Cell culture and transfection
Ovarian cancer cells (SKOV3 and CaOV3) and 293T cell were purchased from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin at 37 °C, 5% CO2. TGFβ1 was purchased from R and D systems and was used to induce EMT in SKOV3 and CaOV3 (10 ng/ml) cells for the indicated time periods.
The TGFβR1 cDNA was subcloned into pCDNA3.1 vector which was transfected into cells using lipofectamine 2000 according to the instruction. For miR-490-3p mimics or miR-490-3p inhibitor transfection, we used LipofectamineVR LTX with PlusTM Reagent (Life Technologies) to transfect them into cells. All siRNAs, miR-490-3p mimics and miR-490-3p inhibitor were synthesized by GenePharma. The sequences are as follows:
miR-490-3p mimics: (sense) 5′-CAACCUGGAGGACUCCAUGCUC-3′; (antisense) 5′-GCAUGGAGUCCUCCAGGUUGUU-3′;
miR-490-3p inhibitor: 5′-CAGCAUGGAGUCCUCCAGGUUG-3′.
Patients and samples
A cohort of 25 ovarian tumor tissues and adjacent normal ovarian tissue samples were obtained from patients aged 25–55 undergoing wedge biopsy of the ovaries or adnexectomy due to myoma or adenomyosis, between 2016.6 and 2017.5. No patients had received chemotherapy or radiotherapy prior to surgery. Consent from all patients were obtained. Ovarian cancer was validated by histological examination in all cases according to World Health Organization criteria. Ovarian cancer and normal ovarian tissue specimens excised surgically from patients were immediately snap-frozen and stored in liquid nitrogen until use. This experiment was approved by ethic committee of the 2nd Affiliated Hospital of Harbin Medical University, and the tissues were acquired with the consent of patients.
Plasmid transfection and lentivirus package
The short hairpin RNAs (shRNA CCAT1) were cloned into PLKO.1 vector. To make lentiviruses, the packaging vectors (pPAX2 and pVSVG) and PLKO. shRNAs were co-transfected into 293T cells. The supernatant was harvested at 48 h after transfection. For virus infection, the virus supernatant was added to medium at 1:5 ratio, after 24 h, 2 μg/ml puromycin was used to select the positive cells.
Wound healing assay
Migration of cells were measured by a wound healing assay in vitro. Briefly, 2 × 105 SKOV3 or CaOV3 cells were seeded onto 6-well plates, with either sh-CCAT1 or sh-NC, and incubated in appropriate complete culture medium for 16 h under normoxic conditions at 37 °C. The monolayer was scratched and incubated in medium without FBS for 24 h. The wound width was measured after 24 h. Three different locations were visualized and photographed under inverted microscope.
Invasion assay was performed using chambers with 8.0-μm pore membranes (Millipore). Ovarian cancer cells (1 × 105 cells) were resuspended in 200 µl of FBS-free medium, and then seeded into the top chamber with Matrigel-coated membrane. Next, 500 µl medium with 10% FBS was added to the bottom chamber as a chemoattractant. After 48 h of incubation, the invaded cells were fixed, stained with 0.005% crystal violet, and counted under the inverted microscope.
Luciferase reporter assays
CCAT1 or TGFβR1 mutant was generated using site-directed mutagenesis. Then, the sequence of the CCAT1 or TGFβR1 was cloned into the firefly luciferase-expressing vector pGL3-luciferase plasmid. As for luciferase assay, the SKOV3 or CaOV3 cells were seeded for triplicates in 24-well plates at the day before transfection, and co-transfected with the CCAT1 or TGFβR1 reporter vector and miR-490-3p. Then, the cells were harvested and lysed, and the luciferase activities were assayed using the Dual-Luciferase Reporter System (Promega). Three independent experiments were performed.
The cells were harvested and washed with PBS buffer, then lysed by 1 × SDS loading buffer. The lysates were boiled at 100 °C for 5 min. The samples were centrifuged at 10,000 rpm for 1 min. Around 50 μg of total proteins was loaded onto SDS-PAGE gel and resolved. After that, the proteins were transferred to PVDF membrane at 300 mA for 1.5 h. The membrane was blocked with 5% non-fat milk in 1× TBST for 1 h at room temperature, the membrane was then incubated with primary antibodies at 4 °C overnight. The following day, the membrane was washed with 1× TBST for three times, 5 min each time. The membrane was incubated with secondary antibodies at room temperature for 1 h. Finally, the membrane was incubated with ECL solution and then exposed. The following antibodies were used: anti-TGFβR1 (cell signaling technology, USA), anti-E-cadherin (cell signaling technology, USA), anti-N-cadherin (cell signaling technology, USA), anti-Claudin (cell signaling technology, USA), anti-β-actin (Proteintech, USA), anti-MMP9 (Abcam, USA), anti-GAPDH (Proteintech, USA).
We extracted the RNA using Trizol method. Cells were lysed by Trizol buffer and then add chloroform to the mixture. The sample was centrifuged at 12,000 rpm for 10 min and transferred to new EP tube, mixed with equivalent volume of isopropanol, next, the resultant was centrifuged at 12,000 rpm for 10 min. Removing the supernatant and add 75% ethanol to wash the pellet and centrifuge. Finally, discard the ethanol and dry the pellet, use 20–30 μl Rnase-free H2O to resolve the RNA.
For reverse transcription, about 1 μg of total RNA was used for reverse transcription according to manufacturer instruction (TAKARA PrimeScript Kit). The expression of miR-490-3p was quantified by TaqMan miRNA assays (Applied Biosystems, Foster City, CA, USA).
For real time PCR, we used SYBR as probe dye and detected the signal, the GAPDH and U6 were used as internal control. The following primers were used:
Each experiment was performed for three times, all values were presented as mean ± SD, comparison of two groups were performed using the two-tailed unpaired student’s t-test. One-way ANOVA was used for comparison among multiple groups and multiple comparisons were further performed using post hoc Turkey test. *P < 0.05 were considered statistically significant (*P < 0.05, **P < 0.01, and ***P < 0.001).