This study enrolled a consecutive series of 30 patients with breast cancer from the middle area of China. As a control, we used 20 non-tumoral-adjacent tissues. Frozen tissues were collected at the Department of Breast Surgery from Shanxi Tumour Hospital (China). The patients ranged in age from 34 to 75 years with a mean age of 48.62 years. Fresh tissues were frozen in liquid nitrogen within 5 min after excision and transferred to a -80℃ freezer. The entire experiments have already obtained the approval of the Ethics Committee of Shanxi Medical niversity (Ethical code: 201922021).
Human breast cancer cell line (MCF7) was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). MCF7 cells were maintained in DMEM/F12 medium supplemented 10% FBS (fetal bovine serum) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. Culture plates were maintained at 37 ℃ in a 5% CO2 humidified atmosphere.
DNA methylation inhibitor treatment
The same numbers of cells MCF7 cells were cultured in 6-well plates and incubated at 37 ℃ overnight. Cells were treated with 1, 5, or 10 µM Decitabine (Selleck, Houston, TX, USA) or vehicle (DMSO) and incubated for 72 with the purpose of mRNA extraction. The cell culture medium was refreshed daily.
Quantitative real-time PCR (qRT-PCR)
RNA extraction kit (BioTeke Corporation, Beijing, China) was employed to extract total RNAs. Prime Script™ RT Master Mix kit (Takara Botechnology Co., Ltd., Dalian, China )was conducted to reverse transcribe the extracted RNA into complementary DNA. The qRT-PCR was carried out following SYBR® Premix Ex Taq Kit (Takara Bio, Inc., Tokyo, Japan). For PCR amplification, glyceraldehyde –phosphate dehydrogenase (GAPDH) was employed as a suitable internal control. The results were recorded when the cycle was finished. The primers used were as follows: KPNA2, forward, 5′-ATTGCAGGTGATGGCTCAGT-3′ and reverse, 5′-CTGCTCAACAGCATC TATCG-3′; GAPDH, forward, 5'-ACCACAGTCCATGCCATCAC-3'; and reverse, 5'-TCCACCA CCCTGTTGCTGTA-3'. Relative gene expression was calculated with the 2−ΔΔCt method, and Step One Software v2.1 was used to evaluate the results.
Methylation specific PCR
The extraction of genomic DNA from cells was performed by the use of a DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA samples were PCR-amplified and fragmented in a volume of 20 µl comprised of 0.25 µl of Hot-StarTaq Master Mix, 0.5 µM of paired primer, and 2 µg of bisulfite-treated DNA template. The agarose gel (3%) containing GelRed™ Nucleic Acid Gel Stain (10,000×; Biotium, CA) was used to separate MSP products in a 20 µl volume after amplification. DNA-free water was served as negative control. The primers used were as follows: forward M primer, 5′- AAATACGAACGGTTTAGGGAATC − 3′ and reverse M primer, 5′- ACATCGCGAAATAAAAAAACG − 3′; forward U primer, 5′-GGAAAT ATGAATGGTTTAGGGAATT-3′, reverse U primer, 5′-ATAACATCACAAAATAAAAAAACAA C-3′.
Gene Expression Profiling Interactive Analysis
Differential expressions of KPNA2 in breast invasive carcinoma tissues were assessed by GEPIA2 (Gene Expression Profiling Interactive Analysis; http://gepia2.cancer-pku.cn), an online database with fast and customizable features based on TCGA (The Cancer Genome Atlas) . Here, GEPIA database containing 1085 breast invasive carcinoma tissues and 112 normal mammary tissues were downloaded for analyses. Gene expression data of the RNA-seq datasets were transformed to log2 (transcript count per million [TPM] + 1).
Oncomine database analysis
KPNA2 gene expression levels in normal or malignant human tissues were obtained from the Oncomine Cancer Microarray database (http://www.oncomine.org), a web-based data mining platform for collecting, standardizing, analyzing and providing cancer microarray information .
cBioPortal database analysis
cBioPortal (The cBio Cancer Genomics Portal) database is a publicly accessible online database (http://www.cbioportal.org/) [18, 19], which provides visualization and analysis tool for more than 715 datasets and 86,733 samples. The term “KPNA2” was used to search the cBioPortal database and The Breast Invasive Carcinoma (TCGA, Cell 2015, n = 818) cohort was utilized. The search parameters included mutations, putative copy-number, and methylation alterations from GISTIC.
The COSMIC (Catalogue of Somatic Mutations in Cancer) database (http://cancer.sanger.ac.uk), an online accessible network tool was performed to analysis mutations in KPNA2. An overview of the distribution of mutations and substitution types on the codogenic strand in breast cancer specimens was perfomed, and the results are drawn in a pie chart 
Breast Cancer Gene-Expression Miner v4.4
bcGenExMiner v4.4 (Breast Cancer Gene-Expression Miner v4.4), a web-based tool, was performed to analysis the KPNA2 expression in breast cancer . Correlations between KNPA2 and the TPX2 were analyzed using the bcGenExMiner v4.4 correlation module.
Kaplan-Meier plotter analysis
Kaplan-Meier Plotter (http://kmplot.com/analysis/) is an online database of published microarray datasets that assess the effect of 54, 675 genes on survival using 10, 461 cancer samples (6, 234 breast, 2, 190 ovarian, 3, 452 lung, and 1, 440 gastric cancer). We used the Kaplan-Meier plotter to assess the correlation between overall survival, relapse free survival, distant metastasis free survival, post-progression survival and KPNA2 mRNA expression in patients with breast cancer. The hazard ratio (HR) with 95% confidence intervals (CI) and log rank p-value were also computed.
The MEXPRESS tool (http://mexpress.be/), a web tool for the visualization and interpretation of TCGA data, offers clinical researchers an easy way to access TCGA expression (normalized RNASeqV2 value), DNA methylation, and clinical data, as well as the relationship between them for one signale in the specific tumor type. In MEXPRESS tool, it executed the Pearson correlation to evaluate the difference between expression value and methylation level. In our research, the KNPA2 expression and methylation status in breast cancer were assessed using the MEXPRESS tool.
UALCAN cancer database
UALCAN (http://ualcan.path.uab.edu/) is a comprehensive, user-friendly, and interactive online portal for analyzing cancer OMICS data, which provides protein expression analysis option using data from TCGA dataset [22–24]. We evaluated the expression and methylation level of KPNA2 in breast cancer by UALCAN analysis.