Knockdown of KIF26B inhibits breast cancer cell proliferation, migration, and invasion

Figures & data

Figure 1 Lentivirus-mediated gene knockdown of KIF26B in breast cancer cells.

Notes: (A) Boxplot showing relative expression of KIF26B in normal, luminal, HER2-positive, and triple-negative breast cancer patients. ***p<0.001. (B) qRT-PCR analysis of KIF26B in 4 breast cancer cell lines and the normal breast cell line MCF-10A. GAPDH served as the internal control. (C) Microscopic images of MCF-7 and MDA-MB-231 cells infected with control lentivirus or shKIF26B lentivirus (scale bar, 100 μm). (D) qRT-PCR analysis of KIF26B mRNA expression in the indicated cells; GAPDH served as the internal control. **p<0.01 and ***p<0.001. (E) The knockdown efficiency of KIF26B shRNA in MCF-7 and MDA-MB-231 cells was confirmed by western blot analysis. GAPDH served as the loading control.
Abbreviations: Con, control; HER2, human epidermal growth factor receptor 2; KIF26B, kinesin family member 26B; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction; GFP, green fluorescent protein.

Figure 2 Depletion of KIF26B suppressed the proliferation and colony formation abilities of breast cancer cells.

Notes: (A) Cell viability was measured by CCK8 assay in MCF-7 and MDA-MB-231 cells after KIF26B knockdown. (B) Knockdown of KIF26B decreased the colony formation activities of MCF-7 and MDA-MB-231 cells. Cell colonies were stained with crystal violet and photographed. (C) The number of colonies was counted and statistically analyzed. *p<0.05, **p<0.01.
Abbreviations: CCK8, cell counting kit-8; d, days; KIF26B, kinesin family member 26B; NC, negative control.

Figure 3 Downregulation of KIF26B induced cell apoptosis in breast cancer cells.

Notes: (A) Flow cytometry analysis of breast cancer cell apoptosis using annexin V-FITC and PI dual labeling with or without KIF26B knockdown. (B) The percentage of apoptotic cells was calculated. The data are shown as the mean±SD. (C) The expression levels of KIF26B, Bcl-2, Bax, and cleaved caspase-3 in MCF-7 cells (left panel) and MDA-MB-231 cells (right panel) were measured by western blotting. (D) qRT-PCR analysis of KIF26B, Bcl-2, and Bax expression in MCF-7 and MDA-MB-231 cells. Each experiment was repeated three times. *p<0.05 and **p<0.01 compared with the negative control group.
Abbreviations: FITC, fluorescein isothiocyanate; KIF26B, kinesin family member 26B; NC, negative control; PI, propidium iodide; qRT-PCR, quantitative reverse transcription polymerase chain reaction; FITC-A, fluorescein isothiocyanate-Annexin V; PE-A, Phycoerythrin-area.

Figure 4 Knockdown of KIF26B arrested the cell cycle at the G0/G1 phase.

Notes: (A) Flow cytometry analysis of the cell cycle progression in MCF-7 and MDA-MB-231 cells infected with control virus (NC) or shKIF26B virus. (B) The populations of cells in the G0/G1, S, and G2/M phases were calculated. The results are presented as the mean±SD. *p<0.05 compared with the negative control group. (C) Protein expression levels of cyclin D1 and p-Rb were decreased in MCF-7 cells (left panel) and MDA-MB-231 cells (right panel) after KIF26B knockdown. (D) mRNA levels of KIF26B and cyclin D1 were determined by qRT-PCR. **p<0.01.
Abbreviations: KIF26B, kinesin family member 26B; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction; PE-A, Phycoerythrin-area; Dip, diploid.

Figure 5 Knockdown of KIF26B inhibits tumor metastasis and invasion by modulating EMT-related genes.

Notes: (A) Transwell migration and Matrigel invasion assays using MCF-7 cells (left panel) and MDA-MB-231 cells (right panel) infected with KIF26B shRNA or NC. Scale bars, 100 μm and representative images were taken under ×100 magnification. (B) Migrated and invaded cells were counted in five random fields. Three independent experiments were performed, and the results are presented as the mean±SD. *p<0.05, **p<0.001. (C) Western blot analysis of the protein levels of EMT-related genes, including E-cadherin, N-cadherin, vimentin, and Snail1, in MCF-7 and MDA-MB-231 cells infected with KIF26B shRNA or NC. GAPDH served as an internal control.
Abbreviations: Con, control; EMT, epithelial–mesenchymal transition; KIF26B, kinesin family member 26B; NC, negative control.