All patients with ovarian cancer were histologically confirmed. All patients who were diagnosed with ovarian cancer had no additional malignant, other disease, such as inflammatory, ischemic disease, wounds, or ulcers that could affect the numbers of EPCs. Paired tissue samples were harvested from 25 patients with ovarian cancer who underwent primary cytoreductive surgery between March and August 2018. The study obtained the informed consent from all the participating patients and the approval from the Ethics Committee of the Cancer Hospital, Harbin Medical University.
Culture of EPCs
EPCs culture and identification have been performed, as described in our previous protocol, So that total mononuclear cells (MNCs) were isolated from 20 ml samples of human peripheral blood from patients with ovarian cancer by density gradient centrifugation with Histopaque-1077 (density 1.077 g/ml; Sigma Aldrich, St. Louis, MO, USA). MNCs were plated in 1 ml endothelial growth medium (EGM-2; Lonza, Basel, Switzerland) on fibronectin-coated (Sigma Aldrich) 24-well plates. After 24 h, non-adherent cells were discarded and adherent cells were cultured as above protocol. Medium was replaced every 2 days, thereafter, and each colony/cluster was followed up. At 7 days after culture, colony forming cells were identified by attached spindle-shaped cells. The adherent cells were incubated with DiI-acLDL (acLDL; Molecule Probes, Leiden, The Netherlands) and then fixed in 2% paraformaldehyde and counterstained with fluorescein isothiocyanate (FITC)-labeled lectin from Ulex europaeus agglutinin (UEA-1) (Sigma Aldrich).
Ovarian cancer cells culture
The IOSE80, SKOV-3 and OVCAR-3 cell lines were established in our laboratory. IOSE80, SKOV-3 and OVCAR-3 cells were grown in RPMI-1640 medium (Sigma, Oakville, ON, Canada) supplemented with 10% fetal calf serum (Hyclone Laboratories Inc., Logan, UT, USA). Cultures were maintained at 37 ℃ in a humidified 5% CO2 atmosphere in air.
miRNA microarray and clustering analysis
miRNA expression between EPCs with ovarian cancer patients and EPCs with healthy controls is unclear, gene expression profiling was examinated using the PrimeView Human Gene Expression Array (Affymetrix), which includes 530,000 probes covering more than 36,000 transcripts and variants. Total RNA hybridization was conducted according to the manufacturer’s instructions. Three repeats were performed from each sample to guarantee consistency of RNA hybridization. All subsequent technical procedures and quality controls were performed by Shanghai Genechem Co. Ltd, China. The arrays were scanned by a GeneChip Scanner 3000 (Affyme-trix, inc, Santa Clara, CA, USA). GeneSpring GX software version 12.0 (Agilent Technologies, Palo Alto, CA, USA) was used to analyse the raw data obtained from each probe. Next, the data were normalized using the PLIER default protocols. Additionally, an unpaired t-test was applied to analyze the significantly differentially expressed genes. Hierarchical clustering analysis was used to reveal the relationship between significantly altered miRNAs in samples for each identified gene set with Euclidean distance and average linkage statistical methods.
The lentiviral vectors were purchased from Shanghai Genechem Company Ltd, China, which composed of the vectors pGCSIL-GFP which stably expressed siRNA and a marker (GFP-RFP fusion protein), and pHelper1.0 (gag/pol element) and Helper2.0 (VSVG element). A non-silencing siRNA (5’-GCCTAACTGTGTCAGAAGGAA-3’) was used as the negative control (NC). The siRNA sequences targeting miR-133a-5p gene were 5’-CGTAATTCCGATTATACGTCCATC-3’ and 5’-GTGATGCGATTATGCTCCCT-3’. The siRNA sequences targeting TRIM59 gene were 5’ -AGTATTACCCTTCATACCATCAAA-3’ and 5’-GTCATGCGCTTTTCCAGCCC-3’. The overexpression sequences targeting miR-133a-5p gene were 5’-TGTGATTACCATGATCCTCCCTTG-3’ and 5’-ATCAAGGGGTAATTCTCTCT-3’. The overexpression sequences targeting TRIM59 gene were 5’ -TATGTTAGCATGCATGCTATCAGA-3’ and 5’-GACTTCCTCTATGCGAGTCG-3’. All the EPCs were planted on a six-well plates at 5 × 104 cells per well and incubated, respectively. Appropriate volumes of lentivirus were added to the cells according to the recommendation of manufacturer, when cell fusion reached to 60%.
A mutant construct of TRIM59 3’-UTR or TRIM22 3’-UTR was obtained by introducing a mutation into the seven nucleotides (CCCGUAA) of the seed region for miR-133a-5p. The miR-133a-5p target sequence in the coding region of TRIM59 or TRIM22 was amplified by PCR and cloned into GV143 that contained a firefly luciferase reporter gene. Wild-type TRIM59/TRIM22 3’-UTR or mutant TRIM59/TRIM22 3’-UTR and the empty 3’-UTR vector were cotransfected into HEK293 cells, with Renilla luciferase vector transfection as reference. After incubation for 48 hrs, the cells were harvested and assayed for Renilla and firefly luciferase activities using the dualluciferase reporter assay system (Promega). The relative luciferase activities were calculated by normalizing to Renilla luciferase. Cells were transfected with the/an empty 3’-UTR vector as a negative control (NC).
Transwell Migration Assay
At 7 days after incubation, the cultured medium were removed and replaced with EBM-2 without any supplements cells were cultured for 12 hours for the migration assay. EPCs migration was evaluated using a transwell migration assay. Briefly, 5× 104 cells were suspended in 100 µL of EBM-2 supplemented with 0.1% BSA and placed in the upper chamber of an 8.0-mm pore size transwell (Costar, Cambridge, MA). 600 µL of the final dilution was placed in the lower chamber. After incubating for 6 hours at 37oC in 5% CO2, the cells that had not migrated were removed from the upper surface of the filters using cotton swabs and those that migrated to the lower surface of the filters were fixed in methanol and stained with Giemsa’s Stain Solution. Migration was determined by counting the cells with a microscope. Five visual fields were randomly chosen for each assay. The average count of the migrating cells in these 5 fields was taken as the migrated cells of the each group.
In vitro tube formation
In vitro tube formation assay was performed using the Matrigel basement membrane matrix (BD Biosciences). 1 ml/well Matrigel, kept on ice, was placed in 4-well culture plates. The plates were then incubated at 37°C for 30 min to allow Matrigel to solidify. About 4×104 EPCs were cultured on the preplated Matrigel and incubated for 48h. To quantitate the in vitro angiogenesis, the average tubular area (enclosed spaces) and the average of tube area per total field area in 5 serial microscopic fields were calculated. The tube area and tube length were expressed as µm2 and µm using image-analyzing software and AxioVision Version Rel 4.8 Software.
Real-time quantitative polymerase chain reaction( qPCR)
Total RNA was extracted from EPCs using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA was quantified by absorption at 260 nm. The isolated RNA was then DNase-treated and reverse-transcribed according to the manufacturer’s recommended protocols. Briefly, miRNAs were reversely transcribed using the Primescript Reverse Transcription kit, miScript syBRGreen PCR kit and miScript Primer Assays according to the manufacturer’s instructions (Qiagen, Valencia, CA. USA). Quantitative real-time PCR was performed using an ABI 7500 sequence detection system. The Cycling parameters were 2 min. at 50°C and 10 min. at 95°C, followed by a total of 40 cycles of 15 sec. at 95°C and 1 min. at 60°C. All of the reactions were performed in triplicate. The gene expression ΔΔCT values of miRNAs were calculated by normalizing U6 to an internal control.
Western blot analysis
EPCs were collected in sample buffer and then incubated in lysis buffer and protease inhibitors for 30 min. Kept on ice. Next, the supernatants were collected following centrifugation at 1.3×104×g at 4°C for 15 min. Total cell proteins were extracted using the mammalian protein extraction reagent including halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentrations were measured by BCA protein assay kit (Thermo Fisher Scientific) and equal amount of proteins were subjected to SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After being blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against VEGF, Id1, TRIM59 and Actin (Santa Cruz), respectively, and followed by incubation with the HRP-conjugated secondary antibodies. The signals were detected using an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA)
All experiments were repeated at least three times. Data were shown as means ± standard deviation (S.D.). P- values less than 0.05 were considered to be statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001). Statistical comparison between groups was performed using Student’s t-test.