Circular RNA HIPK3 plays a carcinogenic role in cervical cancer progression via regulating miR-485-3p/FGF2 axis

Tissue samples

The tumor tissues and matched non-tumor tissues of 70 patients with CC in Linyi Cancer Hospital from December 2016 to August 2019 were obtained. All patients with CC did not receive chemotherapy or radiation before surgery. After the tissues were removed during surgery, all samples were immediately stored at −80°C for further analysis.

Cell culture

The China Center for Type Culture Collection (Wuhan, China) was the provider of human CC cell lines (SiHa and HeLa). CC cells were cultured in RPMI-1640 medium (HyClone, Logan, Utah, USA) containing 10% inactivated fetal bovine serum (FBS; HyClone), 100 U/mL penicillin and 0.1 mg/mL streptomycin (HyClone) at 37℃ in 5% CO₂ and saturated humidity, and passaged at the interval of every 2–3 days. The cells in the logarithmic growth phase were harvested for the following experiments.

Quantitative real-time PCR assay

Total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen, Shanghai, China). RNA was reversely transcribed into complementary DNA using PrimeScript RT Reagent kit (Invitrogen) in accordance with manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) assay was performed using the Bio-Rad CFX96 qPCR system and SYBR Green Premix Ex Taq II (TaKaRa, Dalian, China) in compliance with the manufacturer’s protocol. GAPDH and U6 were used as the internal reference of the expressions of circHIPK3 and miR-485-3 p, respectively, and the quantification was performed by the 2^−ΔΔCt method. The sequences of specific primers were as follows:

circHIPK3-F: 5'-CTACAGATCCGACCAGGAGTTC-3';

circHIPK3-R: 5'-TGTGAACCAGCCACACTCTCAG-3';

miR-485-3 p-F: 5'-GCCGAGGUCAUACACGGCUCU-3';

miR-485-3 p-R: 5'-CTCAACTGGTGTCGTGGA-3';

GAPDH-F: 5'-GGAATCCACTGGCGTCTTCA-3';

GAPDH-R: 5'-GGTTCACGCCCATCACAAAC-3';

U6-F: 5′-GCTTCGGCAGCACATATACTAA-3′;

U6-R: 5'-AACGCTTCACGAATTTGCGT-3′.

Cell transfection

The circRNA overexpression vector pLCDH-circRNA was purchased from Guangzhou Geneseed Biotech, and the circHIPK3 sequence was obtained from the circBank database. Then circHIPK3 sequence fragment was inserted into pLCDH-circRNA. Small interfering RNA (siRNA) targeting circHIPK3, miR-485-3 p mimics and miR-485-3 p inhibitors were constructed by GenePharma (Shanghai, China). Cells were transfected with pLCDH-circHIPK3, siRNAs, miRNA mimics and inhibitors with Lipofectamine 3000 (Invitrogen; ThermoFisher Scientific) according to the supplier’s instructions. Finally, the transfection efficiency was detected by qRT-PCR assay.

Cell proliferation assay

Cells in the logarithmic growth phase were trypsinized with 0.25% trypsin to prepare single-cell suspension. Cells were counted, inoculated in a 96-well plate at the density of 2000 cells/well and cultured for 12, 24, 48, 72 and 96 hours, respectively. On each day, after 10 μL/well cell counting kit-8 (Dojindo, Kumamoto, Japan) was added, the cells were incubated for 2 hours. After that, the absorbance of each well was detected at the wavelength of 450 nm. Ninety-six hours later, the proliferation curve was plotted.

BrdU staining

BrdU assay was also used to detect CC cell proliferation. Cells in each group were inoculated into 24-well plates (with a cover glass) and cultured at a density of 2.5×10⁵ cells/well for 24 hours. Then BrdU kit (RiboBio, Guangzhou, China; final concentration: 50 μmol/L) was loaded and incubated at 37°C for 2 hours. After the medium was discarded and the slide was gently washed three times with phosphate-buffered saline (PBS), and the cells were fixed with 4% paraformaldehyde for 30 min. Next, anti-BrdU antibody (Beyotime, Shanghai, China) was added, with which the cells were incubated in the dark for 30 min at room temperature. After that, DAPI staining solution was added and incubated for 30 min, and then the cells were gently washed three times with PBS. Eventually, the images were obtained under fluorescence microscope (Olympus, Tokyo, Japan).

Cell migration and invasion assay

The migration and invasion of CC cells were determined by Transwell assay. To be specific, in the migration assay, 2×10⁴ cells resuspended in 200 µL serum-free medium were added into upper compartment of the Transwell Chambers (Corning, Beijing, China) and 600 µL medium containing 20% FBS was added to the lower upper compartment. After being cultured for 24 hours, the cells in the upper compartment were removed, and cells passing through the membrane were fixed in 4% paraformaldehyde, followed by being stained with 0.1% crystal violet, washed by tapped water, photographed and counted. In the invasion assay, Transwell membrane needed to be coated with Matrigel before the experiments, and in the migration assay, Matrigel was not used.

Dual-luciferase activity assay

Dual-luciferase activity assay was used to verify the target relationship between circHIPK3 and miR-485-3 p, and miR-485-3 p and the 3’UTR of FGF2. HEK-293T cells were inoculated to 24-well plates (5×10⁵ cells/well). Wild-type (WT) or mutant-type (MUT) circHIPK3 sequence or FGF2 3’UTR sequence was subcloned to pGL3 basic vector (Promega, Madison, Wisconsin, USA) and then the reporter vectors were transfected into cells. MiR-320 mimics or negative controls were co-transfected into the cells, respectively. Forty-eight hours later, luciferase activity was determined by using the dual-luciferase reporter system (Promega) in line with the manufacturer’s instructions.

Western blot analysis

After the cells were rinsed three times with cold PBS, and RIPA lysate was loaded, with which the cells were sonicated, and the protein concentration was quantified by Bradford assay. The same amount of protein from each group was separated by SDS-PAGE and then the protein on the gel was transferred to PVDF membrane (Beyotime). After that, the membrane was blocked with defatted milk at room temperature for 1 hour, and the primary antibody anti-FGF2 (ab92337, Abcam, 1:1000) and anti-GAPDH (ab181602, Abcam, 1:3000) were loaded and incubated at 4°C overnight. Then the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (ab205718) (1:2000; Abcam, UK) was added and incubated for 1 hour at room temperature. After that, the membrane was washed three times with TBS+Tween (TBST) and each time lasted for 5 min. Then, super-sensitive enhanced chemiluminescence substrate (Hubei Biossci Biotechnology, Wuhan, China) was added on the membrane to develop the bands employing chemiluminescence method.

Statistical analysis

Statistical analysis was conducted using SPSS V.13.0 statistical software (SPSS, Chicago, Illinois, USA). The difference between the two groups was compared by student’s t-test. P value <0.05 was regarded as a significant difference.