Patients and specimens. Ninety-eight paired specimens of CRC tissues and adjacent normal tissues were provided by Clinical Biobank in the First Hospital of Hebei Medical University. Written informed consent was obtained from all patients. CRC and normal tissues were collected from CRC resection specimens, and none of patients had received radiotherapy or chemotherapy prior to surgery. Study protocols were approved by the Ethics Committee of the First Hospital of Hebei Medical University and adhered to the principles of the Declaration of Helsinki.
Cell lines culture. Human CRC cell lines HCT116, SW480, LOVO, SW1116, Caco2, and SW1463 cell lines were obtained from Prof. Jun Yu (The Chinese University of Hong Kong, Hong Kong, China). HCT116 were cultured in McCoy’s 5A medium (Gibco, Gaithersburg, MD, USA) and the other cells lines were cultured in DMEM medium (Gibco, Gaithersburg, MD, USA, supplemented with 10% fetal bovine serum (FBS; FBS; Gibco, Gaithersburg, MD, USA) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) in a 37°C humidified incubator with 95% air and 5% CO2.
Transfection and plasmid construction. The hsa-miR-17-5p mimic, inhibitor, HSPB2 overexpression plasmid or shRNA-HSPB2 and a respective negative control (GeneCopoeia, MD, USA) were transfected into cells in 6-well plates using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) when the cells were approximately 60–80% confluent. After the cells were cultured in medium without FBS for 6 h, the medium was replaced by that supplemented with 10% FBS. Cells were harvested 48 h after transfection and the effect of transfection was assessed by real-time quantitative polymerase chain reaction (qRT-PCR).
RNA extraction and qRT–PCR. Total RNA was extracted from the transfected cells and tissues using Trizol Reagent (Invitrogen, Carlsbad, USA). To quantify miR-17-5p, All-in-One miRNA qRT-PCR kits (GeneCopoeia, MD, USA) were used with miR-17-5p-specific primers (HmiRQP0230, GeneCopoeia, MD, USA) according to the manufacturer’s instructions. To quantify HSPB2, cDNA was reverse transcribed using PrimeScript RT reagent kit (Takara, Beijing, China). Then, a qRT-PCR was performed by using SYBR Green Master Mix (Vazyme, NJ, USA). U6 was used as internal control of miRNA, and GAPDH was used as internal control of mRNA. The primer sequences were as follows: HSPB2(forward): 5′-ATGTCGGGCCGCTCAGTGCC-3′; HSPB2(reverse): 5′-GTCACCTCGTCTGGGGTAAA-3′ (22); GAPDH (forward):5′-GAGTCAACGGATTTGGTCGT-3′; GAPDH (reverse) 5′-CATGGGTGGAATCATATTGGA-3′. All the experiments were performed in triplicate on an ABI7500 Sequence Detection System, and the mean cycle threshold (CT) data were obtained. The relative amount normalized to the internal control was calculated with the Eq. 2−ΔΔCT.
Cell apoptosis assay. The transfected cells were stained using the Annexin V-FITC/ PI Apoptosis Detection Kit (Beyotime, Jiangsu, China) in accordance with the manufacturer’s instructions. 1 × 105 cells were analyzed for apoptosis using a flow cytometry (BD, MA, USA), and the percentage of the apoptotic cells was quantified using Cell Quest software.
Cell proliferation assay. The transfected cells were plated at 2 × 103 cells per well in 96-well plates and incubated overnight in medium supplemented with 10% FBS. Cell proliferation was measured using a Cell Counting Kit-8 (Dojindo, Tokyo, Japan) at 24, 48, and 72 h post-transfection following the manufacturer’s instruction. The absorbance at 450 nm was measured using a Promega GloMax Luminescence detector (Promega, MD, USA).
Colony formation assay. 1 × 103 transfected cells were plated in cultured dishes and grown for 2 weeks. Subsequently, the cells were washed twice with phosphate buffer saline (PBS), fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for 1 h. The dishes were scanned and the average number of colonies was achieved.
Transwell invasion and migration assay. Invasion and migration assay were performed using Transwell chambers with 8-µm pores (Corning, MA, USA). For invasion assay, 2 × 105 transfected cells suspended in 500 µl serum-free medium were placed in the upper chambers coated with Matrigel, and medium containing 10% FBS was added to the lower chamber as a chemoattractant. For migration assay, cells in 100 µl serum-free medium were added to the upper chambers, and medium containing 10% FBS was placed in the lower chamber as a chemoattractant. Following incubation at 37℃ for 24 h, cells on the upper surface of the chamber were gently wiped off with cotton swabs. The cells were stained using a Diff-Quick stain kit according to the manufacturer's protocol and counted blindly (five random fields per chamber).
RNA sequencing and data analysis. Total RNA was extracted from cells transfected with hsa-miR-17-5p mimic or inhibitor and the negative control cells, and then subjected to commercial RNA-sequencing (RNA-seq) analysis (Novogene). Preparation of library and sequencing of transcriptome were performed using NEB Next Ultra RNA Library Prep Kit for Illumina (Novogene, Beijing, China) following the manufacturer’s protocols, and the attribute sequence of each sample was added with an index code. The clustering of the index-coded samples was achieved on a cBot Cluster Generation System. After cluster generation, 125-bp paired-end reads to genes were mapped using HTSeq v0.6.0 software. Afterwards, fragments per kilobase of transcript per million fragments mapped (FPKM) were also calculated. Analysis of differential expression was performed using the DEGSeq R package through the one scaling normalized factor. Corrected P values of 0.005 and log2 (fold changes) of 1 were set as the threshold for significantly differential expression after adjusted using the Benjamini-Hochberg method. Moreover, the hierarchical clustering and heatmaps were generated to show the normalized expression among the samples.
Luciferase reporter assay. Four sets of luciferase reporter plasmids were constructed, and each set of plasmids contained a 60–80 bp fragment of HSPB2 3'-untranslational region (3'-UTR) with a conserved miR-17-5p binding site (Sangon Biotech, Shanghai, China). For the luciferase reporter assays, HCT116 and LOVO cells were cultured in 96-well plates, and each well was co-transfected with firefly luciferase reporter plasmids, miR-17-5p mimic, inhibitor or the negative control using Lipofectamine 2000 (Invitrogen, Carlsbad, USA). The firefly luciferase and renilla luciferase activities were evaluated using the Dual Luciferase Reporter Assay Kit (Promega, MD, USA) following the manufacturer's instructions after transfection for 48 h.
Western blot analysis. Total protein was isolated from the cultured cells using RIPA lysis buffer (Beyotime, Shanghai, China). Proteins were separated by 12% SDS-PAGE (Bio-Rad) and transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). The immunoreactive protein bands were detected by the Odyssey Scanning System (Li-Cor, Lincoln, USA) after using antibodies against HSPB2 (Proteintech, Wuhan, China) and β-actin (Solarbio, Beijing, China).
Databases analysis. We used the online databases TargetScan48, microRNA.org49 and mirtarbase50 to predict target genes of miR-17-5p, and confirmed the specific binding sites for miR-17-5p on the 3'-UTR region of the target gene. In addition, the online database The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Ineractive Analysis (GEPIA) were used to further ascertain the mRNA expression of the target gene.
Statistical analysis. The results of normal distribution data are expressed as mean ± SD and the results of non-normal distribution data are expressed as median and quartile spacing. Student’ s t-test, one-way ANOVA and two-way ANOVA were used in this study. All statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, CA, USA) and SPSS 21 (IBM, NY, USA). P < 0.05 was considered as statistically significant.