Clinical samples

Tumor tissues from CC patients and adjacent normal tissues (> 2 cm from the margin of the tumor) (n = 34) were collected and recruited from patients at the First Affiliated Hospital of Zhengzhou University during May 2018 to December 2019 and our protocol was approved by Research Ethics Committee of our hospital and conducted following the Declaration of Helsinki. All pathological specimens were diagnosed by two pathologists. The clinicopathologic parameters of these patients were provided in Table 1.

Table 1 Correlation analyses of cric-ABCB10 expression and clinicopathologic parameters of CC patientsCell culture

Siha, CaSki, and Hela cells were ordered from Cell Bank of Chinese Academy of Sciences (Shanghai, China). C33A, SW756 and normal human cervical epithelial cell (HUCEC) lines were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). CaSki cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS). C33A, Siha and Hela cells were grown in Minimum Essential Medium containing 10% FBS. SW756 cells were maintained in Leibovitz’s L-15 medium containing 10% FBS in a 100% air incubator at 37 °C. Normal primary cervical epithelial cells were maintained in cervical epithelial cell basal medium (ATCC) added with the contents of cervical epithelial cell growth kit (ATCC). All cells except for SW756 cells were maintained in the incubator containing 5% CO2 at 37 °C. FBS and all CC cell medium were ordered from Thermo Scientific lnc. (Waltham, MA, USA).

Plasmids and cell infection

Lentiviral vector plasmids were purchased from Ke Lei Biological Technology Co., Ltd (Shanghai, China). The lentiviral plasmids overexpressing circ-ABCB10 (pcDNA-circ-ABCB10), miR-128-3p and plasmid interfering circ-ABCB10 were constructed. These plasmids were co-transfected with auxiliary plasmids into 293 T cells to gain viruses expressing these plasmids referring to the manufacturer’s instructions. Finally, the viruses (MOI = 10) were used to infect Hela and C33A cells.

RT-qPCR assay

Total RNA was isolated from CC tumor tissues and cells by TRIzol reagent (Thermo Scientific). Next, the synthesis of first strand cDNA was conducted using M-MLV Reverse Transcriptase (Thermo Scientific). Subsequently, the quantitative PCR analysis of cDNA was conducted using the SYBR Green Master Mix (Thermo Scientific) on Applied Biosystems 7500 Real-Time PCR System (Thermo Scientific). Circ-ABCB10 or miR-128-3p levels were normalized by GAPDH or U6 snRNA, respectively. Expression patterns of circ-ABCB10 and miR-128-3p were analyzed using the 2−ΔΔCt method.

MTT assay

The infected cells were seeded into 96-well plates. At 0, 24, 48, or 72 h, 10 µl of MTT solution (5 mg/ml, Beyotime Biotechnology, Shanghai, China) was added into per well. Four hours later, cells were co-incubated with DMSO (150 μl per well). Finally, optical density (OD) values were detected at the wavelength of 490 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Transwell invasion assay

Cell invasive potential was assessed through a transwell chamber (24-well, 8-µm pore size of filter membranes) pre-coated with matrigel (Corning, Corning, NY, USA). Briefly, the upper or low chamber was added with cells (2 × 105) suspended in serum-free medium or medium containing 20% FBS, respectively. Twenty-four hours later, the invaded cells were fixed using the methanol and stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA).

Western blotting assay

The collected cells and tissues samples were prepared using RIPA lysis buffer (Beyotime Biotechnology) added with protease inhibitor (Thermo Scientific). The total content of protein in the lysates was determined using Pierce BCA Protein Assay Kit (Thermo Scientific). Protein (20 µg/lane) was separated through 10% SDS-PAGE and electro-transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocked with 5% skimmed milk, the membranes were incubated overnight at 4 °C with primary antibody against zinc finger E-box binding homeobox 1 (ZEB1) (1/500 dilution, ab203829), E-cadherin (1/1000 dilution, #3195), N-cadherin (1/1000 dilution, #4061), Vimentin (1/1000 dilution, #5741), snail (1/1000 dilution, #3895) and β-actin (1/2000 dilution, ab8227). After washed, the membranes were probed with HRP-labeled secondary antibody (1/5000 dilution, ab672 or ab6789) for 1 h at 25 °C. ZEB1/β-actin antibody and secondary antibodies were ordered from Abcam lnc. (Cambridge, UK). Other antibodies against EMT markers were obtained from Cell Signaling Technology lnc. (Danvers, MA, USA). Protein bands were developed using Pierce ECL Western Blotting Substrate (Thermo Scientific).

Cell apoptosis assay

The apoptotic capacity of cells was examined using the Annexin V-PE/7-AAD Apoptotic Detection kit (Sigma). Briefly, cells re-suspended in binding solution were co-incubated with Annexin V-PE and 7-ADD solutions for 10–30 min at 25 °C under the dark conditions. Subsequently, cell apoptotic pattern was analyzed through flow cytometry (BD Biosciences, San Jose, CA, USA).

Luciferase reporter assay

The corresponding fragment of circ-ABCB10 or ZEB1 3’UTR covering predicted miR-128-3p complementary sites were constructed into pmirGLO vector plasmids by GenePharma Co., Ltd. and generated recombinant plasmids was named as circ-ABCB10-WT or ZEB1-WT reporter. Also, circ-ABCB10-MUT and ZEB1-MUT reporters containing mutant miR-128-3p complementary sequence were also constructed. Next, luciferase reporter was introduced into Hela and C33A cells transfected with miR-Ctrl or miR-128-3p mimics. Forty-eight hours later, dual luciferase reporter assay (Promega, Madison, WI, USA) was used to detect the luciferase activities.

Pull-down assay

The pull-down assay with biotinylated RNA was performed according to the reported method [18]. Biotinylated miR-128-3p (Bio-miR-128-3p) or miR-Ctrl (Bio- miR-Ctrl) mimics were transfected into Hela and C33A cells using Lipofectamine 2000. The cells were treated with lysis buffer (Life Technologies, Carlsbad, CA, USA) and incubated with C-1 magnetic beads (Life Technologies) at 4 °C overnight. Finally, interacted RNAs were purified and detected by qRT-PCR.

In vivo experiments

The animal experiments were approved by Experimental Animal Ethics Committee of our hospital. BALB/c nude mice (female, 5 weeks-old) were ordered from Laboratory Animal Center of Henan Province (Zhengzhou, China) and randomly separated into sh-ctrl or sh-circ-ABCB10 group. Each group contained 6 mice. C33A cells with sh-ctrl or sh-circ-ABCB10 (5 × 106 / mouse) were injected into the subcutaneous tissues of mice in sh-ctrl or sh-circ-ABCB10 group, respectively. Xenograft volume was monitored every other 4 days and calculated by the formula: volume = (length × width2)/2. Tumor tissues were resected and weighed at day 35 after inoculation. MiR-128-3p level in CC xenograft tumors was examined through RT-qPCR assay. ZEB1, E-cadherin, N-cadherin, Vimentin and Snail expression patterns in CC xenograft tumors were measured by western blotting assay. Ki67 protein expression pattern in xenograft tumors was analyzed through immunohistochemistry (IHC) assay. IHC assay was carried out as previously described [19] with the Ki67 antibody (1/ 50 dilution, ab15580, Abcam).

Statistical analysis

Data were analyzed by GraphPad Prism software (version 7.0, GraphPad, San Diego, CA, USA) with the outcomes expressing as mean ± standard deviation. Student’s t-test or one-way ANOVA along with Tukey’s post-hoc test was used to test the statistical difference between/among groups. Difference was defined as statistically significant at P < 0.05.