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. 2022 Jun 10;14(11):4699-4713.
doi: 10.18632/aging.203474. Epub 2022 Jun 10.

Hsa_circ_0063804 enhances ovarian cancer cells proliferation and resistance to cisplatin by targeting miR-1276/CLU axis

Jun You  1 , Yuwen Han  1 , Haifeng Qiao  1 , Yun Han  1 , Xiaoyan Lu  1 , Yiling Lu  1 , Xiaoyu Wang  1 , Haili Kai  1 , Yanli Zheng  1
Affiliations

Hsa_circ_0063804 enhances ovarian cancer cells proliferation and resistance to cisplatin by targeting miR-1276/CLU axis

Jun You et al. Aging (Albany NY). .

Abstract

Purpose: This article researched circ_0063804 effects on ovarian cancer (OC) development and resistance to cisplatin, aiming to provide a new target for OC therapy.

Methods: A total of 108 OC patients participated in this study. The circle structure of circ_0063804 was investigated using RNase R. Circ_0063804 expression in OC cells were up-regulated or down-regulated by transfection. Cell proliferation was assessed by cell counting kit-8 assay and colony formation assay. Flow cytometry was used to detect apoptosis. OC cells resistance to cisplatin was explored through MTT assay. Luciferase reporter assay was performed. qRT-PCR and Western blot was applied to research genes expression. Xenograft tumor experiment was conducted using nude mice. Ki67 expression in xenograft tumor was detected by immunohistochemistry.

Results: Circ_0063804 expression was up-regulated in OC patients and indicated poor prognosis (P < 0.05). Circ_0063804 had a stable circle structure. Circ_0063804 enhanced proliferation, resistance to cisplatin and reduced apoptosis of OC cells (P < 0.01). miR-1276 was down-regulated in OC patients and sponged by circ_0063804. CLU was directly inhibited by miR-1276 and up-regulated in OC patients. Circ_0063804 exacerbated malignant phenotype and resistance to cisplatin of OC cells in vitro by enhancing CLU expression via sponging miR-1276 (P < 0.01). Circ_0063804 silencing inhibited OC cells growth, resistance to cisplatin and Ki67 expression in vivo (P < 0.01).

Conclusion: Circ_0063804 promoted OC cells proliferation and resistance to cisplatin by enhancing CLU expression via sponging miR-1276.

Keywords: OC; circ_0063804; drug resistance; miR-1276/CLU; proliferation.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Circ_0063804 expression in OC patients was up-regulated. (A) Circ_0063804 expression in tumor tissues of OC patients was aberrantly increased than that in normal tissues. (B) The stable circle structure of circ_0063804 was confirmed by using RNase R. (C) High circ_0063804 expression indicated low survival rate of OC patients. (D) Circ_0063804 expression in human OC cell lines was significantly higher than that in human ovarian epithelial cell line. ** P < 0.01. *** P < 0.001. OC: ovarian cancer.
Figure 2
Figure 2
Circ_0063804 silencing promoted OC cells apoptosis and weakened proliferation and resistance to cisplatin. (A) Circ_0063804 expression in OC cells was successfully regulated via transfection. (B, C) According to CCK-8 assay and colony formation assay, circ_0063804 silencing inhibited OC cells proliferation, and circ_0063804 overexpression promoted OC cells proliferation. (D) Flow cytometry revealed that circ_0063804 silencing promoted OC cells apoptosis, and circ_0063804 overexpression inhibited OC cells apoptosis. (E) MTT assay indicated that circ_0063804 silencing reduced the IC50 of OC cells, and circ_0063804 overexpression elevated the IC50 of OC cells. (F) Circ_0063804 silencing reduced OC cells resistance to cisplatin, and circ_0063804 overexpression enhanced OC cells resistance to cisplatin. ** P < 0.01. CCK-8: cell counting kit-8. MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. OC: ovarian cancer.
Figure 3
Figure 3
miR-1276 was sponged by circ_0063804 and down-regulated in OC patients. (A) The binding site of circ_0063804-WT or -Mut for miR-1276. (B) Luciferase reporter assay identified that miR-1276 was a target gene of circ_0063804. (C) Circ_0063804 silencing elevated miR-1276 expression, and circ_0063804 overexpression reduced miR-1276 expression in OC cells. (D) miR-1276 expression was down-regulated in tumor tissues of OC patients. (E) The expression of circ_0063804 and miR-1276 was negatively correlated in tumor tissues of OC patients. ** P < 0.01. OC: ovarian cancer.
Figure 4
Figure 4
CLU was directly inhibited by miR-1276 and up-regulated in OC patients. (A) The binding site of CLU-WT or -Mut for miR-1276 was shown. (B) Luciferase reporter assay showed that CLU was a target gene of miR-1276. (C) miR-1276 up-regulation decreased CLU expression, and miR-1276 down-regulation increased CLU expression. (D) circ_0063804 down-regulation reduced CLU expression, and circ_0063804 up-regulation elevated CLU expression. (E) CLU expression was dramatically increased in tumor tissues than that in normal tissues of OC patients. (F) In OC tissues, CLU expression level was positively correlated with circ_0063804 expression, and negatively correlated with miR-1276 expression. ** P < 0.01. OC: ovarian cancer.
Figure 5
Figure 5
Circ_0063804 promoted OC development in vitro by enhancing CLU expression via sponging miR-1276. (A) circ_0063804 could enhance CLU expression via sponging miR-1276. (B, C) CCK-8 assay and colony formation assay revealed that, circ_0063804 promoted OVCAR3 cells proliferation through promoting CLU expression via sponging miR-1276. (D) circ_0063804 inhibited OVCAR3 cells apoptosis by enhancing CLU expression via sponging miR-1276. (E) circ_0063804 elevated the IC50 of OVCAR3 cells by enhancing CLU expression via sponging miR-1276. (F) circ_0063804 enhanced CLU, p-gp and PARP proteins expression, and weakened c-PARP protein expression through promoting CLU expression via sponging miR-1276. ** P < 0.01. CCK-8: cell counting kit-8. OC: ovarian cancer.
Figure 6
Figure 6
Circ_0063804 silencing inhibited OC cells growth and resistance to cisplatin in vivo. (A) Circ_0063804 silencing and cisplatin decreased xenograft tumor volume. (B) The xenograft tumor in nude mice of each group was presented. (C) Circ_0063804 silencing and cisplatin decreased xenograft tumor weight. (D) Circ_0063804 silencing and cisplatin reduced Ki76 expression in xenograft tumor. (E) Circ_0063804 silencing and cisplatin reduced CLU mRNA expression in xenograft tumor. (F) Circ_0063804 silencing and cisplatin increased miR-1276 expression in xenograft tumor. * P < 0.05. ** P < 0.01. OC: ovarian cancer.
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