Cancer tissue collection
Forty-one cases of EOC tissues and 13 cases of noncancerous normal ovarian tissues were gathered from the Department of Gynecology of the First Affiliated Hospital of China Medical University during the surgical process. None of the patients had undergone chemotherapy or radiotherapy prior to surgery. Informed consent was obtained from all the patients or their families and the project was approved by the Ethics Committee of China Medical University (No:2018-132).
Cell culturing and transfection
The EOC cell lines A2780 and CAOV3 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100U/mL of penicillin/streptomycin and 10% fetal bovine serum (FBS). The OVCAR3 and HO8910 were cultured in RPMI 1640 supplemented with 100U/mL of penicillin/streptomycin and 10% FBS. All cells were cultured in 37℃ atmosphere containing 5% CO2.
RiboBio (Guangzhou, China) designed specific small interfering RNAs (siRNA) against AC022306.2 (si-AC022306.2#1, si-AC022306.2#2 and si-AC022306.2#3) and GALK2 (si-GALK2#1, si-GALK2#2) and si-GALK2#3) and their corresponding NC (siNC). The pCDNA3.1(+)-CMV-AC022306.2 plasmid and the control empty vector were designed and synthesized by Syngentech (Guangzhou, China). The miR-369-3p mimics and the negative control were purchased from Biomics. According to the instructions, Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was utilized for cell transfection, and qRT-PCR was used to analyze the transfection efficiency.
QRT-PCR
Total RNA was extracted from EOC cells and tissues using the RNAeasy™ Animal RNA Isolation Kit with Spin Column (Beyotime, China). After measuring OD260/280 with a spectrophotometer (Unico, Shanghai, China), the cDNA was reverse-transcribed using Hifair III 1st strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus, YEASEN, Shanghai, China). 2x SYBR Green qPCR Master Mix (Bimake, Shanghai, China) was used to carry out the qRT-PCR. The relative expression level was calculated by 2-ΔΔCt method with 18S or U6 as internal reference.
CCK-8 assay
After the cells were maintained in a 37°C humidified/5% carbon dioxide atmosphere for 0h, 24h, 48h, and 72h, cell proliferation was examined with Cell Counting Kit‐8 Reagent Kit. The absorbance at 450nm wavelength was detected by microplate reader.
Cell clonogenesis assay
Cells were planted in a six-well plate with a density of 500 cells per well and cultured in complete medium. On the 14th day, the medium was discarded and cells were washed with PBS. Subsequently, a 0.1% crystal violet-methanol solution was used for fixation and dyeing for 15min, and photographs were taken for observation.
Scratch healing assay
Cells were cultured in six‐well dishes in complete medium. A wound was scratched along the diameter of the well with a 200μL pipette tip. The size of the wound (w0) was measured. The cells were then incubated in the serum‐free medium for 48h. After 48h, the size of the wound (w48) was detected. The wound healing rate was measured by the way as follows: (size of 0h − size of 48h)/ size of 0h.
Trans-well assay
Twenty-four-well trans-well chambers (3422, Corning) were coated with Matrigel (Corning) in cell invasion assay, while the chambers were with no coating in cell migration assay. 25 000 cells in 100μL medium without serum were added to the upper chamber, and medium containing 10% FBS was implanted to the lower one. The cells were grown for 48h. Cotton balls were used to help gently removing the cells that did not pass through, and a 0.1% crystal violet-methanol solution was utilized to fix and stain the cells that passed through. The stained cells were counted with a microscope.
Western Blot
In short, the total protein was electrophoresed by 10% SDS-PAGE. Enhanced chemiluminescence (ECL) chromogenic reagent was utilized for probing GALK2 successively. An antibody against GALK2 was purchased from Proteintech (Wuhan, China). The anti‐GAPDH antibody was served as a loading control.
Subcutaneous tumor formation experiment
BALB/c nude mice (Vital River Laboratories, Beijing, China) were raised in a specific sterile environment. In order to detect the tumorigenic ability of AC022306.2 in vivo, 1.0x107 AC022306.2 overexpression and control OVCAR3 cells were injected into the right axillary area of 4-week-old female nude mice. With the growth of the tumor, the long and short diameters of the subcutaneous tumor were measured with vernier calipers twice a week, and the tumor volume was calculated as follows: tumor volume =1/2 x long diameter x short diameter2. Four weeks later, the tumor was peeled off and the long diameter and short diameter of the tumor were measured. Animal experiments are in accordance with the Guidelines for the Protection and Use of Laboratory Animals published by the National Institutes of Health and were supported by China Medical University Animal Care and Use Committee.
Dual luciferase reporter assay
The reporter gene vector and reporter gene point mutation vector were constructed by Shanghai genechem Co., Ltd. The vector is GV272. The luciferase reporter vectors were named mutant-type dual luciferase reporter plasmid (MUT) and wild-type dual luciferase reporter plasmid (WT), respectively. MiRNA mimics or miR-NC was co-transfected with above MUT and WT plasmids. After 48h of transfection, luciferase activity was measured by dual luciferase reporter gene detection system (Promega).
RNA immunoprecipitation (RIP) assay
The IP lysis buffer was used for cell lysis. The obtained lysates were incubated with magnetic beads conjugated with anti-CREB1 antibody (Proteintech) or IgG antibody (Proteintech) to adhere to the immunoprecipitation product. The RNA was extracted from immunoprecipitation after beads were washed. QRT-PCR was used for analysis.
Statistical analysis
With SPSS 20.0 (Chicago, IL, USA), all statistical analyses were performed. A two‐tailed Student’s t test was implemented to compare data between the two groups. The results were considered statistically significant only if P<0.05.