Cell culture and borax exposure
The HepG2 cell line was purchased from Wuhan University. Cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum in a 5% CO2 humidified incubator at 37°C. Cells were cultured for 2-3 days. Subsequently, the original medium was discarded, and new medium containing borax (Tianjin Bodi Chemical Co. Ltd., Tianjin, China) solution at concentrations of 0 (control) and 4mM was added to the culture plate. Cells were cultured for either 2 or 24 h. After borax exposure, the cells were washed with PBS to remove borax and then subjected to RNA extraction.
Exiqon Agilent Human miRNA array
Agilent Human miRNA Array (Exiqon A/S; Agilent Technologies, Inc.) contains >2,549 capture probes covering all human microRNAs annotated in the miRBase 21.0 (http://www. mirbase.org/). Exiqon gene chips are designed using Agilent's unique microRNAs detection technology, which allows the detection of specific mature miRNAs and the differentiation of highly homologous miRNAs.
RNA extraction and labeling
After 2 or 24 h borax exposure, total RNA was extracted from cells and purified using miRNeasy Mini Kit (Cat 217004, Qiagen, GmbH) following the manufacturer’s instructions. The RIN number was evaluated to assess RNA integration using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.). The miRNAs present in the total RNA were labeled with miRNA Complete Labeling and Hybridization Kit (cat. no. 5190-0456; Agilent Technologies, Inc.) following the manufacturer’s instructions.
Each slide was hybridized with 100 ng cyanine 3-labeled RNA using miRNA Complete Labeling and Hybridization Kit (cat. no. 5190-0456; Agilent Technologies, Inc.) in a hybridization oven (cat. no. G2545A; Agilent Technologies, Inc.) at 55°C, 20 rpm for 20 h according to the manufacturer’s instructions. After hybridization, the slides were washed in staining dishes (cat. no.121; Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (cat. no. 5188-5327; Agilent technologies, Santa Clara, CA, US). Slides were scanned with Agilent Microarray Scanner (cat. no. G2565CA; Agilent technologies, Inc.) using Feature Extraction software 10.7 (Agilent technologies, Inc.) with default settings. The raw data were normalized by the quantile algorithm using Gene Spring software 12.6 (Agilent Technologies, Inc.).
Reverse transcription-quantitative PCR (RT-qPCR) analysis
Single-stranded cDNA was synthesized using miRNA cDNA Kit (Clontech Laboratories, Inc.). The expression levels of miRNAs in HepG2 cells were determined by qPCR using Real-Time PCR Assay Kit (Takara Bio Inc.) according to the manufacturer’s instructions. The upstream sequences of the miRNA specific primers, which were designed using miRBase, are presented in Table I. The downstream primers were obtained from the Real-Time PCR Assay Kit. Primer sequences targeting specific miRNAs were designed using Primer Express 3.0® software (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers were synthesized by Sangon Biotech Co., Ltd. The thermocycling conditions were as follows: 95˚C for 15 min, followed by 45 cycles of 95˚C for 15 sec, 55˚C for 30 sec and 72˚C for 30 sec. The miRNA expression levels were normalized against U6 (which served as an internal control), and were determined using the 2-ΔΔCq method.
Data analysis, target prediction, and GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses
Following normalization, the miRNA expression levels were evaluated via volcano plot and heatmap. The differential expressed miRNAs between borax-treated and control cells were compared using the DEGseq R package (Novogene). P<0.01 and fold change>2 served as the threshold for significantly differential expression. miRanda was used to predicted the target gene of miRNAs (http://www.microrna.org/microrna/home.do). GO enrichment analysis was used for evaluation of the target gene candidates of differentially expressed miRNAs based on a public database of bioinformatics resources (http://www.geneontology.org/). KEGG is a database resource for predicting high-level functions (http://www. genome.jp/kegg/).
Establishment and analysis of protein-protein interaction network
To better understand the relationship among these screened genes, the protein-protein interaction (PPI) network was established using the STRING database (Szklarczyk et al., 2017). The hub genes in the PPI network were identified according to degree using Cytoscape software (version 3.6.1).
miRNA data were analyzed with GeneSpring v12.0 (Agilent Technologies, Inc.). Data are expressed as the mean ± standard error of the mean. Differences in mean values were evaluated by Student's t-test (two means comparison). A threshold of fold-change ≥2.0 and P<0.05 was used to estimated differentially expressed miRNAs.