Human BC samples
A total of 99 paraffin-embedded BC tissue samples were collected from patients undergoing mammectomy and needle biopsy at Qilu Hospital of Shandong University during 2005-2010. Pathomorphological observation was performed according to the criteria established by the World Health Organization. This study was approved by the Ethical Committee of Shandong University, China (code 2012028).
Immunohistochemistry (IHC)
Paraffin-embedded sections were immunostained with antibodies against KIF26B
(1:100, Proteintech, Wuhan, China, 17422-1-AP), ER (MaxVision, Fujian, China, Kit-0012), PR (MaxVision, Fujian, China, Kit-0013), HER2 (MaxVision, Fujian, China, Kit-0012) and Ki67 (Maxvision, Fujian, China, Kit-0002), and were assessed by two experienced pathologists. For negative controls, the primary antibody was replaced with PBS. IHC was scored using a semi-quantitative scoring system. Intensity was scored as 1, 2 and 3 for weak, moderate, and strong staining, respectively. Percentage (P) and intensity (I) were multiplied to generate numerical score (S=P*I). The median score was used as cut-off to distinguish between KIF26B high and low expression.
RNA extraction and RT-qPCR
Total RNA was extracted from cultured cells using TRIzol (Invitrogen) according to manufacturer’s instructions, then reverse transcribed into cDNA using ReverTraAce qPCR RT Kit (Toyobo, Japan). RT-qPCR was then performed using SYBR Green PCR Kit (Roche Diagnostic GmbH, Mannheim, Germany). Relative expression was evaluated using the 2-△Ct method using GAPDH as the reference gene. Primers for RT-qPCR were as follows: KIF26B forward, 5’-AGGCCATGTGCTTAATGCAAA-3’ and reverse 5’-ATCCAGCATCAGATACTGTTTGGT-3’; GAPDH forward 5’-AGAAGGCTGGGGCTCATTTG-3’ and reverse 5’-AGGGGCCATCCACAGTCTTC-3’.
Cell lines
Human BC cell lines MDA-MB-231, MDA-MB-468, MDA-MB-453, MCF-7, SKBR3, MCF-10A and 293T cells were purchased from the Chinese Academy of Science Cell Bank (Shanghai, China). BC cell lines were cultured in Leibovitz’s L15 medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco). 293T cells were cultured in DMEM (Gibco) containing 10%FBS. All cells were cultured in a humidified atmosphere at 37°C with 5% CO2.
SiRNA and cell transfection
KIF26B small interfering RNA (si-KIF26B) and negative control (si-NC) were synthesized by RiboBio (Guangzhou, China). The sequence of si-KIF26B was 5’-CGGACAGCCTCTCCTATTA-3’ and the si-NC sequence was 5’-TTCTCCGAACGTGTCACGT-3’ [10]. After reaching 90% confluence, MDA-MB-231 and MDA-MB-468 cells were seeded in 6- or 12-well plates and transiently transfected with siRNA using the X-treme GENE transfection reagent (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s instructions. The examination of KIF26B expression level and subsequent experiments were performed 48 h later.
MTS assay
MDA-MB-231 and MDA-MB-468 cells transfected with si-KIF26B or si-NC for 24 h were seeded in 96-well plates. MTS (5 mg/ml, Promega, Madison, WI, USA) was added into the culture medium at 24, 48,72 and 96 h, respectively, followed by an incubation of 2 h. The absorbance at 490 nm was then read. Three independent experiments were performed.
EdU assay
MDA-MB-231 andMDA-MB-468 cells transfected with si-KIF26B or si-NC were cultured in 6-well plates for 24 h. The cells were then collected and cultured in 12-well plates for 24 h. The EdU assay was performed in according to the manufacturer’s instructions (RiboBio, Guangzhou, China). Cells were visualized under a fluorescent microscope (Olympus, Tokyo, Japan). The experiment was repeated three times.
Flow cytometry
MDA-MB-231 and MDA-MB-468 cells transfected with si-KIF26B or si-NC were cultured in 6-well plates for 48 h. For the cell cycle assay, the harvested cells were stained with propidium iodide (PI, Beyotime). For apoptosis detection, cells were double-stained with annexin V-fluorescein isothiocyanate (FITC) and PI using an FITC annexin V Apoptosis Detection kit (401003, BestBio, China). The cells were immediately examined by flow cytometry (FACScan; BD Biosciences), according to the manufacturer’s protocol. Each experiment was performed in triplicate.
Dual-luciferase reporter assays
293T cells were seeded in 24-well plates overnight, then co-transfected using 0.5 μg/well of the KIF26B promoter regions (-2,000/0, -1,043/0, -530/0 and -110/0) overexpression plasmids and pGL3-basic vector and 0.01 μg of pRL-TK plasmid (as the internal control) using Lipofectamine 2000 (11668019, Invitrogen). About 48 h after transfection, the cells were subjected to luciferase activity analysis using a Dual-Luciferase Reporter Assay Systems (Promega) to decide the core promoter of KFI26B gene according to the manufacturer’s instructions. Similaly, the plasmids with decided core promoter of KFI26B gene and alternative transcription factor overexpression plasmids(ELK1, STAT4, JUN, pCDNA3.1 basic vector) and 0.01 μg of pRL-TK plasmid were co-transfected to 293T cells to establish the relevant transcriptional factor of KIF26B. Each experiment was performed in triplicate.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were carried out using the EZ-Magna ChIP Chromatin Immunoprecipitation Kit (17-10086, Merck). Briefly, the MDA-MB-231 and MDA-MB-468 cells were crosslinked with 1% formaldehyde for 10 min at room temperature before immunoprecipitation reaction. After incubation with 5 μl anti-ELK1 antibody (ab32106, Abcam) or IgG antibody (rabbit anti- IgG, as the negative control) and protein A/G magnetic beads overnight, immunoprecipitation was performed in accordance with the manufacturer’s instructions. Co-precipitated DNA was quantified using qPCR. A volume of 10 μl chromatin was used as input control prior to immunoprecipitation. This experiment was repeated three times.
Vector construction
The KIF26B promoter regions (-2,000/0, -1,043/0, -530/0 and -110/0) were amplified from genomic DNA extracted from MDA-MB-231 cells, then cloned into the pGL3-Basic vector (Promega). The plasmids containing ELK1, STAT4, JUN, and pcDNA3.1 vector originated from our research group.
Western blotting
Western blotting was carried out using standard protocols. The primary antibodies used were as follows: KIF26B (1:500, 17422-1-AP, Proteintech), ELK1 (1:500, ab32106, Abcam), Wnt6 (1:1,500, ab50030, Abcam), GAPDH (1:1,000, #5174, CST), β-catenin (1:2,000, ab32532, Abcam), c-Myc (1:1,000, #18583, CST), cyclin D1 (1:1,000, #55506, CST), CDK4 (1:1,000, #12790, CST), p27 (1:1,000, #3686, CST),cyclin E1 (1:1,000, #20808, CST), CDK2 (1:1,000, #18048, CST), p21 (1:1,000, #2947, CST, CDK6 (1:1,000, #13331, CST), p53 (1:1,000, #9282, CST), p-Rb (1:1,000, #8516, CST). Each experiment was performed in triplicate.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Differences between the two groups were analyzed using Student’s t test. The χ2 test and Spearman’s correlation were used to evaluate the associations between KIF26B expression levels and clinicopathological parameters in BC. Survival analysis was performed using the Kaplan-Meier method and log-rank test. Data are presented as the mean ± SD. P<0.05 was considered statistically significant.