br Because most of the miRNAs contains the
Because most of the miRNAs contains the miRNA response element, it thus might function as competing endogenous RNA (ceRNA) that fi-nally releases the inhibition of miRNA on the target gene and thus upregulates the expression of the target genes. MiRanda was used to predict the AEB071 of the miRNAs as well as the miRNA targets. Finally, Cytoscape was used to visualize the network.
2.3. RNA extraction, reverse transcription (RT) and quantitative real-time PCR (qRT-PCR)
Total RNA was firstly separated by TRIzol (Invitrogen, Carlsbad, USA) and subjected to cDNA synthesis with random primers according to the instructions for the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, USA). For nuclear and cytoplasmic RNA separation, the PARIS Kit (Invitrogen, Carlsbad, USA) was used to partition cells into nuclear and cytoplasmic fractions. qRT-PCR was subsequently conducted using an Applied Biosystems ViiA™ 7 DX ma-chine (Life Technologies, Carlsbad, USA) and PrimerScript RT Master Mix (Takara, Takara Island, Japan). qRT-PCR was performed under the following protocol: initial denaturation (2 min at 50 °C, 10 min at 95 °C), followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. We analyzed the data using the 2- CT formula and β-actin as the internal control. The primers are listed in Table S2.
2.4. Reverse-transcription PCR (RT-PCR) and Sanger sequencing
Fig. 1. Expression profiles of circRNAs in normal ovarian
tissues (NOT) and epithelial ovarian cancer (EOC). A: Scatter-plot of the diﬀerentially expressed circRNA between NOT and EOC. The values on the x- and y-axes correspond to the normalized signal values of the two types of tissues (log2 scale). The red dots represent upregulated circRNAs, and the green dots indicate downregulated circRNAs with fold change > 2 and p < 0.05. B: Number of circRNA identified in NOT and EOC by RNA sequencing. Only 3414 circRNAs were observed in both groups. C: Length distribution of circRNAs identified in NOT and EOC. Most circRNAs were less than 1500 bp. D: Number of circRNA detected in circbase and our sequencing result. Almost half of the total circRNAs were newly identified circRNA in our sequencing data.
of the RNAse R. The RNA was purified using the RNeasy MinElute Cleanup Kit. Subsequently, DNase was used to remove genomic DNA according to the manufacture’s protocol. The control group was treated with nuclease-free water.
Then the RNase R and DNase treated RNA and control RNA were subjected to cDNA synthesis with random primers according to the instructions for the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, USA). Subsequently, PCR was performed and the PCR products of the circRNA were analyzed by Sanger se-quencing using the primers listed in Table S2.
2.5. siRNA transfection
SiRNA were synthesized by Ribobio (Guangzhou, China) according to the sequence described by Zheng et al (Zheng et al., 2016). The siRNA targeting the back-splicing junction of circHIPK3 was 5'-CUAC AGGUAUGGCCUCACA-3'. The siRNA was transfected using Lipofecta-mine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions. All of the experi-ments were conducted in triplicate and repeated at least three times.
2.6. Wound healing assay
The wound healing assay was performed according to the protocol described before (Memczak et al., 2013). A2780 and SKOV3 cells were seeded in a six-well plate and scratched using 200 μL pipette tips after reaching 95–100% confluency, and after injury, the cells were cultured in serum-free DMEM or RPMI-1640 medium respectively. The cells were photographed at 0 h and 24 h after injury under an EVOS XL Core digital microscope (Thermo Fisher Scientific, Waltham, USA).
2.7. Transwell assay
The migration and invasion assays were conducted using the transwell (Corning, NY, USA) assay with and without Matrigel (BD Science, Bedford, USA), respectively, according to the protocol de-scribed before (Xu et al., 2013). Approximately 3–5 × 104 cells were seeded in the upper chambers with 200 μL serum-free medium. Medium containing 20% FBS was added to the lower chambers. After 24–72 h