replicates. (F) EMSA analysis of the association of PC4-CCND1 5′UTR in Huh7 cells and in vitro. In the in vitro assay, the transcribed 5′UTR fragment of CCND1 mRNA was incubated with different concentrations of recombinant GST-tagged PC4 protein and separated on a non-denaturing PAGE. The recombinant GST-PC4 proteins were examined by Coomassie brilliant blue staining. (G) EMSA analysis showing the association of various DNA and RNA molecules with recombinant GST-tagged PC4 protein including (1) the association of WDR74, LINC00869, and CCND1 dsDNA with PC4 protein. Lane 1–4 corresponds to synthesized DNA sequences of WDR74, LINC00869, and CCND1, which were derived from PC4 ChIP-seq peaks for WDR74 and LINC00869, and H3K27ac ChIP-seq peak for CCND1. (2) The association of CCND1-5′UTR ssDNA with PC4 protein. Lane 5–7 represents the interaction between PC4 protein and CCND1-5′UTR-forward sequence (CCND1-ssDNA-F) and CCND1-5′UTR-reverse sequence (CCND1-ssDNA-R). (3) The association of CCND1-5′UTR RNA with PC4 protein. Lane 8–9 indicates the binding of PC4 protein with CCND1-5′UTR RNA. (H) qPCR showing the mRNA stability of CCND1 in Huh7 and HepG2 cells with stable knockdown of PC4 upon treatment with ActD. Data are generated from n = 3 biological replicates. (I) qPCR showing the mRNA stability of CCND1 in Huh7 and HepG2 cells with stable overexpression of PC4 upon treatment with ActD. Data are generated from n = 3 biological replicates. (J) qPCR showing CCND1 mRNA expression in Huh7 and HepG2 cells with stable knockdown of PC4. Data in each group were normalized to that in NC. Data were generated from n = 3 biological replicates. (K) qPCR showing CCND1 mRNA expression in Huh7 and HepG2 cells with stable overexpression of PC4. Data in each group were normalized to that in NC. Data are generated from n = 3 biological replicates. (L) Western blot showing the indicated protein expressions in Huh7 and HepG2 cells with stable knockdown of PC4. The protein expressions are quantified and normalized, and the values are listed below each band. The PC4 and β-actin blots are duplicated in Fig. S1 L. (M) Western blot showing the indicated protein expressions in Huh7 and HepG2 cells with stable overexpression of PC4. The protein expressions are quantified and normalized, and the values are listed below each band. All the data were shown as means ± SD (one-way ANOVA test); error bars represent SD. **P < 0.01, ***P < 0.001, ****P < 0.0001. F, G, L, and M are representative of three independent experiments. Source data are available for this figure: SourceData F1.

PC4 acts as an RBP that stabilizes CCND1 mRNA. (A) Schematic representation showing the screening process used to identify PC4 as an essential RBP that promotes HCC. In the initial step, a comprehensive analysis of differential expression and survival data was conducted using the Liver Cancer Institute (LCI), TCGA-Liver Hepatocellular Carcinoma (LIHC), and Stanford datasets, as described by Dang et al. (2017). This analysis led to the identification of 148 RBPs that were clinically relevant in HCC. Among these RBPs, the top 20 candidates with the highest clinical relevance were selected. Notably, four of these candidates had previously unknown roles in HCC, making them particularly interesting for further investigation. To evaluate the potential impact of these four RBP candidates on cell proliferation, an siRNA screening was performed. Huh7 cells were treated with siRNAs targeting each candidate, and their effects on cell growth were assessed at day 1 and day 20. (B) Dot plot of gene ontology (GO) enrichment showing significant GO terms for differentially expressed genes after PC4 knockdown (left) and RNAs that are bound with and regulated by PC4 (right) in Huh7 cells. Color indicates P value and dot size denotes the number of genes enriched. Dots are not shown for terms with no statistically significant (P < 0.05) enrichment. (C) Venn diagrams showing the overlapping sets of data from three different categories related to PC4-bound RNA transcripts with a significant alternation upon PC4 knockdown. The first category represents differential expression transcripts identified by RNA-seq upon PC4 knockdown in the three cell lines, with the number of genes indicated. The second category represents PC4-bound RNA transcripts identified by RIP-seq in two independent experiments. The third category represents the intersection of both datasets. In the Venn diagrams, five PC4-upregulated RNA targets that are related to the cell cycle are shown in red, while five PC4-downregulated RNAs involved in the cell cycle are represented in blue. Furthermore, among the five RNAs shown in red, they belong to G1/S genes, whereas the other five RNAs in blue are categorized as G2/M genes. (D) Heatmap representing the mRNA half-life of five indicated genes in Huh7 cells with knockdown of NC or PC4, following treatment with actinomycin D (ActD) at different timepoints (h). The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. (E) Distribution of PC4-binding peaks across CCND1 mRNA from RIP-seq and eCLIP-seq, which were performed by anti-PC4 antibody in Huh7 cells. Data are generated from n = 2 biological replicates. (F) EMSA analysis of the association of PC4-CCND1 5′UTR in Huh7 cells and in vitro. In the in vitro assay, the transcribed 5′UTR fragment of CCND1 mRNA was incubated with different concentrations of recombinant GST-tagged PC4 protein and separated on a non-denaturing PAGE. The recombinant GST-PC4 proteins were examined by Coomassie brilliant blue staining. (G) EMSA analysis showing the association of various DNA and RNA molecules with recombinant GST-tagged PC4 protein including (1) the association of WDR74, LINC00869, and CCND1 dsDNA with PC4 protein. Lane 1–4 corresponds to synthesized DNA sequences of WDR74, LINC00869, and CCND1, which were derived from PC4 ChIP-seq peaks for WDR74 and LINC00869, and H3K27ac ChIP-seq peak for CCND1. (2) The association of CCND1-5′UTR ssDNA with PC4 protein. Lane 5–7 represents the interaction between PC4 protein and CCND1-5′UTR-forward sequence (CCND1-ssDNA-F) and CCND1-5′UTR-reverse sequence (CCND1-ssDNA-R). (3) The association of CCND1-5′UTR RNA with PC4 protein. Lane 8–9 indicates the binding of PC4 protein with CCND1-5′UTR RNA. (H) qPCR showing the mRNA stability of CCND1 in Huh7 and HepG2 cells with stable knockdown of PC4 upon treatment with ActD. Data are generated from n = 3 biological replicates. (I) qPCR showing the mRNA stability of CCND1 in Huh7 and HepG2 cells with stable overexpression of PC4 upon treatment with ActD. Data are generated from n = 3 biological replicates. (J) qPCR showing CCND1 mRNA expression in Huh7 and HepG2 cells with stable knockdown of PC4. Data in each group were normalized to that in NC. Data were generated from n = 3 biological replicates. (K) qPCR showing CCND1 mRNA expression in Huh7 and HepG2 cells with stable overexpression of PC4. Data in each group were normalized to that in NC. Data are generated from n = 3 biological replicates. (L) Western blot showing the indicated protein expressions in Huh7 and HepG2 cells with stable knockdown of PC4. The protein expressions are quantified and normalized, and the values are listed below each band. The PC4 and β-actin blots are duplicated in Fig. S1 L. (M) Western blot showing the indicated protein expressions in Huh7 and HepG2 cells with stable overexpression of PC4. The protein expressions are quantified and normalized, and the values are listed below each band. All the data were shown as means ± SD (one-way ANOVA test); error bars represent SD. **P < 0.01, ***P < 0.001, ****P < 0.0001. F, G, L, and M are representative of three independent experiments. Source data are available for this figure: SourceData F1.

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