CircMUC16 encourages self-cleaning in ovarian cancer cells by interacting with ATG13 and miR-199a

The ovarian and serum samples were obtained from the Department of Obstetrics and Gynecology, Second Affiliated Hospital, Chongqing Medical University. The Ethics Committee of Chongqing Medical University approved this study. Three pathologists confirmed the pathological diagnosis of ovarian cancer.

First, we characterized circRNA transcripts via sequencing analysis of ribosomal RNA and linear RNA. Total RNA was extracted from three EOC tissues and four healthy ovarian tissues. Each sample was sequenced on Illumina HiSeq and yielded an average of 57.74 million reads, which were mapped to the human reference genome (GRCh38/hg38) by TopHat2. Qualified reads were sent to CIRCexplorer v2.2.3 pipeline to identify and quantify circRNAs with default parameters. A circRNA was called with the support of a minimum of two unique back-spliced reads. The whole process of library construction, sequencing and data analysis was performed at Shanghai Lifegenes Technology Co., Ltd.

SKOV3 human ovarian cancer cell line derived from the ascites from a 64 year old caucasian female with an ovarian tumor. The ES-2 cell line was established from a surgical tumor specimen taken from a 47 year old black woman. The A2780 cell line was established from tumor tissue from an untreated patient. CAOV-3 human ovarian cancer cell line derived from a 54 years old caucasian female with ovarian adenocarcinoma. Human ovarian cancer cells were cultured in RPMI 1640 medium (Sigma-Aldrich, R8758), with 10% fetal bovine serum and streptomycin. The cells were incubated under 5% CO2 at 37 °C [12]. Lentiviral shRNA interference vectors targeting circMUC16 (named LV2–1 and LV2–2) and the circMUC16-lentiviral expression vector (named LV6) were purchased from Genepharma. MIR199A mimics and inhibitors were synthesized by Genepharma. We constructed pCMV5-Beclin1 for overexpression of Beclin1. The siRNA was synthesized by Genepharma (Shanghai, China). The sequences are as follows: CircMUC16–1: 5′-TGACCTTGCTCAGGCCCGA-3′; CircMUC16–1: 5′-ACCTTGCTCAGGCCCGAGA-3′; RUNX1: 5′-GGAUUUCUGUUGUGUUUAAAU-3′; BECLIN1: 5′-GGUGUUUGAUACUGUUUGAGA-3′; ATG5: 5′-CAAUCCCAUCCAGAGUUGCUUGUGA-3′; and NC (negative control) siRNA: 5′-UUCUUCGAAGGUGUCACGUTT-3′.

We used a tandem mRFP-GFP-tagged LC3 to monitor autophagy flux based on our previous study [12]. SKOV3 cells were infected with mRFP-GFP-LC3 and LV2–1, LV2–2 or LV2-NC. A2780 cells were infected with mRFP-GFP-LC3 and LV6 or LV6-NC. mRFP-GFP-LC3 distribution in cells was analyzed by confocal microscope. Image pro plus 6.0 software was used to quantify the LC3 dots. All experiments were repeated three times [12].

Biotin-labelled circMUC16 probe was purchased from Genepharma. The specific operation protocol was in accordance with a previous study [13]. Finally, the expression of miRNA was detected using qPCR.

The specific steps of circRNA pulldown are described in our previous study [14]. Briefly, the specific proteins were identified using SDS-PAGE gel electrophoresis, silver stain and MALDI-TOF-MS [14].

Total RNA was isolated using a high-purity total RNA rapid extraction kit (Bioteke Corporation, RP1201) following the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using the iSCRIPT cDNA synthesis kit (Bio-Rad Laboratories, 4,106,228). The forward primer for circMUC16 sequence was 5′-CTGCTCAGGCCTGTGTTC-3′, and the reverse primer sequence was 5′-GGGGCCCAGCTCTTCA-3′. The real-time PCR was performed using the All-in-One qPCR mix kit. Each sample was analyzed in triplicate. The primers were purchased from Genepharma. The 2^-ΔΔCT method was used to quantify the gene expression [14].

We used ChIP assay kit (Cell Signaling Technology, 9003) to conduct a ChIP assay according to the manufacturer’s instructions. Briefly, cross-linked chromatin was sonicated into 200–1000-bp fragments. Then, the chromatin was immunoprecipitated using anti-RUNX1 antibody (ab23980, Abcam). qPCR was performed as described earlier [12].

Luciferase reporter gene assay was performed using the dual-luciferase reporter assay system (Promega Corporation, E1910) according to the manufacturer’s instructions. The wild-type or mutant reporter of circMUC16, Beclin1 and RUNX1 was constructed. Then, the reporter was co-transfected into SKOV3 cells in 24-well plates with 100 nM miR-199a or 100 nM miR-NC and Renilla plasmid using Endofectin-Plus (Gene-Copeia, Z01010A). All experiments were repeated three times [12].

The expressions of Beclin1, RUNX1, ATG14, and GAPDH proteins were detected by western blot. The primary antibodies used included rabbit monoclonal anti-Beclin1 (Abcam, ab210498); rabbit polyclonal RUNX1 (Proteintech, 25,315–1-AP); rabbit polyclonal ATG14 (Abcam, ab227849), and rabbit monoclonal anti-GAPDH (Abcam, ab181602). The band density was analyzed using a gel imaging system and compared with the internal control.

The cell proliferation was detected using Cell-Light Apollo567 in vitro kit (RiboBio, Guangzhou, China). Ovarian cancer cells were cultured in 1640 medium. EdU was applied at 20 μM. The cells were fixed with 4% paraformaldehyde and stained with Apollo 567 and Hoechst 33342 [12].

Matrigel invasion assays were used to determine the cellular invasion ability as described in our previous study [12]. A total of 5 × 104 cells were seeded into the top chamber of the transwell filter and incubated. After 48 h, the cells on the lower side of the chamber were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet (Beyotime Institute of Biotechnology, C0121), and counted using a microscope [12].

Cy3-labelled probe against circMUC16 (5’CY3-TCTTTCTCGGGCCTGAGCAAGGTCAGT- 3’CY3) and FITC-labeled probe against miR-199a were obtained from Genepharma. The FISH assay was performed following the manufacturer’s guide and instructions (F03401↗, Genepharma). Cell nuclei were stained with Hoechst 33342. Finally, images were obtained on a confocal microscope [15, 16]. All experiments were repeated three times.

The xenograft model was established as described in our previous study [12]. All animal experimental procedures were conducted with the approval of the Committee on the Use and Care of Animals (Chongqing Medical University, Chongqing, China), based on the institution’s guidelines. SKOV3 cells were infected with LV2-NC and LV2–1, and intraperitoneally injected into six-week-old BALB/c nude mice (5 × 106 cells). After five weeks, the animals were examined. Then, the number of mice with ascites were counted and weighed [12].

All statistical analyses were performed with SPSS version 17.0 software (Chicago, IL). Statistical analysis was conducted by Student’s t-test or analysis of variance (ANOVA). The chi-square test was used to compare the associations between circMUC16 expression and the clinicopathological variables of ovarian cancer samples. Data were presented as mean ± standard deviation. A two-sided p < 0.05 was considered significant.

Since circMUC16 was found to regulate autophagy, we investigated whether circMUC16-mediated autophagy was involved in cellular proliferation and migration. The expression of LC3-II was decreased in SKOV3 cells after knockdown of circMUC16. However, ectopic expression of ATG5 or Beclin1 partly reversed this effect (Fig. 3a). The LC3-II expression was elevated in A2780 cells infected with LV6-circMUC16. Consequently, when ATG5 or Beclin1 was silenced, this effect was partly reversed (Fig. 3b). The cellular proliferation and invasion of A2780 cells infected with LV6 was increased. Also, when autophagy was inhibited via silencing ATG5 or Beclin1, this effect was partly retarded (Fig. 3c-f). Hence, circMUC16-mediated autophagy promoted proliferation and invasion of ovarian cancer.