CIMB, Vol. 44, Pages 5848-5865: Checkpoint Kinase 1 (CHK1) Functions as Both a Diagnostic Marker and a Regulator of Epithelial-to-Mesenchymal Transition (EMT) in Triple-Negative Breast Cancer

Figure 1. Gene expression pattern with each GSE dataset and clustering of DEGs heat map of overlapping genes. (A) The volcano plot shows gene expression distribution of the microarray data in GSE36295, (B) GSE36693, and (C) GSE65216. The data was cut-off based on p-value < 0.01 and fold change (FC) log > 1. X-axis and y-axis present fold change log and log-transformed p-value, respectively. (D) Venn diagram for intersection of all up-regulated and down-regulated DEGs of GSE36295, GSE36693, and GSE65216 using the FunRich program. (E) Heat map exhibiting expression changed genes of up-regulated and down-regulated DEGs using GraphPad.

Figure 1. Gene expression pattern with each GSE dataset and clustering of DEGs heat map of overlapping genes. (A) The volcano plot shows gene expression distribution of the microarray data in GSE36295, (B) GSE36693, and (C) GSE65216. The data was cut-off based on p-value < 0.01 and fold change (FC) log > 1. X-axis and y-axis present fold change log and log-transformed p-value, respectively. (D) Venn diagram for intersection of all up-regulated and down-regulated DEGs of GSE36295, GSE36693, and GSE65216 using the FunRich program. (E) Heat map exhibiting expression changed genes of up-regulated and down-regulated DEGs using GraphPad.

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Figure 2. PPI network of DEGs. (A) PPI network of up-regulated and (B) down-regulated genes created by the STRING site. (C) Clustering analysis of up-regulated DEGs and (D) down-regulated DEGs for selecting hub genes using the MCODE plug-in in cytoscape.

Figure 2. PPI network of DEGs. (A) PPI network of up-regulated and (B) down-regulated genes created by the STRING site. (C) Clustering analysis of up-regulated DEGs and (D) down-regulated DEGs for selecting hub genes using the MCODE plug-in in cytoscape.

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Figure 3. Survival rate analysis between TNBC patients and up-regulated and down-regulated genes. (A) The correlation of survival rate and up-regulated MCM4, CDC7, CCNB2, and CHEK1 gene expression in TNBC patients. (B) The correlation of survival rate and down-regulated CXCL12, IL6ST, and IGF1 gene expression in TNBC patients.

Figure 3. Survival rate analysis between TNBC patients and up-regulated and down-regulated genes. (A) The correlation of survival rate and up-regulated MCM4, CDC7, CCNB2, and CHEK1 gene expression in TNBC patients. (B) The correlation of survival rate and down-regulated CXCL12, IL6ST, and IGF1 gene expression in TNBC patients.

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Figure 4. Comparison of DEGs expression between non-TNBC and TNBC patients or cell lines. (A) The comparison of up-regulated DEGs, MCM4, CDC7, CCNB2, and CHEK1, in Healthy, non-TNBC, and TNBC from GSE65216. (B) The comparison of down-regulated DEGs, CXCL12, IL6ST, and IGF1, in Healthy, non-TNBC, and TNBC. (C) The mRNA expression comparison of up-regulated DEGs, MCM4, CDC7, CCNB2, and CHEK1, in non-TNBC cells and TNBC cells, which are MCF-7 and MDA-MB231. (D) The mRNA expression comparison of down-regulated DEGs, CXCL12, IL6ST, and IGF1, in non-TNBC cells and TNBC cells, which are MCF-7 and MDA-MB231. Columns are presented with the mean of SEM. Statistical analysis using one-way ANOVA was performed in (A,B), and t-test performed in (C,D); * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001 vs. control in each group, n.s.: nonsignificant.

Figure 4. Comparison of DEGs expression between non-TNBC and TNBC patients or cell lines. (A) The comparison of up-regulated DEGs, MCM4, CDC7, CCNB2, and CHEK1, in Healthy, non-TNBC, and TNBC from GSE65216. (B) The comparison of down-regulated DEGs, CXCL12, IL6ST, and IGF1, in Healthy, non-TNBC, and TNBC. (C) The mRNA expression comparison of up-regulated DEGs, MCM4, CDC7, CCNB2, and CHEK1, in non-TNBC cells and TNBC cells, which are MCF-7 and MDA-MB231. (D) The mRNA expression comparison of down-regulated DEGs, CXCL12, IL6ST, and IGF1, in non-TNBC cells and TNBC cells, which are MCF-7 and MDA-MB231. Columns are presented with the mean of SEM. Statistical analysis using one-way ANOVA was performed in (A,B), and t-test performed in (C,D); * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001 vs. control in each group, n.s.: nonsignificant.

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Figure 5. The expression of CHK1 protein according to breast cancer subtypes. (A) CHK1 protein expression level with basal-like subtype of breast patients versus HER2-positive, luminal A, and luminal B from ‘The cancer proteome atlas’. (B) CHK1 protein expression comparison graph from ‘The cancer proteome atlas’. (C) The survival rate of breast cancer patients according to CHK1 protein expression using the KM plotter website. (D) Western blotting of CHK1 in non-TNBC cell lines, including MCF-7 and T47D, and TNBC cell lines, including MDA-MB453, MDA-MB231, BT549, and Hs578T. (E) The graph showing quantification of CHK1 expression normalized with endogenous GAPDH through Graphpad. Columns are presented with the mean of SEM. Statistical analysis using t-test was performed by comparing non-TNBC and TNBC; ** p < 0.005 vs. control in each group.

Figure 5. The expression of CHK1 protein according to breast cancer subtypes. (A) CHK1 protein expression level with basal-like subtype of breast patients versus HER2-positive, luminal A, and luminal B from ‘The cancer proteome atlas’. (B) CHK1 protein expression comparison graph from ‘The cancer proteome atlas’. (C) The survival rate of breast cancer patients according to CHK1 protein expression using the KM plotter website. (D) Western blotting of CHK1 in non-TNBC cell lines, including MCF-7 and T47D, and TNBC cell lines, including MDA-MB453, MDA-MB231, BT549, and Hs578T. (E) The graph showing quantification of CHK1 expression normalized with endogenous GAPDH through Graphpad. Columns are presented with the mean of SEM. Statistical analysis using t-test was performed by comparing non-TNBC and TNBC; ** p < 0.005 vs. control in each group.

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Figure 6. CHK1 induced epithelial to mesenchymal transition (EMT) in breast cancer cells. (A) The correlation of CHK1 with CDH1 and OCLN, which are epithelial marker genes. (B) Western blotting of EMT marker proteins in control and CHK1-overexpressing MCF-7 cells. (C) Fluorescence of phalloidin for observation of the morphology in control and CHK1-overexpression MCF-7 cells. (D) Migration assay with control and CHK1-overexpression MCF-7 cells for 48 h after scratch. (E) Transwell invasion assay with control and CHK1-overexpression MCF-7 cells for 48 h after incubation. (F) Western blotting of EMT marker proteins in control and CHK1-knockdown MDA-MB231 cells. (G) Fluorescence of phalloidin for observation of the morphology in control and CHK1-knockdown MDA-MB231 cells. (H) Migration assay with control and CHK1-knockdown MDA-MB231 cells for 16 h after scratch. (I) Transwell invasion assay with control and CHK1-knockdown MDA-MB231 cells for 12 h after incubation. Columns are presented with the mean of SEM. Statistical analysis using t-test was performed by comparing HA-Con and HA-CHK1 or siCon and siCHK1; * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001 vs. control in each group.

Figure 6. CHK1 induced epithelial to mesenchymal transition (EMT) in breast cancer cells. (A) The correlation of CHK1 with CDH1 and OCLN, which are epithelial marker genes. (B) Western blotting of EMT marker proteins in control and CHK1-overexpressing MCF-7 cells. (C) Fluorescence of phalloidin for observation of the morphology in control and CHK1-overexpression MCF-7 cells. (D) Migration assay with control and CHK1-overexpression MCF-7 cells for 48 h after scratch. (E) Transwell invasion assay with control and CHK1-overexpression MCF-7 cells for 48 h after incubation. (F) Western blotting of EMT marker proteins in control and CHK1-knockdown MDA-MB231 cells. (G) Fluorescence of phalloidin for observation of the morphology in control and CHK1-knockdown MDA-MB231 cells. (H) Migration assay with control and CHK1-knockdown MDA-MB231 cells for 16 h after scratch. (I) Transwell invasion assay with control and CHK1-knockdown MDA-MB231 cells for 12 h after incubation. Columns are presented with the mean of SEM. Statistical analysis using t-test was performed by comparing HA-Con and HA-CHK1 or siCon and siCHK1; * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001 vs. control in each group.

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Table 1. Primer sequence used in RT-PCR.

Table 1. Primer sequence used in RT-PCR.

Name of GeneDirectionNucleotide SequenceReferenceMCM4Forward
Reverse5′-GGCAGACACCACACACAGTT-3′
5′-CGAATAGGCACAGCTCGATA-3′[38]CDC7Forward
Reverse5′-TCAAACACCTCCAGGACAATAC-3′
5′-GTACCTCATTCCAGCCTTCTAAA-3′[39]CCNB2Forward
Reverse5′-AAAGCTCAGAACACCAAAGTTCCA-3′
5′-ACAGAAGCAGTAGGTTTCAGTTGT-3′[40]CHEK1Forward
Reverse5′-GGTCACAGGAGAGAAGGAAT-3′
5′-TCTCTGACCATCTGGTTCAGG-3′[41]CXCL12Forward
Reverse5′-ATGAACGCCAAGGTCGTGGTCG-3′
5′-TGTTGTTGTTCTTCAGCCG-3′[42]IL6STForward
Reverse5′-TGTAGATGGCGGTGATGGTA-3′
5′-CCCTCAGTACCTGGACCAAA-3′[43]RUNX1T1Forward
Reverse5′-ACGAACAGCTGCTTCTGGAT-3′
5′-TGCTTGGATGTTCTGAGTGC-3′[44]IGF1Forward
Reverse5′-CCATGTCCTCCTCGCATCTC-3′
5′-TTGAGGGGTGCGCAATACAT-3′[45,46]

Table 2. Microarray datasets of TNBC employed in present study.

Table 2. Microarray datasets of TNBC employed in present study.

GEO SeriesTNBC SamplesNon-TNBC SamplesPlatformReferenceGSE 362951139Affymetrix Human Gene 1.0 ST ArrayMerdad A et al. Anticancer Res (2014)GSE 366932166Illumina HumanHT-12 V4.0 expression beadchipLee ST et al. Proc Natl Acad Sci USA (2013)GSE 6521653100Affymetrix Human Genome U133 Plus 2.0 ArrayMaire V et al. Cancer Res (2013)

Table 3. Enrichment analysis of up-regulated DEGs. (A) Biological pathway. (B) Cellular component.

Table 3. Enrichment analysis of up-regulated DEGs. (A) Biological pathway. (B) Cellular component.

(A)Biological PathwayNo. of Genes in the DatasetPercentage of GenesFold Enrichmentp-Value (Bonferroni Method)Genes Mapped (from Input Data Set)Mitotic M-M/G1 phases1127.57.1521530.000313MCM2; CDC20; AURKB; MCM4; CDC7; BIRC5; KIF2C; MCM10; UBE2C; BUB1; CENPF; 2/M Checkpoints61521.967860.000383MCM2; MCM4; CDC7; MCM10; CCNB2; CHEK1; Cell Cycle, Mitotic12305.9559750.000568MCM2; CDC20; AURKB; MCM4; CDC7; BIRC5; KIF2C; MCM10; CCNB2; UBE2C; BUB1; CENPF; DNA Replication1127.56.6315180.000672MCM2; CDC20; AURKB; MCM4; CDC7; BIRC5; KIF2C; MCM10; UBE2C; BUB1; CENPF; Cell Cycle Checkpoints82010.670790.000939MCM2; CDC20; MCM4; CDC7; MCM10; CCNB2; CHEK1; UBE2C; G2/M DNA damage checkpoint512.527.150180.001423MCM2; MCM4; CDC7; MCM10; CHEK1; Mitotic Prometaphase717.511.130680.003929CDC20; AURKB; BIRC5; KIF2C; UBE2C; BUB1; CENPF; Activation of ATR in response to replication stress512.521.281460.005033MCM2; MCM4; CDC7; MCM10; CHEK1; FOXM1 transcription factor network512.518.748560.009595AURKB; BIRC5; FOXM1; CCNB2; CENPF; (B) Cellular ComponentNo. of Genes in the DatasetPercentage of GenesFold Enrichmentp-Value (Bonferroni Method)Genes Mapped (from Input Data Set)Chromosome57.46268718.43848870.005756MCM2; AURKB; MCM4; BIRC5; BUB1; Nucleoplasm1014.925374.841626390.028453CHAF1B; MCM2; CDC20; MCM4; CDC7; FANCD2; WDR4; MCM10; CHEK1; UBE2C; Chromosome passenger complex22.985075145.0249660.048965AURKB; BIRC5;

Table 4. Enrichment analysis of down-regulated DEGs. (A) Biological pathway. (B) Cellular component. (C) Molecular function.

Table 4. Enrichment analysis of down-regulated DEGs. (A) Biological pathway. (B) Cellular component. (C) Molecular function.

(A)Biological PathwayNo. of Genes in the DatasetPercentage of GenesFold Enrichmentp-Value (Bonferroni Method)Genes Mapped (from Input Data Set)Epithelial-to-mesenchymal transition1731.4814810.707461.34E-10SFRP4; ECM2; DCN; SPARCL1; CXCL12; F13A1; COL14A1; LHFP; ZCCHC24; RUNX1T1; AKAP12; EFEMP1; DPT; SRPX; JAM2; MFAP4; IGF1; (B) Cellular ComponentNo. of Genes in the DatasetPercentage of GenesFold Enrichmentp-Value (Bonferroni Method)genes MAPPED (from Input Data Set)Extracellular3639.560443.155161445.15E-08SCGB2A2; SCGB1D2; TFF1; TFF3; HTRA1; AGR3; SFRP4; PIP; ECM2; CPB1; LAMA2; IGFBP4; NTN4; CCDC80; DCN; SPARCL1; CXCL12; SMOC2; SCUBE2; IL6ST; F13A1; STC2; COL14A1; PDGFD; SEPP1; APOD; SEMA3C; GHR; EFEMP1; DPT; SRPX; OGN; CFD; MFAP4; IGF1; ZBTB16; Extracellular space1314.285715.14930830.001012SCGB1D2; ABI3BP; HTRA1; SFRP4; DCN; IL6ST; COL14A1; APOD; GHR; EFEMP1; DPT; OGN; IGF1; Extracellular matrix88.79120910.67281040.000636ABI3BP; HTRA1; DCN; COL14A1; EFEMP1; DPT; OGN; MFAP4; (C) Molecular FunctionNo. of Genes in the DatasetPercentage of GenesFold Enrichmentp-Value (Bonferroni Method)Genes Mapped (from Input Data Set)Extracellular matrix structural constituent88.5106389.302560.000534ECM2; LAMA2; NTN4; DCN; COL14A1; EFEMP1; DPT; MFAP4; Carboxypeptidase activity33.19148927.621330.037609CPB1; CPA3; CPE;

Table 5. KEGG pathway analysis of total DEGs. (A) Up-regulated DEGs. (B) Down-regulated DEGs.

Table 5. KEGG pathway analysis of total DEGs. (A) Up-regulated DEGs. (B) Down-regulated DEGs.

(A)KEGG PathwaysNo. of Genes in the DatasetPercentage of Genesp-Value (Bonferroni Method)Genes Mapped (from Input Data Set)Cell cycle810.810813.22e-06TTK; MCM2; CDC20; BUB1; CDC7; CHEK1; MCM4; CCNB2Rheumatoid arthritis56.756750.0011HLA-DOB; TNFSF13B; ICAM1; CXCL8; MMP1 (B) KEGG PathwaysNo. of Genes in the DatasetPercentage of Genesp-Value (Bonferroni Method)Genes Mapped (from Input Data Set)Pathways in cancer1010.638290.026FZD4; IGF1; PIK3R1; CXCL12; IL6ST; ZBTB16; RUNX1T1; LPAR1; LAMA2; GSTM3Regulation of lipolysis in adipocytes44.255320.026XGLL; FABP4; PIK3R1; PLA2G16

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