Cell culture and transfection
Human breast cancer cell lines in this study, including MCF-7 and ZR-75-30, were purchased from China Infrastructure of Cell Line Resource and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 mg/ml penicillin and streptomycin at 37°C in 5% CO2. Hsa-miR-105-3p inhibitor and its corresponding negative control (NC inhibitor) were synthesized by Sangon Biotech (Shanghai Co., Ltd) and transfected into these MCF-7 and ZR-75-30 cell lines by Lipofectamine 2000 (Thermo Fisher Scientific, Inc.; USA) according to the manufacturer's instruction. The successfully transfected cell lines were further studied in the following experiments. To silence the expression of GOLIM4 in MCF-7 and ZR-75-30 cell lines, shRNA targeted for GOLIM4, was synthesized by Sangon Biotech (Shanghai, Co., Ltd), and transfected into these MCF-7 and ZR-75-30 cell lines by Lipofectamine 2000.
Cancer tissues and paired adjacent tissues of 80 patients with breast cancer were collected from our hospital. MagMAX™ RNA isolation kit and VetMAX™-Plus One-Step RT-PCR Kit (USA; Thermo Fisher Scientifc, Inc.) were applied to isolate total RNA and miRNAs according to the instruments. After the total RNA was extracted from tumor tissues, TaqMan microRNA assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) was carried out to measure the expression of miR-105-3p. The reagent components in the reaction system were showed as follows: 20×TaqMan miRNA assay (1µL), 2× TaqMan Universal PCR Master mix (10 µL; USA; Thermo Fisher Scientific, Inc.), cDNA (1.33 µL), forward primer (1 µL;) and reverse primer (1 µL) and double distilled water (5.67 µL). The RT-qPCR was performed in the ABI 7500 Real-Time PCR System and the results of the threshold cycle (Ct) were calculated by 2−ΔΔCt method after being normalized by the endogenous controls U6 snRNA (Forward primer: 5’-ATTGGAACGATACAGAGAAGATT-3’; Reverse primer:5’-GGAACGCTTCACGAATTTG-3’). The expression level of GOLIM4 was detected with the methods as described above in breast cancer tissue and cell lines. miR-105-3p: Forward primer: 5’- CCACGGACGTTTGAGCAT -3’; Reverse primer:5’-TATGGTTGTTCACGACTCCTTCAC-3’. GOLIM4: Forward primer: 5’-CAGAGCCAATCCAACAAG-3’; Reverse primer: 5’- ATTGCCGACTCCACGACAC-3’. GAPDH: Forward primer: 5’-TGACTTCAACAGCGACACCCA-3’; Reverse primer: 5’CACCCTGTTGCTGTAGCCAAA-3’.
Colony formation assay
ZR-75-30 and MCF-7 cells were cultured into logarithmic growth phase, and then the cells were collected and suspended into a cell suspension. The concentration of suspended cells was adjust to 1×104 cell/mL, and a total of 5×104 cells were inoculated into 10 cm dish. The cells were incubated for 2 weeks and then methanol was added to fix the formed cells colonies. Subsequently, the cells were with crystal violet dye. The number of colonies containing more than 50 cells was counted.
Cell Counting Kit-8 (Dojindo, Inc.; Japan) was used to detected the proliferation capacity of the transfected ZR-75-30 and MCF-7 cells according to the instruments. Briefly, a total of 5×103 cells was seeded into the 96-well plates with 10 µL CCK-8 solution. The cells were incubated for 24, 48 and 72 h, and then the absorbance was measured at 490 nm with a microplate reader.
The ZR-75-30 and MCF-7 cells were transfected with miR-105-3p inhibitor and cultured into the logarithmic growth phase. The bottom of 6-well plate was rowed with five straight lines with 0.5 cm intervals. After the cells were suspended and adjusted into 1 × 105 cells/mL, 2 mL cell suspension was added into the well and cultured for 24 h. Subsequently, the scratch that was perpendicular to the base lines above was made by 10-μl pipetting spear. The cells were cultured for 48 h with serum-free medium, and wound closure (%) was finally calculated.
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay
TUNEL assay was performed to detect the apoptosis of transfected cells using One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology Co., Ltd; China) following the manufacturer's instructions. Briefly, a total of 5×103 cells was seeded into the 96-well plates for 48 h culture. Then, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Subsequent to washing by PBS twice, 0.1% TritonX-100 was added to permeabilize the cell membrane. The cells were successively treated with TUNEL reaction mixture, converter-POD and DAPI substrate. The cell images were then obtained under the fluorescence microscope (Olympus, Tokyo, Japan).
Transwell chamber assay
Transwell chamber assay was applied to detect the invasion ability of ZR-75-30 and MCF-7 cells after transfected with NC or miR-105-3p inhibitor. The upper and lower chamber were added with FBS-free culture medium and culture medium containing 10% FBS, respectively. The transfected cells were cultured in FBS-free culture medium for 12 h, and then a density of 2×104 cells was seeded into the upper chamber with 10 mg/mL matrigel. After cultured for 48 h, the top of the filter was carefully wiped with cotton swabs to remove the remained cells. Those cells that migrated through the membrane were fixed with 95% methanol, stained with 0.5% crystal violet and finally counted with microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan).
Western blot assay
After the transfected cells were cultured for 72 h, the cells were washed with PBS for three times and then centrifuged to obtain cell pellet. ProteoPrep® Total Extraction Sample Kit was used to extracted total protein according to the instruments followed by the detection of protein concentration using a BCA assay kit (USA; Thermo Fisher Scientifc, Inc.). Equal quality of protein was separated on 12 % SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientifc, Inc.; USA). After blocked with 5% milk in PBST for 1 h at room temperature, the membrane was incubated with proper dilution of primary antibodies for 1 h at 37℃ including anti-BAX, anti-Bcl-2, anti-Cleaved caspase-3, anti-Cleaved caspase-9, anti-ICAM-1 and anti-VCAM-1. β-actin was chosen as an internal control in the assay. Subsequent to washing with TBST for three times, HRP labeled secondary antibodies corresponding to primary antibodies were used to probe the expression of target protein in the membrane. The protein bands were visualized using Novex™ ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientifc, Inc. USA). Densitometry quantification of protein bands was conducted using Image J pro plus software and then normalized with β-actin.
Luciferase reporter assay
In order to study the relationship between miR-105-3p and GOLIM4, the ZR-75-30 and MCF-7 cells were co-transfected with miR-NC or miR-105-3p and the psiCHECK2 vector containing the wild type or mutant GOLIM4 fragment (psiCHECK2- GOLIM4-3’ UTR WT or psiCHECK2- GOLIM4-3’ UTR MUT) with lipofectamine2000 (Thermo Fisher Scientific, Inc.; USA). After cultured for 48 h and three times washes, the cells were lysed with harvest buffer for 10 min at 0℃. The mixture of ATP buffer and luciferin buffer (1:3.6) was added into the cell lysate followed by the detection of the absorbance.
The measurement data that conforms to the normal distribution was showed as mean ± standard deviation. The difference between two groups was analyzed by Student's t test. One-Way ANOVA was used to analyze the differences among group followed by Bonferroni post‐hoc analysis. The 80 patients were divided into the high and low miR-105-3p expression group according to the median miRNA expression, and then life table methods were used to analyze the difference of survival data between the two groups, after which the differences in survival time were tested by the Mantel-Cox log-rank method. All the data were analyzed by SPSS software, version 20 (SPSS, Inc., Chicago, IL, USA). A P value < 0.05 was considered to indicate a significant effect.