Fig 1.
The correlation between CHRNA5 and the fraction of total genomic alterations.
A-B. Linear regression and correlation coefficients between the CHRNA5 expression level and the fraction of genomewide alterantions in CCLE (A) and TCGA (B) datasets. The colors blue, red, and green, respectively, indicate MCF7, BT20, and MDA-MB-231 cell lines in (A).
Fig 2.
Downregulation of CHRNA5 expression by RNAi.
A. Schematic representation of target sites of siRNA molecules and primers for CHRNA5 isoforms. B-D. Downregulation of CHRNA5 isoforms in MCF7 cells upon transient transfection with siRNA-1 (10nM) (B), siRNA-2 (50nM) (C), and siRNA-3 (50nM) (D) in comparison with the corresponding siRNA-CN at 72h of treatment (n = 2 per group). Student’s t-test was applied. E-F. Depletion of CHRNA5 expression at protein level (Western Blotting together with densitometry analyses, loading control, GAPDH) with siRNA-1 (10nM) (n = 4 per group; Student’s t-test) (E), siRNA-2 and siRNA-3 (n = 4 for siRNA-CN; n = 3 for siRNA-2 and siRNA-3; One-Way ANOVA followed by Tukey’s HSD tests) (F). (*: p < 0.05; **: p < 0.01; ***, #: p < 0.001; **** p < 0.0001).
Fig 3.
Functional analysis and comparative transcriptomics of CHRNA5 RNAi data.
A. Normalized Enrichment Scores (NES) of GSEA analysis of microarray results (FDR q value < 0.25). B. Correlation between logFC values of nutlin-3a and siRNA-1 treated MCF7 cells for TP53 targets (confirmed by chip-seq, i.e., chip-seq peak = yes) significantly moduled by siRNA-1. C. Correlation profile of CCLE breast cancer cell lines versus CHRNA5 RNAi logFC after filtration. D. Correlation between CHRNA5 RNAi logFC values in MCF7 with CHRNA5 RNAi logFC values in lung cancer cell line A549. For C and D, genes significant in both categories are colored black; genes insignificant (or significant in one type of data) are shown in gray; and TP53 targets are shown in red.
Table 1.
Chromosome enrichment (Top 5) of CHRNA5 modulated genes (depleted by the siRNA-1 in MCF7 and positively correlated with CHRNA5 in CCLE).
Fig 4.
RT-qPCR validation of CHRNA5 RNAi microarray data.
A. RT-qPCR validation of selected genes modulated by siRNA-1 in MCF7 cells. B. RT-qPCR validation of selected genes in siRNA-2 and siRNA-3 treated MCF7 cells. C. Heatmap of RT-qPCR analysis results of selected genes in MCF7, BT20 and MDA-MB-231 in the presence of siRNA-CN or siRNA-1. For siRNA-1, and siRNA-2 and -3 treatment RT-qPCR results, student’s t-tests in comparison with their corresponding siRNA-CN group, i.e., 10nM for siRNA-1 and 50nM for siRNA-2 and -3, and for microarray data limma were used. (*: p < 0.05, **: p < 0.01, #: p < 0.001). (n = 2 per group for all cell lines).
Fig 5.
The effect of CHRNA5 depletion on cell cycle arrest and apoptosis in MCF7 cells.
A. Relative cell viability measured with MTT assay (n = 3 per group). B. BrdU incorporation assay (n = 2 per group). BrdU positive (pink) and DAPI (blue) labelled cells were shown in representative images of treatments. C. PI staining and FACS analysis (n = 3 per group). (*: p < 0.05, **: p < 0.01, ***: p < 0.001).
Fig 6.
Western blotting and RT-qPCR analyses relevant to effects of RNAi on cell cycle, apoptosis and DDR in MCF7 cells.
A-B. Western Blot results for pRB, total CASP7, cleaved CASP7, BCL2, BAX, total CHEK1, pCHEK1, and pH2AX in siRNA-1 (A) and siRNA-2 and siRNA-3 (B) treated MCF7 cells. C-F. Densitometry measurements of pRB (C), BAX/BCL2 ratio (D), total CHEK1 (E), and pCHEK1 (F). One-Way ANOVA was used in comparison with corresponding control groups, siRNA-CN (10nM) vs. siRNA-1 and siRNA-CN (50nM) vs. siRNA-2 and -3. G. DFA plot for control and RNAi treatment groups. H. Vector weights of variables and their correlation with LD1 and LD2 of DFA. (n = 2 per group for siRNA-CN (10nM) and siRNA-1; n = 3 per group for siRNA-CN (50nM) and siRNA-2 and -3). I. RT-qPCR results of FAS, BAX, BCL2, CCND1, CCNE2, and CHEK1 in siRNA-1-3 treated MCF7 cells (n = 2 per group). J. BAX/BCL2 ratio from RT-qPCR results (n = 2 per group). One-way ANOVA followed by Tukey’s multiple test correction was used. (+: p < 0.1, *: p < 0.05, **: p < 0.01, #: p < 0.001).
Fig 7.
Analysis of DDR genes in METABRIC and TCGA data A, C. METABRIC (A) and TCGA (C) patient clustering with DDR genes and CHRNA5; red cluster includes CHRNA5. B, D. METABRIC (B) and TCGA (D) patient clustering for genes in red cluster shown in Fig 7A and 7C. Bottomline refers to TP53 mutation status: red, mutation; blue, wildtype. E-F. Correlation between logFC response to siRNA-1 treatment and correlation coefficient of CHRNA5 and DDR genes from METABRIC (E) and TCGA (F). Genes whose expression levels did not change significantly with siRNA-1 treatment are shown in gray (p < 0.05).
Fig 8.
Association between CHRNA RNAi and topoisomerase inhibitors.
A. Comparison of log fold changes (logFC) in response to DOXO and SN38, siRNA-1 and SN38, siRNA-1 and DOXO treatment in MCF7 (from left to right) and the corresponding statistical analysis results (Fisher’s Exact Test counts and p-value; bottom). Genes significant in both categories are colored black; genes insignificant (or significant in one type of data) are shown in gray; and TP53 targets are shown in red. B-E Relative cell viability for CPT and siRNA-1 (B), DOXO and siRNA-1 (C), CPT and siRNA-2 (D), and DOXO and siRNA-2 (E) treatments at increasing concentrations of CPT (B, D) and DOXO (C, E). One-Way ANOVA followed by Tukey’s multiple test correction were used for statistical analysis of MTT assays (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig 9.
The effect of CHRNA5 siRNA in drug sensitivity.
A-C. Western blot results in DMSO, CPT (0.125μM), and DOXO (0.125μM) treated MCF7 cells in the absence or presence of siRNA-1 (A), siRNA-2, (B) and siRNA-3, (C). D-G. Two-Way ANOVA of densitometry measurements for CHRNA5 (D), BAX/BCL2 (E), CHEK1 (F), pCHEK1 (G). H. DFA of densitometry measurements for control and treatment groups in A-C. I. Vector weights of variables and their correlation with LD1 and LD2 of DFA analysis. (n = 3 for DMSO siRNA-CN (50nM) and siRNA-2 and -3; n = 2 for other groups). (+: p < 0.1; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig 10.
Effects of CHRNA5 RNAi in drug sensitivity in BT-20 cell line.
Relative cell viability of siRNA-1 treated BT-20 cells to A. CPT and siRNA-1, B. DOXO and siRNA-1 treatments at increasing concentrations of CPT and DOXO. C-F. Western blots (C, D for siRNA-1 or siRNA-CN in the CPT (0.125μM) and DOXO (0.125μM) groups, respectively) and densitometry results with statistical analysis in CPT (E), DOXO (F) treatment groups with or without siRNA-1, respectively for CHRNA5, CHEK1, pCHEK1, pH2AX. (n = 2 for Western blotting and n = 3 for MTT assays; +: p < 0.1; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig 11.
Effects of CHRNA5 RNAi in drug sensitivity in MDA-MB-231 cell line.
Relative cell viability of siRNA-1 treated MDA-MB-231 cells to A. CPT and siRNA-1, B. DOXO and siRNA-1 treatments at increasing concentrations of CPT (0.015μM) and DOXO (0.06μM). C-F. Western blots (C, D for siRNA-1 or siRNA-CN in the CPT and DOXO groups, respectively) and densitometry results with statistical analysis in CPT (E), DOXO (F) treatment groups with or without siRNA-1 respectively for CHRNA5, CHEK1, pCHEK1, pH2AX. (n = 2 for Western blotting and n = 3 for MTT assays; +: p < 0.1; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).
Fig 12.
Correlation between CHRNA5 and CHEK1 expression levels.
A-C. CCLE (A, n = 58), TCGA (B, n = 1100), METABRIC (C, n = 1904). The colors blue, red, and green, respectively, indicate MCF7, BT20, and MDA-MB-231 cell lines in (A).