Hsa_circ_0061179 inhibits DNA damage in ovarian cancer cells via miR-143-3p/TIMELESS

doi:10.21203/rs.3.rs-1961746/v1

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Hsa_circ_0061179 inhibits DNA damage in ovarian cancer cells via miR-143-3p/TIMELESS

https://doi.org/10.21203/rs.3.rs-1961746/v1

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Ovarian cancer (OC) is among the most common and deadly solid malignancies in women. Despite many advances in OC research, the incidence of OC continues to rise, and its pathogenesis remains largely unknown. Herein, we elucidated the function of hsa_circ_0061179 in OC. The levels of hsa_circ_0061179, miR-143-3p, TIMELESS, and DNA damage repair-related proteins in OC or normal ovarian tissues and cells were measured using RT-qPCR and immunoblotting. The biological effects of hsa_circ_0061179 and miR-143-3p on proliferation, clone formation, DNA damage, and apoptosis of OC cells were detected by the cell counting kit-8 assay, 5-methylethyl-2’-deoxyuridine, flow cytometry, the comet assay, and immunofluorescence staining combined with the confocal microscopy. The interaction among hsa_circ_0061179, miR-143-3p, and TIMELESS was validated by the luciferase reporter assay. Mice tumor xenograft models were used to evaluate the influence of hsa_circ_0061179 on OC growth in vivo. We found that human OC biospecimens expressed higher levels of hsa_circ_0061179 and lower levels of miR-143-3p. Hsa_circ_0061179 was found to bind with miR-143-3p, which directly targets TIMELESS. Hsa_circ_0061179 knockdown or miR-143-3p overexpression suppressed the proliferation and clone formation of OC cells and increased DNA damage and apoptosis of OC cells via the miR-143-3p/TIMELESS axis. Taken together, hsa_circ_0061179 knockdown inhibited OC growth in vivo. Our findings suggest that hsa_circ_0061179 acts as the sponge of miR-143-3p to activate TIMELESS signaling and inhibits DNA damage and apoptosis in OC cells.

Ovarian cancer

hsa_circ_0061179

miR-143-3p

TIMELESS

DNA damage

Ovarian cancer (OC) is among the most common and deadly solid malignancies in females, and is associated with the highest mortality rate of all gynecological tumors [1]. Targeted therapy with molecules involved in DNA damage repair pathway is recognized as a new therapy for OC [2]. Tumor cells repair the DNA damage induced by targeted drugs and increase the stability of DNA replication forks, which is an important reason for relapse or failure of treatment [3, 4]. Therefore, a better understanding of how to accumulate DNA damage to tumor cells, reduce the stability of DNA replication forks, and enhance the sensitivity of targeted drugs to the treatment of OC is of prime importance to ensuring effective treatment without relapse.

Circular RNA (circRNA) is single-stranded, closed non-coding RNA (ncRNA) that is produced by special reverse splicing of precursor mRNA (pre-mRNA) [5]. Circular RNA lacks the 5ʹ cap and the 3ʹ poly (A) tail and forms a covalent ring, and thus has higher stablility than normal linear RNA [6]. Recent researches confirmed that circRNAs are widely present in various tissues and may have significant biological functions [7], such as transcriptional regulation, molecular metabolism, RNA modification, and protein translation [8]. Some circRNAs contain multiple binding sites of microRNA (miRNA) and act as competitive endogenous RNAs (ceRNAs). These RNAs are defined as “molecular sponges” that bind to miRNAs and prevent the binding and action of miRNA and target mRNA [9]. The most striking example is of ciRS-7 (also known as CDR1as), which functions as a sponge for miR-7 in the animal nervous system [10, 11]. Therefore, the function of circRNAs are becoming new research targets for precision medicine and translational medicine.

In this study, we compared the circRNAs expressions between OC and normal ovarian tissues and found that hsa_circ_0061179 (also known as circZBTB46) was highly expressed in OC cells. Hsa_circ_0061179 downregulation inhibited the proliferation and clone formation and induced cellular DNA damage and cell apoptosis of OC cells. Hsa_circ_0061179, as a ceRNA, competitively binds to miR-143-3p, activates the ATR/CHK1 pathway through TIMELESS, and inhibits DNA damage in OC cells. These results not only uncovered the regulatory mechanisms of the hsa_circ_0061179/mir-143-3p/TIMELESS axis on OC cell survival but also provided significant insights in the early intervention and targeted molecular therapy of OC.

2.1 Subjects

Patients with OC who received diagnosis and treatment in the Department of Gynecology of the First Affiliated Hospital of Soochow University from July 2019 to June 2021 were selected as the research participants, and none of the patients received any preoperative treatment. Patients who underwent oophorectomy for other benign tumors of the female reproductive system and were diagnosed as having normal ovaries by postoperative pathology were selected as control subjects. All tissue specimens were cryopreserved at -80°C. All patients were aware of and agreed to the use of biospecimen, and the study complied with the relevant regulations of the Ethics Committee of the First Affiliated Hospital of Soochow University (approval no. 2021027).

2.2 RNA sequencing (RNA-seq)

Tumor tissues were collected from five patients with OC. Normal ovarian tissues were collected from five benign gynecological diseases patients. Total RNA was extracted from tumor or control tissues with Trizol (Invitrogen, 15596-026). Linear RNA was degraded by adding RNase R (Lucigen, RNR07250) to total RNA. The reaction products were then extracted using the RNeasy Cleanup kit (Qiagen, 74204). Illumina sequencing library was generated using the VAHTS Total RNA seq (H/M/R) Library Prep Kit (Vazyme, NR603-01). Sequencing was performed using Illumina Novaseq6000. Sequencing services were provided by Gene Denovo Biotechnology Co.

2.3 Identification of circRNAs

CircRNAs was identified as described before [12, 13]. Briefly, unmapped reads were identified and 20mers from both ends of these reads were extracted and aligned into hg38. CircRNAs were identified by find_circ (version 1) [11]. Candidate circRNAs were selected if ≥ 2 reads were identified in more than one sample.

2.4 Cell culture and small interfering RNA (siRNA)

Human OC cells, including SKOV3 (Gaining biological, CM-H143), CAOV3 (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, SCSP-570), HO8910 (Cobioer biosciences Co. LTD, CBP61086), JHOS2 (Suer biosciences Co. LTD), OVCAR3 (Gaining biological, CM-H766), and A2780 (Cobioer biosciences Co. LTD, CBP60283), and the normal human ovarian surface epithelial cell line IOSE80 (Gaining biological, CM-H049) were cultured using RPMI-1640 (HyClone, SH30809.01) containing 10% fetal bovine serum (Biosera, 04-007-1A) and 1% penicillin–streptomycin (Beyotime, C0222). Cells were incubated in 5% CO₂ at 37°C.

SiNC, si-RNA, and miR-143-3p inhibitor and mimics were purchased from Gene pharma (Shanghai, China) and transfected into OC cells using Lipofectamine® 3000 (ThermoFisher, L3000001). Detailed sequences are as follows: si-hsa_circ_0061179#1, sense: 5ʹ- GCCGAGACUCAAAGUCUGUTT-3ʹ, si-hsa_circ_0061179#2, sense: 5ʹ-CUCAAAGUCUGUAGAAGAGTT-3ʹ, hsa-miR-143-3p mimics sense: 5ʹ- UGAGAUGAAGCACUGUAGCUC-3ʹ, and hsa-miR-143-3p inhibitor, sense: 5'-GCCGCAUCAUCAAGAACAAUA-3'.

2.5 Reverse transcription-quantitative PCR (RT-qPCR) analysis

Total RNA was extracted using TRIzol (Invitrogen). DNaseI was added to the lysis buffer to avoid DNA contamination. RT was performed by a standard kit (Invitrogen, RR047A) with 1–2µg of total RNA. qPCR was performed on CFX96 (Bio-Rad, USA) by SYBR Premix Ex Taq II (Takara, RR420A) and analyzed using the 2-DDcT2 method [14]. Primer sequences are shown in Supplemental Table 1.

2.6 Cell counting kit‑8 assay (CCK-8)

The proliferation of OC cells was detected with CCK-8 kit (New Cell & Molecular Biotech Co., Ltd., C6005), and three replicates were set for each group. The cell concentration was about 200 cells/well. The cells were stored in a 96-well plate for 1 hour, and the absorbance was measured at a wavelength of 450 nm after 0, 24, 48, 72, and 96 hours of siRNAs transfection.

2.7 5‑Ethynyl‑2'‑deoxyuridine (EdU) assay for cell proliferation

The transfected cells were resuspended in 500µl culture medium, which contained 10µM EdU (Beyotime, C0075S), and incubated 2 hat 37°C with 5% CO₂. The cells were then treated with 4% paraformaldehyde (PFA) (BeyotiMe, P0099) for 15 minutes, 0.2% Triton X-100 (Beyotime, ST677) for 30 minutes, and click reaction solution for 30 minutes (cover from light) at room temperature. Then, the nucleus was stained with Hoechst solution 10 minutes and observed by fluorescence microscope.

2.8 Clone formation assay

After one week of culture, the transfected cells were fixed with 10% methanol at room temperature for 15 minutes, incubated with 1% crystal violet (Beyotime, C0121) for 10 minutes and then imaged with an EVOSFL imaging system (Life Technologies). Colony numbers were analyzed with IMAGEJ.

2.9 Apoptosis analysis

Apoptosis was estimated by Annexin-V-FITC Apoptosis Detection Kit (SigmaAldrich, A9210). Cells were collected, washed with 1 × PBS (Solarbio, P1010), and resuspended with 500 µl of 1 × Annexin-V binding buffer, which contained 5 µl of propidium iodide and Annexin-V-FITC. After 15 minutes of incubation at 4°C (cover from light), the stained cells were detected with Beckman flow cytometer (Beckman, FC500).

2.10 RNA fluorescence in situ hybridization (FISH)

RNA FISH was performed with the Stellaris FISH RNA kit (Biosearch Technologies, R11060.4). After washing with 1 × PBS (Solarbio), cells were treated with 4% PFA (BeyotiMe) for 15 minutes followed by 70% ethanol at 4°C overnight. Cells were stained with RNA FISH probe (diluted in hybridization buffer) (Ribo, R110607) the next day overnight at 37°C in a dark place, then washed 3 times using the wash buffer. DAPI was used to stain the nuclei. Cells were observed under a fluorescence microscope (ZEISS). The commercially available Stellaris FISH probe (Cyst-labeled hsa_circ_0061179) was purchased from Biosearch Technologies (Petaluma, CA, USA).

2.11 Dual-luciferase report assay

The PmirGLO Dual-luciferase vector (Fubio Biological Technology Co., Shanghai, E1330) was used to clone the wildtype (WT) or mutated (MUT) binding sequences of hsa_circ_0061179 or TIMELESS. Transfection was the same as described aforementioned. Luciferase activity was detected with Luciferase Assay System (Promega, E1910) by Synergy H1 Multi-Mode Microplate Reader (Biotek).

2.12 RNA immunoprecipitation (RIP) assay

RIP assays were performed to detect the interaction between hsa_circ_0061179 and TIMELESS or miR-143-3p and TIMELESS with EZ-Magna RIP kit (Merck Millipore, 17–701). HO8910 and SKOV3 cells were lysed with RIP lysis buffer. The cell lysates were then incubated with Argonaute2 (Ago2) antibody-conjugated magnetic beads (Millipore, 04-642) or negative control beads (Millipore, 17–701) at 4°C overnight. The beads were then washed six times with RIA wash buffer and incubated with 10% SDS and proteinase K at 55°C for 30 minutes. Total RNA was finally purified with TRIzol (Invitrogen). The enrichment of different RNAs was measured by RT primer sequences as shown in Table S1. Enrichment analysis was the same as described in 2.5.

2.13 Comet assay

The comet assay was performed with Comet Assay Kit (Cell Biolabs, STA-351). A volume of 10µl cell suspension (2×10⁵/ml) was mixed with 100µl of low melting point agarose on a glass plate. The slide was immediately solidified at 4℃, incubated in pyrolysis buffer at 4°C for 1 hour, and then incubated in alkaline solution for 30 minutes. Next, alkaline electrophoresis was performed for 15 min at 33V. Vista Green DNA Dye (Cell Biolabs, STA-351) was used for staining the glass slides. Finally, the comet image of each sample was taken by fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and analyzed by CASP software.

2.14 Immunofluorescence staining

Cells were fixed with 4% PFA (Beyotime), incubated in 0.5% Triton X-100 (Beyotime) for 10 minutes, blocked with 1 × PBS containing 5% BSA (BioFroxx, GR025) for 1h at room temperature. Then the slides were taken out and buckled upside down on the sealing membrane covered with anti-γ-H2AX antibody (Cell Signaling Technology, 7631) overnight at 4℃. Thereafter, the slides were washed with 1 × PBS three times in a 12-well plate. Then, small round slides were taken out and placed upside down on the sealing film covered with the secondary anti-antibody (Abcam, ab228549). The washing procedure was the same as before. Nuclear staining was performed with DAPI solution for 30 min; any exposure to light was avoided during the process. While avoiding light, small round slides were taken out and placed on slides, on top of which have a drop of anti-fluorescence quenching agent (Beyotime, P0126). Imagining was taken by fluorescence microscope (ZEISS).

2.15 Western blotting

Western blotting was performed as previously described [15]. RIPA lysis buffer (Beyotime, P0013B) was used for extracting total protein. Target proteins were detected by primary antibodies, including: TIMELESS (Genetex, GTX129604), p-ATR (Genetex, GTX128145), p-CHK1 (Genetex, GTX129490), RAD51 (Genetex, GTX70230), and γ-H2AX (Genetex, GTX127340), followed by secondary antibodies (Beyotime, NoA0277 and A0286). GAPDH was used asinternal control. The proteins were then detected by the ECL kit (Shanghai Yeasen Biotechnology, 36208ES76).

2.16 Animal experiments

Four to five weeks of female athymic BALB/c nude mice (body weight, 16–18 g) were obtained from the Model Animal Research Center (Soochow university, China). Animal experiments were approved by the Ethics Committee of Soochow University (approval no. SUDA20201027A01). Total amount of 2 × 10⁶ control HO8910 cells and hsa_circ_0061179 knockdown HO8910 cells were suspended with 100µl 1 × PBS (Solarbio) and inoculated into the root of the right forelimb of female nude mice. Each group has 6 mice (control and sh-hsa_circ_0061179). Tumor growth in mice was monitored every 2 days, and tumor size was detected using calipers. Tumor tissues were weighted after thirty days of tumor cell injection and harvested for further analysis.

2.17 Immunohistochemistry (IHC)

After the specimens were fixed in formaldehyde, they were dehydrated in ethanol to make paraffin-embedded blocks. Then, the specimens were cut into 4-µM-thick tissue sections, then baked in a post oven at 60°C for 30 minutes for later use. After routine dewaxing and hydration, the tissue sections were washed with 1 × PBS (Solarbio), incubated in 0.01M citrate buffer (pH 6.0), kept under pressure for 3 minutes, and then taken out to cool naturally. To stop the activity of endogenous peroxidase, wash with 1 × PBS, add about 50µl of 1:200 diluted TIMELESS antibody (Abcam, UK, EPR5275), and incubate in a humid box at 4°C overnight. 50 µl of secondary antibody (Shanghai South Gene Technology, GK600711) was then added dropwise (30 minutes, room temperature). Thereafter, tissue slides were stained with glucose oxidase-DAB brown (Solarbio, P1010), counterstained with hematoxylin solution (Solarbio, G1080), dehydrated in stages, transparented, sealed with neutral resin (Solarbio, GB590) before observation under a microscope (Olympus CKX31).

2.18 Statistical analysis

All patients with OC were divided into hsa_circ_0061179-low or -high groups (n = 28 and 34, separately) based on its median expression. The chi-square test was used to detect the correlation between clinicopathological characteristics and hsa_circ_0061179 expression of OC patients. The statistical significance of survival was measured by log-rank test. Statistical analysis was performed by unpaired Student's t-test, one-way ANOVA test, or Mann–Whitney U test. P < 0.05 indicates statistically significant difference. GraphPad Prism 7 (IBM, USA) was used to perform these analyses.

3.1 Hsa_circ_0061179 is abnormally expressed in OC

We collected five high-grade serous ovarian adenocarcinoma tissues and five normal ovarian tissues and screened out a large number of differentially expressed circRNAs by high-throughput whole transcriptome sequencing (Fig. 1A, B). As shown in Fig. 1C, hsa_circ_0061179 was significantly highly expressed in OC tissues, suggesting the pathogenic role of hsa_circ_0061179 in OC. As suggested by CIRCBASE (www.circbase.org), hsa_circ_0061179 contains 970 bases and is resulted from the circularization of exon 2 of the ZBTB46 gene on chromosome 20 (chr20:62421173 − 6242214; Fig. 1D, E); it is thus known as circZBTB46 as well. Considering that head-to-tail splicing not only resulted from cDNA reverse splicing but also resulted from genome rearrangement, we further designed divergent and convergent primers for hsa_circ_0061179 and performed RT-PCR using the cDNA of OC HO8910 cells. As shown in Fig. 1F, hsa_circ_0061179 could only be amplified from the cDNA of OC HO8910 cells, but not gDNA of these cells (Fig. 1F). Sanger sequencing further confirmed the splice site sequence and the expected size of hsa_circ_0061179 (Fig. 1G).

3.2 Hsa_circ_0061179 promotes OC cell proliferation, clone formation, and inhibits apoptosis

We estimated the expression of hsa_circ_0061179 in OC tissues to identify its role in OC. Analyses of numerous patient samples further confirmed that OC tissues expressed a significantly higher level of Hsa_circ_0061179 than normal ovarian tissues in patients with benign tumors of the reproductive system (Fig. 2A). Further survival analysis revealed superior survival of patients who expressed low levels of hsa_circ_0061179 than of those who expressed higher levels (Fig. 2B). As shown in Table 1, hsa_circ_0061179 levels were higher in patients with OC having an advanced Federation International of Gynecology and Obstetrics (FIGO) stage or in patients with positive lymph node metastasis. Collectively, hsa_circ_0061179 expression was elevated in patients with OC. Higher hsa_circ_0061179 levels were associated with inferior survival.

Since the high level of hsa_circ_0061179 expression in OC cells and its correlation with OC patient survival, we next elucidate the function of hsa_circ_0061179 in OC cells. To find the best cell line model, we first checked the expression of hsa_circ_0061179 in different OC cells. As shown in Fig. 2C, the hsa_circ_0061179 level was significantly higher in OC cell lines than in human ovarian surface epithelial cells, with the highest expression detected in HO8910 and SKOV3 cell lines. Transfection of siRNA in HO8910 and SKOV3 cells greatly reduced the hsa_circ_0061179 level (Fig. 2D). Further EdU assays (Fig. 2E, F) and CCK-8 assays (Fig. 2G) showed that hsa_circ_0061179 knockdown reduced the proliferation of both OC cell lines. Consistent with these findings, hsa_circ_0061179 knockdown significantly reduced colony formation as well (Fig. 2H, I). Finally, hsa_circ_0061179 knockdown greatly increased the population of apoptotic cells (Fig. 2J, K). Altogether, these results suggest that hsa_circ_0061179 knockdown inhibits the proliferation and colony formation, and triggers apoptosis of OC cells.

3.3 Hsa_circ_0061179 directly binds to miR-143-3p in OC cells

Since the lack of 5’ cap and a 3’ poly(A) tail, circle RNA is more resistant to RNase R than linear RNA[6]. This also applies to hsa_circ_0061179, which has a circular structure unlike linear ZBTB46 (Fig. 3A). Further analysis of cytoplasmic and nuclear RNA revealed the cytoplasmic localization of hsa_circ_0061179 in HO8910 and SKOV3 cells (Fig. 3B), and this was also confirmed by FISH (Fig. 3C). We speculate that hsa_circ_0061179 serves as a miRNA sponge. Hsa_circ_0061179 sequence analysis by Starbase [16] revealed that hsa_circ_0061179 has a complementary binding site of miR-143-3p (Fig. 3D). In addition, the relative expression level of miR-143-3p was significantly decreased in OC cell lines (Fig. 3E) and increased after hsa_circ_0061179 knockdown (Fig. 3F). To verify the regulatory relationship between miR-143-3p and hsa_circ_0061179, we performed a luciferase reporter assay. The miR-143-3p mimics repressed the activity of WT-hsa_circ_0061179 while having no influence on that of MUT-hsa_circ_0061179 with the predicted binding site mutation (Fig. 3G). At last, the Ago2-RIP assay indicated that significantly more miR-143-3p was pulled down by Ago2-specific antibodies than by IgG in OC cells (Fig. 3H). Collectively, we demonstrated that hsa_circ_0061179 directly binds to miR-143-3p in OC cells.

3.4 Hsa_circ_0061179 promotes cancer cell proliferation and clone formation and inhibits DNA damage and apoptosis via miR-143-3p

Subsequently, we explored how hsa_circ_0061179 and miR-143-3p functionally interact with each other in OC. Through EdU detection, we found that downregulating hsa_circ_0061179 reduced OC cell proliferation (Fig. 4A, B). Similar results were detected using the CCK-8 assay as well (Fig. 4C). MiR-143-3p inhibitor rescued the cell proliferation caused by hsa_circ_0061179 knockdown, indicating the regulatory function of hsa_circ_0061179 on cell proliferation is achieved through repressing miR-143-3p. In addition, the suppression of colony formation by hsa_circ_0061179 knockdown was also rescued by miR-143-3p inhibitor (Fig. 4D, E). Furthermore, miR-143-3p inhibitor decreased the percentage of apoptosis induced by hsa_circ_0061179 knockdown (Fig. 4F, G). Similarly, using comet assays, we found that the DNA damage in cancer cells induced by hsa_circ_0061179 knockdown was restored by miR-143-3p inhibitor (Fig. 4H, I). γ-H2AX is a DNA breakpoint marker protein. The number of γ-H2AX foci reflects DNA damage [17]. As shown in Fig. 4J and 4K, the repression of hsa_circ_0061179 increased the number of γ-H2AX foci in OC cells, and miR-143-3p inhibitor rescued the hsa_circ_0061179 knockdown-induced DNA damage in cancer cells (Fig. 4J, K). Taken together, our findings suggest that hsa_circ_0061179 inhibits DNA damage and apoptosis, and increased the proliferation and clone formation of OC cells by targeting miR-143-3p.

3.5 MiR-143-3p affects the ATR/CHK1 signaling by targeting TIMELESS

MiRNA binds to the 3ʹUTR of the target mRNA and represses the expression of its target gene [18, 19]. Using Starbase [16], we identified a miR-143-3p binding site on TIMELESS (Fig. 5A). Further luciferase reporter assay verified that miR-143-3p mimics strongly repressed the activity of WT-TIMELESS while having no influence on that of MUT-TIMELESS (with mutations on miR-143-3P binding sites) (Fig. 5B). Moreover, miR-143-3p strongly repressed TIMELESS protein level, as detected by western blotting (Fig. 5C). TIMELESS is reportedly related to ATR/CHK1 signal transduction [20]. We also verified that the miR-143-3p overexpression strongly repressed the levels of p-ATR, p-CHK1, and RAD51 in cancer cells and activated the expression of γ-H2AX (Fig. 5C, D), indicating that miR-143-3p regulates the ATR/CHK1 signaling pathway through TIMELESS. Since hsa_circ_0061179 repressed miR-143-3p expression, we further detected the influence of hsa_circ_0061179 on the expression of these proteins. Hsa_circ_0061179 knockdown repressed TIMELESS, p-ATR, p-CHK1, and RAD51 expression, and increased γ-H2AX expression, which was rescued by the inhibitor of miR-143-3p (Fig. 5E, F). Taken together, we found that the miR-143-3p overexpression decreased the level of TIMELESS and the phosphorylation levels of ATR and CHK1, indicating that it affects the ATR/CHKI signal transduction by targeting TIMELESS.

3.6 MiR-143-3p represses the proliferation and clone formation, and induces DNA damage and apoptosis through TIMELESS in OC cells

Next, we sought to understand the miR-143-3p/TIMELESS axis function in OC. EdU detection showed that miR-143-3p mimics reduced the proliferation OC cells (Fig. 6A, B), and this was also further confirmed by CCK-8 assays (Fig. 6C). Conversely, the co-expression of TIMELESS increased the proliferation of OC cells. Upregulated miR-143-3p reduced the colony number of OC cells (Fig. 6D, E). In addition, TIMELESS overexpression together with miR-143-3p repressed the percentage of apoptosis induced by miR-143-3p mimics (Fig. 6F, G). Similarly, using comet assays, we found that DNA damage in OC cells triggered by miR-143-3p mimics was diminished by co-overexpression of TIMELESS (Fig. 6H, I). Furthermore, miR-143-3p mimics increased γ-H2AX foci number in OC cells, and TIMELESS co-overexpression reversed hsa_circ_0061179 knockdown-induced DNA damage in cancer cells (Fig. 6J, K). These results indicate that the ectopic expression of miR-143-3p inhibits the proliferation and clone formation, and induces DNA damage and apoptosis of OC cells through TIMELESS.

3.7 Hsa_circ_0061179 knockdown has tumor suppressor effect in vivo

To validate the influence of hsa_circ_0061179 on OC cell survival in vivo, we inoculated sh-hsa_circ_0061179 or sh-NC transduced HO8910 cells into the root of the right forelimb of 4–5 weeks old female nude mice and monitored the tumor size every 2 days. As shown in Fig. 7A-B, hsa_circ_0061179 knockdown significantly repressed the tumor growth. Furthermore, sh-hsa_circ_0061179 reduced the expression level of TIMELESS, p-ATR, p-CHK1, and RAD51, and increased the expression level of γ-H2AX (Fig. 7C, D). TIMELESS expression was significantly repressed after hsa_circ_0061179 knockdown, as validated by immunohistochemistry (Fig. 7E). These results indicate that hsa_circ_0061179 promotes the growth of nest carcinomas in vivo.

Approximately 314,000 new cases of OC are reported every year globally. The early diagnosis of OC is difficult because of its deep location in the pelvis, which requires advanced imaging techniques to visualize, and the atypical or asymptomatic symptoms. Thus most of the patients (70%) were diagnosed in the advanced stage. About 75% of OC patients develop recurrent OC after complete remission after initial treatment, and about 207,000 patients with OC die of this disease every year [1], making it as the top cause of death in all types of gynecological tumors [21, 22]. Therapies targeting molecules involved in DNA damage repair pathways are currently accepted therapies for OC [2]. Resistance to targeted drugs remains an obstacle in clinical practice despite some progress in this regard [2, 23]. Some patients are not sensitive to drugs targeting DNA damage repair pathways, and many patients become resistant to long-term oral targeted drugs [3]. The reasons for drug resistance in OC remain poorly understood. Our current study characterized the function of the hsa_circ_0061179/miR-143-3p/TIMELESS axis in OC. We found that hsa_circ_0061179 was significantly upregulated, whereas miR-143-3p was repressed in OC. High hsa_circ_0061179 expression was associated with inferior prognosis in OC patients. Hsa_circ_0061179 promoted cell proliferation and clone formation, and at the same time, inhibited DNA damage and apoptosis by targeting the miR-143-3p/TIMELESS signaling pathway in OC cells. Downregulation of hsa_circ_0061179 inhibited the growth of OC cells in vivo. These results provide strong evidence that hsa_circ_0061179 activates TIMELESS signal transduction through miR-143-3p to inhibit DNA damage and promote OC growth (Fig. 8).

CircRNA plays essential roles in the initiation and progression of various tumors [7, 24], including OC [25], and functions as tumor suppressor [26]. In line with its role in other solid tumors, circRNA also regulates gene expression through competitive endogenous RNA or miRNA sponge in OC and regulates genes that participate in important pathways, such as autophagy [27] and tumor progression [28]. CircRNA regulates transcription or slicing by interacting with RNA-binding proteins [29, 30]. CircRNAs also reportedly influence the diagnosis and prognosis of certain types of tumors. CircEPSTI1 is a potential therapeutic target for OC because it regulates OC progression [31]. CircRNA expression levels are related with the prognosis of different types of cancers in either positive or negative ways [32] [33]. In this study, we screened the abnormally expressed circle RNAs in OC clinical tissue specimens by RNA sequencing. Consequently, hsa_circ_0061179 expression was increased in OC, and higher hsa_circ_0061179 level was related with low survival rates. In addition, hsa_circ_0061179 knockdown greatly repressed the proliferation and colony formation of OC cells and induced cellular DNA damage and apoptosis, thus reducing the tumor growth rate in vivo. These results indicate the role of hsa_circ_0061179 in the DNA damage repair pathway in OC cells. Given the profound effect of hsa_circ_0061179 in OC, further study of this circle RNA in different types of cancers will be interesting. Although miR-143-3p is the downstream target of hsa_circ_0061179, whether hsa_circ_0061179 interacts with other miRNAs still remains to be clarified.

MiRNAs are associated with cancer occurrence and development, including OC. For example, OC expressed high level of miR-378, which is a marker of anti-angiogenic therapy response [34]. MiR-330-3p is a key determiner of mesenchymal properties of OC and may indicate poor prognosis in OC patients [35]. Li et al. [36] reported that miR-30b-3p targets CTHRC1 and influences epithelial-to-mesenchymal transition. MiR-143-3p is located at the 33rd position of chromosome 5 in the human genome. It is reported to be abnormally expressed in various malignant tumors, e.g., lung cancer [37], chondrosarcoma [38], and pancreatic cancer [39]. MiR-143-3p is also involved in several malignant tumor-related events, e.g., proliferation, invasion, and migration, and has been shown to have antitumor activity. Haijuan Shi et al. [40] verified that miR-143-3p repressed the OC cell proliferation. Although miR-143-3p inhibits the initiation and development of OC, the underlying upstream and downstream regulatory mechanisms remain unclear. We found that miR-143-3p is lower expressed in OC, and thus might be a tumor suppressor. MiR-143-3p upregulation inhibited the proliferation and colony formation and induced cellular DNA damage and apoptosis of OC cells. Given the important role of miR-143-3p in various cancers, as described by previous studies and the current study, other upstream regulator of miR-143-3p besides hsa_circ_0061179 may also be involved. On the other hand, other targets of miR-143-3p have been reported in addition to TIMELESS, including CDK1 [41], VASH1 [37], and FGF1 [42]. It is unclear whether these targets are also involved in OC development, which needs to be verified in the future.

The TIMELESS gene is located in chromosome 12q13.3 and is highly conserved in evolution. TIMELESS is mainly involved in biological clock regulation and maintaining the stability of DNA replication fork in mammals [43, 44]. Studies have shown that TIMELESS and PARP-1 form a complex and play a role in DNA damage repair [45], and can also promote DNA repair, especially homologous recombination repair [46]. Julien N Bianco et al. [47] found that overexpressed CLASPIN and TIMELESS improved replication stress tolerance in cancer cells by protecting the replication fork. In this research, we demonstrated that miR-143-3p directly binds with the TIMELESS mRNA, and hsa_circ_0061179/miR-143-3p regulates OC cell DNA damage through TIMELESS signaling, which is consistent with previous researches reporting that the upregulation of TIMELESS suppresses DNA damage and tumor progression in OC cells [48].

In conclusion, by combining multilayers of experiments, we uncovered the regulation mechanism of hsa_circ_0061179 on DNA damage and apoptosis of OC cells through the miR-143-3p/TIMELESS axis. The importance of the hsa_circ_0061179/miR-143-3p/TIMELESS axis suggests its therapeutic target potential in the future.

Acknowledgments

This work was supported by the Project of Suzhou Minsheng Science and Technology (No. SYSD2019204) and the Project of Suzhou Science and Technology Development Plan (No. SLJ202006).

Author contributions

YW and YZ performed the experiment and wrote the paper. XS and JZ analyzed the data. JZ and FS prepared the manuscript. YC and JW designed the study, and they have seen and can confirm the authenticity of the raw data. All authors have read and approved the final manuscript.

Ethics approval

The procedures for the collection and use of tissues were performed in accordance with the guidelines of the Helsinki Declaration of 2013. All patients were fully informed of the study before seeking their consent, which all participants gladly provided; the study has been approved by the ethics committee of the First Hospital Affiliated Soochow University (approval no. 2021027, 10 May 2019). All animal experiments have been approved by the Animal Care and Use Committee of the First Hospital Affiliated Soochow University (approval no. SUDA20201027A01, 27 October 2020).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Table I Correlation of hsa_circ_0061179 expression with clinicopathological parameters in ovarian cancer patients.

Clinical parameters	N(62)	hsa_circ_0061179 expression (%)		P-value
Clinical parameters	N(62)	Low	High	P-value
Age (years)				0.058
≤50	43	16 (37.21)	27 62.79)
>50	19	12 (63.16)	7 (36.84)
FIGO staging				0.026*
Ⅰ-Ⅱ	28	17 (60.71)	11 (39.29)
Ⅲ-Ⅳ	34	11 (32.35)	23 (67.65)
Pathological type				0.374
Serous	37	15(40.54)	22(59.46)
Others	25	13(52.00)	12(48.00)
Tumor size				0.284
≤5cm	29	11(37.93)	18(62.07)
＞5cm	33	17(51.52)	16 (48.48)
Tumor differentiation				0.105
Low	42	16 (38.10)	26 (61.90)
Medium-high	20	12(60.00)	8 (40.00)
Lymph node metastasis				0.001*
Yes	32	7(21.88)	25(78.12)
No	30	21 (70.00)	9(30.00)

FIGO: Federation International of Gynecology and Obstetrics. * : P<0.05.

No competing interests reported.

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Hsa_circ_0061179 inhibits DNA damage in ovarian cancer cells via miR-143-3p/TIMELESS

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Subjects

2.2 RNA sequencing (RNA-seq)

2.3 Identification of circRNAs

2.4 Cell culture and small interfering RNA (siRNA)

2.5 Reverse transcription-quantitative PCR (RT-qPCR) analysis

2.6 Cell counting kit‑8 assay (CCK-8)

2.7 5‑Ethynyl‑2'‑deoxyuridine (EdU) assay for cell proliferation

2.8 Clone formation assay

2.9 Apoptosis analysis

2.10 RNA fluorescence in situ hybridization (FISH)

3 Results

3.1 Hsa_circ_0061179 is abnormally expressed in OC

3.2 Hsa_circ_0061179 promotes OC cell proliferation, clone formation, and inhibits apoptosis

3.3 Hsa_circ_0061179 directly binds to miR-143-3p in OC cells

3.5 MiR-143-3p affects the ATR/CHK1 signaling by targeting TIMELESS

3.7 Hsa_circ_0061179 knockdown has tumor suppressor effect in vivo

4 Discussion

5. Conclusions

Declarations

References

Tables

Additional Declarations

Supplementary Files

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