MicroRNA array
SKOV3 cells were transfected with RAC1 siRNA or siRNA NC. Total RNA was then isolated by TRIzol regent (Takara, Japan) in accordance with the manufacturer’s instructions. RNA quality was determined by a spectrophotometer at 260 and 280 nm, and RNA integrity was evaluated by 1.5% formaldehyde denaturing gel. Flash Tag Biotin HSR labeling was performed. Biotin-labeled RNA was hybridized to the Affymetrix GeneChip miRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA). Transcriptome Analysis Console (Affymetrix, USA) was used to analyze differentially expressed miRNAs.
Cells and clinical specimens
Ovarian cancer cell lines SKOV3 and OVCAR3 were obtained from the Cell Resource Center of Chinese Academy of Medical Sciences and maintained in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin–streptomycin solution (Beyotime, China) in a 5% CO2 humidified incubator at 37 °C. Fresh tumor tissues and adjacent normal tissues were collected immediately after surgery in Shandong Provincial Hospital Affiliated to Shandong First Medical University. A written informed consent was obtained from each patient. Ethical approval from the Shandong Provincial Hospital Affiliated to Shandong First Medical University was received before the studies were performed in accordance with the Declaration of Helsinki.
qRT-PCR
The total RNA of the cells was extracted with Trizol (Sigma, USA) in accordance with the instructions. After quantification, 2 ug of RNA was used for reverse transcription with miR-3613 specific reverse transcription primers to obtain cDNA. The PCR primers of miR-3613 were then employed for amplification and quantification using the 2-△△ct method. U6 snRNA was applied as the control. The primer sequences were as follows: miR-3613-5p-RT, 5ʹ-GTCGTATCCAGTGCAGGG TCCGAGGTGCACTGGATACGACGAACAAA-3ʹ, miR-3613-5p-forward, 5ʹ-TGCGGTGTTG TACTTTTTTTTT-3ʹ, miR-3613-5p-reverse, 5ʹ-CCAGTGCAGGGTCCGAGGT-3ʹ, U6-RT, 5ʹ-GTCGTATCCAGTGCAGGGTCCGAGGTGCACTGGATACGACAAAATATGGAAC-3ʹ, U6- forward, 5ʹ-TGCGGGTGCTCGCTTCGGCAGC-3ʹ, and U6- reverse, 5ʹ-CCAGTGCAGGGTCCGA GGT-3ʹ. Each experiment was performed in triplicate.
MTT
The cells were resuspended in a culture medium containing 10% FBS and inoculated in a 96-well plate with 1,000 cells per well. After 24 hours, lipofectamin3000 was used to transfect miRNA mimics and DNA plasmids into the cells, which were then cultured for 4 days. After transfection, cell viability was detected by MTT method every day. Each well was added with 20 ul of 5 mg/ml MTT solution (Beyotime, China). After incubation for 4 hours, the medium was discarded, 150 ul of DMSO was added, and the mixture was shaken for 10 minutes. The absorption value of each hole was measured at a wavelength of 450 nm. Each experiment was performed in triplicate.
Colony formation assay
The cells in the logarithmic growth phase were digested with 0.25% trypsin and pipetted into single cells. The cells suspended in culture medium containing 2% FBS were seeded into a six-well plate at a density of 1000 cells per well. All cells were maintained in an incubator containing 5% CO2 at 37 °C. The medium was changed every 3 days until visible clones were detected. Afterward, the medium was discarded, and the cells were carefully washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with GIMSA for 10–30 minutes, and slowly washed with running water. Images were capture to record the number of clones. Each experiment was performed in triplicate.
Wound healing
Approximately 5x105 cells were seeded in a 24-well plate and culture for 24 hours until the cell density was more than 90%. A10 ul pipette tip was then used to make a straight scratch. The cells were washed three times with PBS to remove the suspended cells. A serum-free medium was added, and the cells were placed in a 37 °C 5% CO2 incubator for culture. After 24 hours, images were captured under a microscope, and the cell migration rate was calculated. Each experiment was performed in triplicate.
Transwell assay
In brief, 5 μg/μl Matrigel (BD, USA) was added to the upper chamber (Corning, USA) and placed at 37 °C for 30 min. The digested cells were washed with PBS, resuspended in a serum-free medium containing BSA, and seeded at a concentration of 105 per well. The lower chamber was added with 500 ul of medium containing 10% fetal bovine serum and cultured for 24 hours. The medium was then discarded, and the cells in the chamber were removed and fixed with 90% alcohol at room temperature for 30 minutes. The cells were then stained with 0.1% crystal violet at room temperature for 10 minutes. After being washed with water, the invasive cells were recorded under a microscope (Olympus, DP50, Japan). Each experiment was performed in triplicate.
Western blot
The total protein of the cells was harvested with RIPA lysate (Beyotime, China), quantified by BCA method, and then separated by 10% SDS-PAGE. The separated protein was transferred to the PVDF membrane. After being washed three times with TBST, the membrane was blocked in 5% BSA overnight at 4 °C and then incubated with the following diluted primary antibody for 2 hours at room temperature: RAC1 polyclonal antibody (Abcam, ab155938, UK), N-cadherin polyclonal antibody (Affinity, AF4039, China), vimentin polyclonal antibody (Affinity, AF7013, China), and GAPDH polyclonal antibody (Affinity, AF0911, China). HRP-labeled secondary antibody was then added to react at room temperature for 2 hours, and the specimens were finally visualized by ECL luminescence reagent (Beyotime, China) until the protein is clear. Each experiment was performed in triplicate.
Plasmid, oligo, and transfection
Pre-miR-3613 genomic sequence was synthesized and inserted into pcDNA3.1 vector to construct the miR-3613 expression plasmid. RAC1 expression plasmid was purchased from SinoBiological Inc. (HG10535-CF). RAC1 siRNA sequence was 5ʹ-CAAACAGUUGGAGA AACGUACGGUA-3ʹ.
Luciferase activity assay
The wild type and mutant 3ʹ-UTR of RAC1 were synthesized and cloned into the pmirGLO vector. The cells were seeded into a 48-well plate. After 24 hours, the luciferase reporter plasmids were transfected when the cell density reached approximately 70%. After 48 hours, the culture medium was discarded, and the cells were washed with 100 μl of PBS. Each well was then added with 50 μl of diluted lysis buffer and then shaken for 20 minutes on a shaker to ensure that the cells were completely lysed. Firefly luciferase and renilla luciferase activities were detected following the instructions of Dual-Lumi™ dual luciferase reporter gene detection kit (Beyotime, China). Each experiment was performed in triplicate.
In vivo experiments
Eighteen 5-week-old female BALB/c nude mice were purchased from Charles River Laboratories, Inc. and randomly classified into three groups, namely, vector control, miR-3613 overexpression group, and miR-3613 and RAC1 overexpression group. The prepared cells were injected subcutaneously into the nude mice at a concentration of 5x106 cells per mouse. Tumor volumes were monitored every 3 days. After 1 month, all animals were euthanized through the intravenous injection of sodium pentobarbital at a final concentration of 100 mg/kg. The surgically harvested solid tumor tissues were fixed in 4% formalin, and their volume was calculated as follows: volume= (length×width2)/2. All the animals were well taken care of, and all the experiments were performed in accordance with the ethical standards of the Institutional Animal Use and Care Committee of the Shandong Provincial Hospital Affiliated to Shandong First Medical University. Ethical approval was obtained prior to the study. Each experiment was performed in triplicate.
Immunohistochemistry
Tumor tissues was embedded in paraffin and cut into 4 μm-thick sections. After heating in a microwave oven and blocking the antigen with 3% H2O2 solution, non-specific binding was blocked with 5% bovine serum albumin at 37°C for 30 minutes. Then the sections were incubated overnight at 4°C with the following primary antibody, RAC1 (Abcam, USA, 1:50). The signal was detected by the DAB method (Beyotime, China) and examined under a microscope (Nikon, Japan). The immunohistochemical staining score was evaluated by two pathologists. The scoring system used is as follows: no positive cells are scored as 0; no positive cells are scored as 0. The yellow, light brown and dark brown staining of positive cells are 1-3 respectively. Each experiment was repeated three times.
Statistical analysis.
All statistical analyses were performed using SPSS 19.0 (SPSS, Chicago, IL, USA). Significant differences among two groups were compared using Student’s t test. Comparisons among three or more groups were analyzed using ANOVA with post hoc Student–Newman–Keuls test. Statistical significance was considered at P < 0.05 and labeled with∗.