Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

CircRNA_101505 sensitizes hepatocellular carcinoma cells to cisplatin by sponging miR-103 and promotes oxidored-nitro domain-containing protein 1 expression

CircRNA_101505 sensitizes hepatocellular carcinoma cells to cisplatin by sponging miR-103 and... Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and a leading cause of cancer-related deaths worldwide. Emerging studies have shown that circular RNAs (circRNAs) are differentially expressed in HCC and play an important role in HCC pathogenesis and metastasis. However, the mechanism of circRNA in the chemoresistance of HCC remains unclear. In this study, we aimed to investigate the role of circRNA in cisplatin resistance of HCC. We identified a novel circRNA circRNA_101505 that was decreased in cisplatin-resistant HCC tissues and cell lines, and associated with a poor survival outcome. Gain-of-function investigations showed that overexpression of circRNA_101505 suppressed cancer cell growth in vivo and in vitro, and enhanced cisplatin toxicity in HCC cells. Mechanistic studies found that circRNA_101505 could sensitize HCC cells to cisplatin by sponging miR- 103, and thereby promoting oxidored-nitro domain-containing protein 1 (NOR1) expression. In conclusion, the significant inhibitory effects indicate circRNA_101505 to be a potential therapeutic target for HCC treatment. Our findings provide significant evidence to further elucidate the therapeutic use of circRNA in HCC. Introduction CircRNAs are a tissue-specific class of noncoding RNAs Hepatocellular carcinoma (HCC) is one of the most molecules. They are characterized by a covalently closed common malignant tumors and a leading cause of cancer- continuous loop without poly(A) tail . Recently, thou- related deaths worldwide . Owing to the high frequency of sands of endogenous circRNAs have been discovered. tumor metastasis, recurrence, and drug resistance, the CircRNAs mediate gene expression by sponging micro- survival rate of patients with HCC is low. Additionally, the RNAs or interacting with other molecules and then effective therapies for advanced HCC patients are lim- inhibit their function . For example, circRNA, circ- ited . Thus, identifying novel targets for HCC therapy is ZNF652 promotes migratory and invasive capabilities of urgently required. Emerging studies have found that cir- HCC cells by sponging miR-203 and miR-502-5p and cular RNAs (circRNAs) are differentially expressed in enhancing epithelial-mesenchymal transition (EMT) . HCC and play an important role in HCC pathogenesis CircRNA circSETD3 was significantly downregulated in and metastasis . HCC tissues and cell lines and associated with unfavor- able prognosis of HCC patients . Matboli et al showed that the combination of hsa_circ_00156, hsa_circ _00224 and hsa_circ _00520 acted as biomarkers with higher Correspondence: Fengxia Liu (592111443@qq.com)or sensitivities and specificities than alpha-fetoprotein in Rong Gui (aguirong@163.com) HCC . Multidrug resistance occurs frequently in HCC Department of Blood Transfusion, the Third Xiangya Hospital of Central South during the long-term chemotherapy, leading to cancer University, Tongzipo Road 138, 410013 Changsha, China Edited by I. D'Agnano © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Luo et al. Cell Death Discovery (2019) 5:121 Page 2 of 9 relapse. An increasing number of studies have highlighted tumor tissues that expressing circRNA_101505 compared the key roles of ncRNAs, including miRNAs, long with control (Fig. 2g). ncRNAs (lncRNAs) in chemoresistance of HCC . How- ever, the mechanism of circRNA in chemoresistance of CircRNA_101505 sponges miR-103 in HCC HCC remains unclear. We tested whether circRNA_101505 binds to miRNAs In this study, we aimed to investigate the role of cir- in HCC cells. The bioinformatics tool predicted two cRNA in cisplatin resistance of HCC. We identified a binding sites on circRNA_101505 for miR-103 (Fig. 3a). novel circRNA circRNA_101505, that was decreased in Further luciferase assay confirmed that circRNA_101505 cisplatin-resistant HCC tissues and cell lines. Gain-of- targeted to miR-103 (Fig. 3b), suggesting that cir- function investigations showed that circRNA_101505 cRNA_101505 may function as a sponge to miR-103. In overexpression suppressed cancer cell growth in vivo and addition, RIP assay revealed that miR-103 directly inter- in vitro. Subsequent studies displayed that cir- acted with circRNA_101505 (Fig. 3c). To investigate the cRNA_101505 could sensitize HCC cells to cisplatin by downstream mechanism by which circRNA_101505 targeting the miR-103/oxidored-nitro domain-containing exerted its functions in HCC cells, the mimic of miR-103 protein 1 (NOR1) signaling axis. was co-transfected with circRNA_101505. The results showed that miR-103 mimic could significantly reversed Results circRNA_101505-mediated induction of apoptosis Downregulation of CircRNA_101505 is associated with (Fig. 3d, e) and promotion of cisplatin toxicity in Hep3B-R cisplatin resistance in HCC and Huh7-R cells (Fig. 3f). To investigate the role of circRNAs in cisplatin-resistant HCC, we performed circRNAs array to identify the dif- MiR-103 confers cisplatin resistance in HCC cells by ferentially expressed circRNAs. Several circRNAs were targeting NOR1 differentially expressed in cisplatin-resistant and -sensi- We further investigated the role and downstream tive HCC (Fig. 1a). qPCR results further confirmed that mechanism that miR-103 affects cisplatin resistance in circRNA_101505 was significantly decreased in HCC tis- HCC. NOR1 was a predictive target of miR-103 (Fig. 4a). sues compared with adjacent tissues, and its expression MiR-103 wild-type and mutant sequences were con- was lower in cisplatin-resistant HCC tissues than in the structed into luciferase reporter gene and co-transfected cisplatin-sensitive tissues (Fig. 1b). We also found that with NOR1, and luciferase assay confirmed that miR-103 circRNA_101505 was significantly decreased in cisplatin- targeted to NOR1 (Fig. 4a, b), and negatively regulated resistant HCC cell lines (Hep3B-R and Huh7-R) com- NOR1 expression (Fig. 4c). We expressed NOR1 in pared with the parental HCC cell lines (Fig. 1c). In addi- Hep3B-R and Huh7-R cells (Fig. 4d), and found that tion, further survival analyses revealed that the HCC NOR1 could significantly reversed miR-103-mediated patients with low circRNA_101505 level had shorter inhibition of apoptosis and suppression of cisplatin toxi- overall survival than the patients had high cir- city in Hep3B-R and Huh7-R cells (Fig. 4e, f). In addition, cRNA_101505 level (p = 0.0005, Fig. 1d). These results we also found that the levels of miR-103 were significantly suggest that circRNA_101505 is closely associated with increased and NOR1 was decreased in HCC tissues cisplatin resistance in HCC. compared with adjacent tissues (Fig. 5a), and miR-103 levels were higher but NOR1 levels were lower in Overexpression of circRNA_101505 sensitizes HCC cells to cisplatin-resistant HCC tissues than in the cisplatin- cisplatin sensitive tissues (Fig. 5a). Pearson analysis showed that To investigate the role of circRNA_101505 in cisplatin circRNA_101505 were negatively correlated with miR-103 resistance in HCC, we overexpressed circRNA_101505 in (Fig. 5b) and positively correlated with NOR1 (Fig. 5c), Hep3B-R and Huh7-R cells (Fig. 2a). We found that and miR-103 was negatively correlated with NOR1 overexpression of circRNA_101505 significantly inhibited (Fig. 5d). These results demonstrate that cir- cells proliferation (Fig. 2b) and induced apoptosis (Fig. 2c) cRNA_101505 sensitizes HCC cells to cisplatin through in Hep3B-R and Huh7-R cells compared with negative miR-103/NOR1 axis. control. In addition, circRNA_101505 upregulation sen- sitizes Hep3B-R and Huh7-R cells to cisplatin (Fig. 2d, e). Discussion Furthermore, the tumor suppressive effects of cir- In this study, we found that circRNA_101505 down- cRNA_101505 upregulation were also confirmed in vivo. regulation is associated with a poor survival outcome and Our results showed that the tumor volumes in nude mice cisplatin resistance in HCC. Functional investigations injected with Hep3B-R cells expressing circRNA_101505 revealed that circRNA_101505 overexpression inhibited were smaller than in the control nude mice (Fig. 2f), and HCC cell growth and sensitized HCC cells to cisplatin. the proliferative marker ki67 expression was decreased in Further experiments demonstrated that circRNA_101505 Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 3 of 9 Fig. 1 circRNAs expression in HCC. a The heatmap of deferentially expressed circRNAs in HCC. b QPCR was performed to confirm the expression of circRNAs in cisplatin-resistant and cisplatin-sensitive HCC tissues. c QPCR was performed to confirm the expression of circRNAs in cisplatin-resistant and cisplatin-sensitive HCC cell lines. d Kaplan–Meier curves of overall survival in HCC patients with low or high circRNA_101505 expression. *p < 0.05 exhibited tumor suppressive effects by sponging miR-103, decreasing ABCB1 expression . The expression of cir- leading to NOR1 upregulation. cAKT3 was upregulated in cisplatin-resistant gastric Cisplatin is a small-molecule platinum compound. cancer tissues and cells compared with cisplatin-sensitive Cisplatin-based chemotherapeutic regimens are one of samples, and was associated with aggressive character- the most widely used regimens for the treatment of var- istics and poor survival outcomes. CircAKT3 promotes ious solid cancers, including liver cancer, cervical cancer DNA damage repair and inhibits apoptosis through and several others. The anticancer activity of cisplatin PIK3R1 activation by sponging miR-198 . Chi et al. have involves multiple mechanisms . However, drug resis- reported that hsa_circ_0000285 expression is lower in tance of cisplatin limits its application and effectiveness. patients with cisplatin-resistant bladder cancer, and acts Cisplatin resistance may result from reduced drug accu- as an independent prognostic factor for bladder cancer mulation inside cancer cells and drug inactivation by fast patient outcome . In this study, we demonstrated that repairing of DNA lesions . Several efforts have been circRNA_101505 was downregulated in HCC tissues and made to minimize cisplatin resistance . Recently, new associated with poor survival outcomes and cisplatin evidence shows that circRNAs are associated with cis- resistance in HCC. Gain-of-function experiments revealed platin resistance . For example, upregulation of circPVT1 that circRNA_101505 overexpression inhibited HCC cell was observed in osteosarcoma tissues, serums and proliferation, induced apoptosis and sensitized HCC cells cisplatin-resistant cell lines, and was correlated with poor to cisplatin in vitro and in vivo. prognosis of patients with osteosarcoma. circPVT1 Further investigations showed that circRNA_101505 knockdown sensitizes osteosarcoma cells to cisplatin by interacted with miR-103, and miR-103 mimics reversed Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 4 of 9 Fig. 2 CircRNA_101505 sensitizes HCC cells to cisplatin. a QPCR for circRNA_101505 in Hep3B-R and Huh7-R cells after circRNA_101505 transfection. b Cell proliferation analysis showed that overexpressing circRNA_101505 inhibited proliferation of Hep3B-R and Huh7-R cells. c Flow cytometry showed that overexpressing circRNA_101505 induced apoptosis in Hep3B-R and Huh7-R cells. d, e Overexpressing circRNA_101505 sensitizes Hep3B-R and Huh7-R cells to cisplatin. (f) Hep3B-R cells that transfected with circRNA_101505 were injected into nude mice. The tumor volumes were measured every week. g Immunohistochemistry analysis for Ki67 in xenografted tumor tissues. Scale bar, 100 μm. *p < 0.05 vs control circRNA_101505-mediated tumor suppressive effects. are associated with drug transporter-related proteins, cell Noncoding RNAs, including miRNAs, lncRNAs and cir- apoptosis-related proteins, DNA damage repair, and cRNAs, play an important role in the evolution and pro- tumor microenvironment . MiR-103 expression is gression of drug resistance in cancers. The mechanisms upregulated in Adriamycin-resistant acute myeloid Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 5 of 9 Fig. 3 CircRNA_101505 serves as a sponge for miR-103 in HCC cells. a The predictive binding sites of miR-103 on circRNA_101505. b Hep3B-R cells were co-transfected Luc- circRNA_101505 or Luc- circRNA_101505 mutant with miR-103 mimics. Luciferase reporter gene assay was performed to measure luciferase activity. c RNA pull-down assay for the amount of circRNA_101505 and miR-103 in Hep3B-R cells. d, e circRNA_101505-induced apoptosis was reduced by miR-103 mimics in Hep3B-R and Huh7-R cells. (f) miR-103 mimics reversed circRNA_101505-enhance cisplatin toxicity in Hep3B-R and Huh7-R cells. *p < 0.05 leukemia cells and confers K562 cells’ drug resistance via cancer cells . In hepatocellular carcinoma, we previously regulation of COP1 through PI3K/AKT signal pathway . demonstrated that NOR1 increases the expression of MiR-103 is controlled only by the MET oncogene and growth factor receptor-bound protein 2 (Grb2) mRNA in associated with gefitinib-induced apoptosis and epithelial- the human hepatocellular carcinoma cells, and NOR1 mesenchymal transition of non-small cell lung cancers enhanced monofunctional alkylating agent 5-(aziridin-1- in vitro and in vivo . Furthermore, a recent study also yl)-2,4-dinitrobenzamide (CB1954)-induced cell killing in demonstrates that circular RNA circARF3 acts as an HCC cells, but this effect was reversed by stable trans- endogenous miR-103 sponge to alleviate adipose inflam- fection of Grb2 small hairpin RNA . These results indi- mation by promoting mitophagy . cate that circRNA_101505 functions as a tumor In this study, we identified circRNA_101505 as a new suppressor by sponging miR-103, leading to NOR1 interactive molecule of miR-103, also confirmed that upregulation, and finally inhibits cell growth and sensi- NOR1 was a new downstream target of miR-103. NOR1 tizes HCC cells to cisplatin. was cloned in our previous study and was involved in the In conclusion, our study revealed that circRNA_101505 development and progression of nasopharyngeal carci- is frequently inactivated in cisplatin-resistant HCC tissues 21,22 noma . Moreover, it inhibited proliferation and and cell lines and associated with a poor survival outcome. induced apoptosis via the MAPK activation in prostate CircRNA_101505 may sponge miR-103 and thereby Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 6 of 9 Fig. 4 MiR-103 targets to NOR1 in HCC cells. a The predictive binding sites of miR-103 to 3′-UTR of NOR1 (green), the red color showed the mutant bases in binding site. b Hep3B-R cells were co-transfected Luc-miR-103 or Luc- miR-103 mutant with NOR1. Luciferase reporter gene assay was performed to measure luciferase activity. c QPCR for miR-103 and NOR1 in Hep3B-R cells after miR-103 mimics transfection. d QPCR for NOR1 in Hep3B-R and Huh7-R cells after NOR1 transfection. e NOR1 reversed miR-103-mediated reduction of apoptosis in Hep3B-R and Huh7-R cells. f NOR1 reversed miR-103-attenuated cisplatin toxicity in Hep3B-R and Huh7-R cells. *p < 0.05 promote NOR1 expression. Subsequently, this may sup- CircRNA microarray analysis press cell growth and sensitize HCC cells to cisplatin Total RNA was extracted from patients with cisplatin- in vitro and in vivo. The significant inhibitory effects resistant or cisplatin-sensitive HCC using the RNeasy indicate circRNA_101505 as a potential therapeutic target Mini Kit (Qiagen, GmBH, Hilden, Germany) according to for HCC treatment. Therefore, our findings provide sig- the manufacturer’s instructions. The adjacent tissues were nificant evidence to further elucidate the therapeutic use used as control. Patients with cisplatin-resistant HCC of circRNA in HCC. were defined as those with persistent disease more than two months, and those with recurrent disease more than Material and methods 2 months after completion of chemotherapy containing Human tissues collection cisplatin. Patients with cisplatin-sensitive HCC were This project was approved by the Ethic Committee of defined as those without local residual lesions or recur- The Third Xiangya Hospital of Central South University. rence at 2 months after completion of chemotherapy Written informed consent was obtained from each patient. containing cisplatin. Purified total RNA was quantified Hepatocellular carcinoma tissues (N = 120) and the adja- using the NanoDrop 2000 spectrophotometer. The total cent tissues (N = 78) were collected from The Third RNA was sent to Aksomics Co. Ltd. (Shanghai, China) to Xiangya Hospital of Central South University between analyze circRNA expression profiles. Differentially May 2008 and Nov 2018. Each sample was immediately expressed circRNAs were identified as fold change > 2 and flash-frozen in liquid nitrogen until RNA extraction. adjusted p < 0.05. Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 7 of 9 Fig. 5 The relationship of circRNA_101505, miR-103 and NOR1 in patients with HCC. a Scatter plots illustrating qRT-PCR analysis of expression fold change for miR-103 and NOR1 in cisplatin-resistant and cisplatin-sensitive HCC tissues. b circRNA_101505 expression was negatively correlated with miR-103. c circRNA_101505 expression was positively correlated with NOR1. d miR-103 expression was negatively correlated with NOR1. *p < 0.05 vs control Cell culture constructed and purchased from (GenomediTech, The cell lines used in the present study included Hep3B Shanghai, China). miR-103 inhibitors and mimics were and Huh7 cells were cultured in Roswell Park Memorial purchased from RiboBio (Guangzhou, China). Transfec- Institute (RPMI) 1640 medium (Gibco) supplemented tion was performed using Lipofectamine 3000 (Life- with 10% fetal bovine serum and maintained in a humi- Technologies, USA) according to the manufacturer’s dified atmosphere with 5% CO at 37 °C. instructions. The cisplatin-resistant Hep3B (Hep3B-R) and Huh7 (Huh7-R) cells was established in our lab. The Hep3B or Cell proliferation analysis Huh7 cells were treated with the initial concentration of A BrdU Cell Proliferation Assay Kit (CST (Shanghai) cisplatin (0.5 μg/L). The medium was changed once every Biological Reagents Company Limited, Shanghai, China) 3 days. Each dose was maintained for 2 weeks, and then was used to measure the cell proliferation rate according was doubled. to the manufacturer’s instructions. The cells were seeded To test the cell survival and proliferation rates, Hep3B- in a 96-well plate and incubated overnight. The cells then R and Huh7-R cells were exposed to cisplatin at a series of were transfected circRNA_101505, miR-103 or NOR1, concentrations (0, 2.5, 5, 10 and 20 μg/L) for 24 h or at with co-treatment of cisplatin and cells were incubated for 10 μg/L for 24 h. 24 h. Finally, 10 μM BrdU was added to the plate and cells were incubated for 4 h. The absorbance at 450 nm was Cell transfection read and recorded to calculate the relative proliferation circRNA_101505 overexpression plasmids (cir- rate. The experiment was done three times with triplicate cRNA_101505-OE) and NOR1 expressed plasmids were samples. Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 8 of 9 Cell apoptosis analysis Tumor xenograft in nude mice An Annexin V-FITC Apoptosis Detection Kit (CST Animal experiments were approved by the Ethical (Shanghai) Biological Reagents Company Limited, Committee for Animal Research of the Third Xiangya Shanghai, China) was used for analysis of apoptosis Hospital of Central South University. Ten nude mice (5 according to the manufacturer’s instructions. Briefly, cells mice per group, male, 2 months old) were purchased from with indicated treatment were resuspended in 236 μl Animal Center of Central South University. The Hep3B-R Annexin V-FITC binding buffer and then mixed well with cells transfected with circRNA_101505-OE was sub- 1 μl Annexin V-FITC and 13 μl Propidium Iodide for cutaneously injected into mice. The tumor sizes were 30 min incubation. The cells were analyzed by using a BD measured every week. The tumor volume was calculated FacScanto II flow cytometer (BD Biosciences, San Jose, using the formula: 0.5 × L × W where L and W are the CA). Unstained cells, and fluorescence minus one (FMO) long and short diameter of the tumor, respectively. controls were used for cytometry and gating set up. Immunohistochemistry Quantitative PCR analysis The expression of Ki67 in tumor tissues from nude mice Total RNA was extracted with Trizol reagent. The was analyzed by immunohistochemical analysis. Briefly, expression of circRNA_101505 and NOR1 was measured the tissues were fixed with 4% formaldehyde for 24 h, by TaqMan Fast Advanced Master Mix (Thermo Scien- embedded and cut into 4-μm-thick section. The sections tific, Shanghai, China) according to manufacturers’ were treated with 10 mmol/l sodium citrate buffer and instructions. Expression of β-actin was used as an endo- incubated with anti-Ki67 antibody (1: 200 dilution) over- genous control. QPCR was performed at the condition: night at 4 °C. The positive signaling was stained by using a Hold 50 °C for 2 min, 95.0 °C for 20 s, and 40 circles of Mouse and Rabbit Specific HRP/DAB (ABC) Detection 95.0 °C for 1 s and 60 °C for 20 s in 7900HT Fast Real- IHC kit (Abcam Trading (Shanghai) Company Ltd., Time PCR Instrument. The primers were used as fol- Shanghai, China), and counterstained with hematoxylin. lowing: circRNA_101505: forward, 5′-CCGAGTTCCTAA The relative integral optical density (IOD) of positive AAGCAGCC-3′; reverse, 5′-CCATCAGCAGTCCTAG signaling was obtained by ImageJ software. GTCC-3′. β-actin: forward, 5′-CACACTGTGCCCATC- TATGAGG-3′; reverse, 5′-TCGAAGTCTAGGGCGA- Statistical analysis CATAGC-3′. miR-103 expression was measured by Data were analyzed in GraphPad Prism software miRNA QPCR Master Mix (Agilent, Beijing, China) (GraphPad Software Inc., La Jolla, CA). Overall survival according to the manufacturer’s instructions. analysis was performed by Kaplan–Meier curves and log- rank test for significance. Student’s t test with two tails Luciferase reporter assay was used to assess the statistical significance in two The wild-type sequence and mutants in binding sites of groups and one-way ANOVA with post hoc Bonferroni circRNA_101505 was cloned downstream of FL reporter test were used in three or more groups. Correlations were vector. Hep3B cells were seeded and cultured in 96-well analyzed by Pearson correlation test. P < 0.05 was con- plates for 24 h. The cells were co-transfected with FL sidered statistically significant. reporter, Renilla luciferase reporter and miR-103 mimic for 48 h. Luciferase activity was measured using a Luci- Acknowledgements This work was supported by National Natural Science Foundation of China (No. ferase Reporter Assay Kit (BioVision Technologies, Inc, 81573091 and No. 81802668) and Natural Science Foundation of Hunan Exton, PA, USA). Relative luciferase activity was nor- Provincial China (No. 2018JJ3776 and No. 2017JJ3467). malized to Renilla activity. Authors Contributions RNA immunoprecipitation (RIP) F.L., X.N. and R.G. conceived the study and participated in the study design, A Magna RIP™ RNA-Binding Protein Immunoprecipi- performance, coordination and manuscript writing. Y.L., Y.F., R.H. and M.G. performed the research. All authors have read and approved the final tation Kit (Merck Life Science (Shanghai) Co., Ltd. manuscript. In Figure 1, Y.L. generated the data and assembled the figure. In Shanghai, China) was used in this assay as previously Figure 2, R.H. generated the data and labelled the image, Y.L. and X.N. describe (22234798). Briefly, Hep3B cells were fixed with assembled the figure. In Figure 3, Y.F. and R.H. generated the data and labelled the image, Y.L. and X.N. assembled the figure. In Figure 4, F.L. and R.G. formaldehyde, lysed with lysis buffer and sonicated. The generated the data and labelled the image, Y.L. assembled the figure. In Figure supernatant was collected and incubated with a 5, F.Y., F.L. and R.G. generated the data and labelled the image, F.L. and R.G. circRNA_101505-specific probes dynabeads (Invitrogen) assembled the figure. mixture overnight at 30 °C. On the next day, the dyna- beads was washed and incubated with 200 μl of lysis buffer. Finally, RNA was extracted from these complexes Competing interests and was used for qPCR. The authors declare no competing interests. Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 9 of 9 Publisher’s note 13. Su, M. et al. Circular RNAs in cancer: emerging functions in hallmarks, Springer Nature remains neutral with regard to jurisdictional claims in stemness, resistance and roles as potential biomarkers. Mol. Cancer 18,90 published maps and institutional affiliations. (2019). 14. Kun-Peng, Z.,Xiao-Long,M.&Chun-Lin,Z.Overexpressed circPVT1, a potential Received: 23 May 2019 Revised: 24 June 2019 Accepted: 26 June 2019 new circular RNA biomarker, contributes to doxorubicin and cisplatin resis- tance of osteosarcoma cells by regulating ABCB1. Int. J. Biol. Sci. 14, 321–330 (2018). 15. Huang, X. et al. Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression. Mol. Cancer 18,71 (2019). References 16. Chi, B. J. et al. Downregulation of hsa_circ_0000285 serves as a prognostic 1. Wang,M., Yu, F.& Li, P.CircularRNAs: characteristics, function andclinical biomarker for bladder cancer and is involved in cisplatin resistance. Neoplasma significance in hepatocellular carcinoma. Cancers 10,pii:E258(2018). 66,197–202 (2019). 2. Fu, L., Jiang, Z., Li, T., Hu, Y. & Guo, J. Circular RNAs in hepatocellular carcinoma: 17. Feng, W. et al. ncRNAs associated with drug resistance and the therapy of Functions and implications. Cancer Med. 7, 3101–3109 (2018). digestive system neoplasms. J. Cell Physiol.(2019) https://doi.org/10.1002/ 3. Qiu, L. P. et al. The emerging role of circular RNAs in hepatocellular carcinoma. jcp.28551. J. Cancer 9, 1548–1559 (2018). 18. Wan,L.etal. MicroRNA-103 confers the resistance to long-treatment of 4. Hu, J. et al. Progress and prospects of circular RNAs in Hepatocellular carci- adriamycin to human leukemia cells by regulation of COP1. J. Cell Biochem. noma: novel insights into their function. J. Cell Physiol. 233, 4408–4422 (2018). 119, 3843–3852 (2018). 5. Yao, R.,Zou,H.& Liao,W.Prospect ofcircular RNA in hepatocellular carcinoma: 19. Garofalo, M. et al. EGFR and MET receptor tyrosine kinase-altered microRNA a novel potential biomarker and therapeutic target. Front Oncol. 8, 332 (2018). expression induces tumorigenesis and gefitinib resistance in lung cancers. Nat. 6. Guo, J.,Duan, H.,Li, Y.,Yang, L. &Yuan, L. A novel circular RNA circ-ZNF652 Med. 18,74–82 (2011). promotes hepatocellular carcinoma metastasis through inducing snail- 20. Zhang, Z. et al. circARF3 alleviates mitophagy-mediated inflammation by mediated epithelial-mesenchymal transition by sponging miR-203/miR-502- 5p. Biochem. Biophys. Res. Commun. 513,812–819 (2019). targeting miR-103/TRAF3 in mouse adipose tissue. Mol. Ther. Nucleic Acids 14, 7. Xu, L. et al. CircSETD3 (Hsa_circ_0000567) acts as a sponge for microRNA-421 192–203 (2019). inhibiting hepatocellular carcinoma growth. J. Exp. Clin. Cancer Res. 38,98 21. Nie, X. et al. Cloning, expression, and mutation analysis of NOR1, a novel (2019). human gene down-regulated in HNE1 nasopharyngeal carcinoma cell line. J. 8. Matboli, M. et al. circRNAs (hsa_circ_00156, hsa_circ _000224, and hsa_circ Cancer Res. Clin. Oncol. 129,410–414 (2003). _000520) are novel potential biomarkers in hepatocellular carcinoma. J. Cell 22. Wang, W. et al. Oxidored-nitro domain containing protein 1 (NOR1) Biochem. 38, BSR20180915 (2018). expression suppresses slug/vimentin but not snail in nasopharyngeal 9. Ding,B., Lou, W.,Xu, L. &Fan,W.Non-codingRNA in drug resistance of carcinoma: Inhibition of EMT in vitro and in vivo in mice. Cancer Lett. 348, hepatocellular carcinoma. Biosci. Rep. 38, pii: BSR20180915 (2018). 109–118 (2014). 10. Zhu, H. et al. Molecular mechanisms of cisplatin resistance in cervical cancer. 23. Shan, Z. et al. Overexpression of oxidored-nitro domain containing protein 1 Drug Des. Devel. Ther. 10, 1885–1895 (2016). induces growth inhibition and apoptosis in human prostate cancer PC3 cells. 11. Ghosh,S.Cisplatin:the first metal based anticancer drug. Bioorg. Chem. 88, Oncol. Rep. 32,1939–1946 (2014). 102925 (2019). 24. Gui,R., Li,D., Qi, G., Suhad, A. & Nie,X. Inhibitionof Grb2-mediated activation of 12. Sun, C.Y., Zhang, Q. Y.,Zheng,G.J.&Feng, B.Phytochemicals: current strategy MAPK signal transduction suppresses NOR1/CB1954-induced cytotoxicity in to sensitize cancer cells to cisplatin. Biomed. Pharm. 110,518–527 (2019). the HepG2 cell line. Oncol. Lett. 4, 566–570 (2012). Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

CircRNA_101505 sensitizes hepatocellular carcinoma cells to cisplatin by sponging miR-103 and promotes oxidored-nitro domain-containing protein 1 expression

Download PDF
9 pages

Loading next page...
 
/lp/springer-journals/circrna-101505-sensitizes-hepatocellular-carcinoma-cells-to-cisplatin-1gYdJEvZBk

References (25)

Publisher
Springer Journals
Copyright
Copyright © 2019 by The Author(s)
Subject
Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
eISSN
2058-7716
DOI
10.1038/s41420-019-0202-6
Publisher site
See Article on Publisher Site

Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and a leading cause of cancer-related deaths worldwide. Emerging studies have shown that circular RNAs (circRNAs) are differentially expressed in HCC and play an important role in HCC pathogenesis and metastasis. However, the mechanism of circRNA in the chemoresistance of HCC remains unclear. In this study, we aimed to investigate the role of circRNA in cisplatin resistance of HCC. We identified a novel circRNA circRNA_101505 that was decreased in cisplatin-resistant HCC tissues and cell lines, and associated with a poor survival outcome. Gain-of-function investigations showed that overexpression of circRNA_101505 suppressed cancer cell growth in vivo and in vitro, and enhanced cisplatin toxicity in HCC cells. Mechanistic studies found that circRNA_101505 could sensitize HCC cells to cisplatin by sponging miR- 103, and thereby promoting oxidored-nitro domain-containing protein 1 (NOR1) expression. In conclusion, the significant inhibitory effects indicate circRNA_101505 to be a potential therapeutic target for HCC treatment. Our findings provide significant evidence to further elucidate the therapeutic use of circRNA in HCC. Introduction CircRNAs are a tissue-specific class of noncoding RNAs Hepatocellular carcinoma (HCC) is one of the most molecules. They are characterized by a covalently closed common malignant tumors and a leading cause of cancer- continuous loop without poly(A) tail . Recently, thou- related deaths worldwide . Owing to the high frequency of sands of endogenous circRNAs have been discovered. tumor metastasis, recurrence, and drug resistance, the CircRNAs mediate gene expression by sponging micro- survival rate of patients with HCC is low. Additionally, the RNAs or interacting with other molecules and then effective therapies for advanced HCC patients are lim- inhibit their function . For example, circRNA, circ- ited . Thus, identifying novel targets for HCC therapy is ZNF652 promotes migratory and invasive capabilities of urgently required. Emerging studies have found that cir- HCC cells by sponging miR-203 and miR-502-5p and cular RNAs (circRNAs) are differentially expressed in enhancing epithelial-mesenchymal transition (EMT) . HCC and play an important role in HCC pathogenesis CircRNA circSETD3 was significantly downregulated in and metastasis . HCC tissues and cell lines and associated with unfavor- able prognosis of HCC patients . Matboli et al showed that the combination of hsa_circ_00156, hsa_circ _00224 and hsa_circ _00520 acted as biomarkers with higher Correspondence: Fengxia Liu (592111443@qq.com)or sensitivities and specificities than alpha-fetoprotein in Rong Gui (aguirong@163.com) HCC . Multidrug resistance occurs frequently in HCC Department of Blood Transfusion, the Third Xiangya Hospital of Central South during the long-term chemotherapy, leading to cancer University, Tongzipo Road 138, 410013 Changsha, China Edited by I. D'Agnano © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Luo et al. Cell Death Discovery (2019) 5:121 Page 2 of 9 relapse. An increasing number of studies have highlighted tumor tissues that expressing circRNA_101505 compared the key roles of ncRNAs, including miRNAs, long with control (Fig. 2g). ncRNAs (lncRNAs) in chemoresistance of HCC . How- ever, the mechanism of circRNA in chemoresistance of CircRNA_101505 sponges miR-103 in HCC HCC remains unclear. We tested whether circRNA_101505 binds to miRNAs In this study, we aimed to investigate the role of cir- in HCC cells. The bioinformatics tool predicted two cRNA in cisplatin resistance of HCC. We identified a binding sites on circRNA_101505 for miR-103 (Fig. 3a). novel circRNA circRNA_101505, that was decreased in Further luciferase assay confirmed that circRNA_101505 cisplatin-resistant HCC tissues and cell lines. Gain-of- targeted to miR-103 (Fig. 3b), suggesting that cir- function investigations showed that circRNA_101505 cRNA_101505 may function as a sponge to miR-103. In overexpression suppressed cancer cell growth in vivo and addition, RIP assay revealed that miR-103 directly inter- in vitro. Subsequent studies displayed that cir- acted with circRNA_101505 (Fig. 3c). To investigate the cRNA_101505 could sensitize HCC cells to cisplatin by downstream mechanism by which circRNA_101505 targeting the miR-103/oxidored-nitro domain-containing exerted its functions in HCC cells, the mimic of miR-103 protein 1 (NOR1) signaling axis. was co-transfected with circRNA_101505. The results showed that miR-103 mimic could significantly reversed Results circRNA_101505-mediated induction of apoptosis Downregulation of CircRNA_101505 is associated with (Fig. 3d, e) and promotion of cisplatin toxicity in Hep3B-R cisplatin resistance in HCC and Huh7-R cells (Fig. 3f). To investigate the role of circRNAs in cisplatin-resistant HCC, we performed circRNAs array to identify the dif- MiR-103 confers cisplatin resistance in HCC cells by ferentially expressed circRNAs. Several circRNAs were targeting NOR1 differentially expressed in cisplatin-resistant and -sensi- We further investigated the role and downstream tive HCC (Fig. 1a). qPCR results further confirmed that mechanism that miR-103 affects cisplatin resistance in circRNA_101505 was significantly decreased in HCC tis- HCC. NOR1 was a predictive target of miR-103 (Fig. 4a). sues compared with adjacent tissues, and its expression MiR-103 wild-type and mutant sequences were con- was lower in cisplatin-resistant HCC tissues than in the structed into luciferase reporter gene and co-transfected cisplatin-sensitive tissues (Fig. 1b). We also found that with NOR1, and luciferase assay confirmed that miR-103 circRNA_101505 was significantly decreased in cisplatin- targeted to NOR1 (Fig. 4a, b), and negatively regulated resistant HCC cell lines (Hep3B-R and Huh7-R) com- NOR1 expression (Fig. 4c). We expressed NOR1 in pared with the parental HCC cell lines (Fig. 1c). In addi- Hep3B-R and Huh7-R cells (Fig. 4d), and found that tion, further survival analyses revealed that the HCC NOR1 could significantly reversed miR-103-mediated patients with low circRNA_101505 level had shorter inhibition of apoptosis and suppression of cisplatin toxi- overall survival than the patients had high cir- city in Hep3B-R and Huh7-R cells (Fig. 4e, f). In addition, cRNA_101505 level (p = 0.0005, Fig. 1d). These results we also found that the levels of miR-103 were significantly suggest that circRNA_101505 is closely associated with increased and NOR1 was decreased in HCC tissues cisplatin resistance in HCC. compared with adjacent tissues (Fig. 5a), and miR-103 levels were higher but NOR1 levels were lower in Overexpression of circRNA_101505 sensitizes HCC cells to cisplatin-resistant HCC tissues than in the cisplatin- cisplatin sensitive tissues (Fig. 5a). Pearson analysis showed that To investigate the role of circRNA_101505 in cisplatin circRNA_101505 were negatively correlated with miR-103 resistance in HCC, we overexpressed circRNA_101505 in (Fig. 5b) and positively correlated with NOR1 (Fig. 5c), Hep3B-R and Huh7-R cells (Fig. 2a). We found that and miR-103 was negatively correlated with NOR1 overexpression of circRNA_101505 significantly inhibited (Fig. 5d). These results demonstrate that cir- cells proliferation (Fig. 2b) and induced apoptosis (Fig. 2c) cRNA_101505 sensitizes HCC cells to cisplatin through in Hep3B-R and Huh7-R cells compared with negative miR-103/NOR1 axis. control. In addition, circRNA_101505 upregulation sen- sitizes Hep3B-R and Huh7-R cells to cisplatin (Fig. 2d, e). Discussion Furthermore, the tumor suppressive effects of cir- In this study, we found that circRNA_101505 down- cRNA_101505 upregulation were also confirmed in vivo. regulation is associated with a poor survival outcome and Our results showed that the tumor volumes in nude mice cisplatin resistance in HCC. Functional investigations injected with Hep3B-R cells expressing circRNA_101505 revealed that circRNA_101505 overexpression inhibited were smaller than in the control nude mice (Fig. 2f), and HCC cell growth and sensitized HCC cells to cisplatin. the proliferative marker ki67 expression was decreased in Further experiments demonstrated that circRNA_101505 Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 3 of 9 Fig. 1 circRNAs expression in HCC. a The heatmap of deferentially expressed circRNAs in HCC. b QPCR was performed to confirm the expression of circRNAs in cisplatin-resistant and cisplatin-sensitive HCC tissues. c QPCR was performed to confirm the expression of circRNAs in cisplatin-resistant and cisplatin-sensitive HCC cell lines. d Kaplan–Meier curves of overall survival in HCC patients with low or high circRNA_101505 expression. *p < 0.05 exhibited tumor suppressive effects by sponging miR-103, decreasing ABCB1 expression . The expression of cir- leading to NOR1 upregulation. cAKT3 was upregulated in cisplatin-resistant gastric Cisplatin is a small-molecule platinum compound. cancer tissues and cells compared with cisplatin-sensitive Cisplatin-based chemotherapeutic regimens are one of samples, and was associated with aggressive character- the most widely used regimens for the treatment of var- istics and poor survival outcomes. CircAKT3 promotes ious solid cancers, including liver cancer, cervical cancer DNA damage repair and inhibits apoptosis through and several others. The anticancer activity of cisplatin PIK3R1 activation by sponging miR-198 . Chi et al. have involves multiple mechanisms . However, drug resis- reported that hsa_circ_0000285 expression is lower in tance of cisplatin limits its application and effectiveness. patients with cisplatin-resistant bladder cancer, and acts Cisplatin resistance may result from reduced drug accu- as an independent prognostic factor for bladder cancer mulation inside cancer cells and drug inactivation by fast patient outcome . In this study, we demonstrated that repairing of DNA lesions . Several efforts have been circRNA_101505 was downregulated in HCC tissues and made to minimize cisplatin resistance . Recently, new associated with poor survival outcomes and cisplatin evidence shows that circRNAs are associated with cis- resistance in HCC. Gain-of-function experiments revealed platin resistance . For example, upregulation of circPVT1 that circRNA_101505 overexpression inhibited HCC cell was observed in osteosarcoma tissues, serums and proliferation, induced apoptosis and sensitized HCC cells cisplatin-resistant cell lines, and was correlated with poor to cisplatin in vitro and in vivo. prognosis of patients with osteosarcoma. circPVT1 Further investigations showed that circRNA_101505 knockdown sensitizes osteosarcoma cells to cisplatin by interacted with miR-103, and miR-103 mimics reversed Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 4 of 9 Fig. 2 CircRNA_101505 sensitizes HCC cells to cisplatin. a QPCR for circRNA_101505 in Hep3B-R and Huh7-R cells after circRNA_101505 transfection. b Cell proliferation analysis showed that overexpressing circRNA_101505 inhibited proliferation of Hep3B-R and Huh7-R cells. c Flow cytometry showed that overexpressing circRNA_101505 induced apoptosis in Hep3B-R and Huh7-R cells. d, e Overexpressing circRNA_101505 sensitizes Hep3B-R and Huh7-R cells to cisplatin. (f) Hep3B-R cells that transfected with circRNA_101505 were injected into nude mice. The tumor volumes were measured every week. g Immunohistochemistry analysis for Ki67 in xenografted tumor tissues. Scale bar, 100 μm. *p < 0.05 vs control circRNA_101505-mediated tumor suppressive effects. are associated with drug transporter-related proteins, cell Noncoding RNAs, including miRNAs, lncRNAs and cir- apoptosis-related proteins, DNA damage repair, and cRNAs, play an important role in the evolution and pro- tumor microenvironment . MiR-103 expression is gression of drug resistance in cancers. The mechanisms upregulated in Adriamycin-resistant acute myeloid Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 5 of 9 Fig. 3 CircRNA_101505 serves as a sponge for miR-103 in HCC cells. a The predictive binding sites of miR-103 on circRNA_101505. b Hep3B-R cells were co-transfected Luc- circRNA_101505 or Luc- circRNA_101505 mutant with miR-103 mimics. Luciferase reporter gene assay was performed to measure luciferase activity. c RNA pull-down assay for the amount of circRNA_101505 and miR-103 in Hep3B-R cells. d, e circRNA_101505-induced apoptosis was reduced by miR-103 mimics in Hep3B-R and Huh7-R cells. (f) miR-103 mimics reversed circRNA_101505-enhance cisplatin toxicity in Hep3B-R and Huh7-R cells. *p < 0.05 leukemia cells and confers K562 cells’ drug resistance via cancer cells . In hepatocellular carcinoma, we previously regulation of COP1 through PI3K/AKT signal pathway . demonstrated that NOR1 increases the expression of MiR-103 is controlled only by the MET oncogene and growth factor receptor-bound protein 2 (Grb2) mRNA in associated with gefitinib-induced apoptosis and epithelial- the human hepatocellular carcinoma cells, and NOR1 mesenchymal transition of non-small cell lung cancers enhanced monofunctional alkylating agent 5-(aziridin-1- in vitro and in vivo . Furthermore, a recent study also yl)-2,4-dinitrobenzamide (CB1954)-induced cell killing in demonstrates that circular RNA circARF3 acts as an HCC cells, but this effect was reversed by stable trans- endogenous miR-103 sponge to alleviate adipose inflam- fection of Grb2 small hairpin RNA . These results indi- mation by promoting mitophagy . cate that circRNA_101505 functions as a tumor In this study, we identified circRNA_101505 as a new suppressor by sponging miR-103, leading to NOR1 interactive molecule of miR-103, also confirmed that upregulation, and finally inhibits cell growth and sensi- NOR1 was a new downstream target of miR-103. NOR1 tizes HCC cells to cisplatin. was cloned in our previous study and was involved in the In conclusion, our study revealed that circRNA_101505 development and progression of nasopharyngeal carci- is frequently inactivated in cisplatin-resistant HCC tissues 21,22 noma . Moreover, it inhibited proliferation and and cell lines and associated with a poor survival outcome. induced apoptosis via the MAPK activation in prostate CircRNA_101505 may sponge miR-103 and thereby Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 6 of 9 Fig. 4 MiR-103 targets to NOR1 in HCC cells. a The predictive binding sites of miR-103 to 3′-UTR of NOR1 (green), the red color showed the mutant bases in binding site. b Hep3B-R cells were co-transfected Luc-miR-103 or Luc- miR-103 mutant with NOR1. Luciferase reporter gene assay was performed to measure luciferase activity. c QPCR for miR-103 and NOR1 in Hep3B-R cells after miR-103 mimics transfection. d QPCR for NOR1 in Hep3B-R and Huh7-R cells after NOR1 transfection. e NOR1 reversed miR-103-mediated reduction of apoptosis in Hep3B-R and Huh7-R cells. f NOR1 reversed miR-103-attenuated cisplatin toxicity in Hep3B-R and Huh7-R cells. *p < 0.05 promote NOR1 expression. Subsequently, this may sup- CircRNA microarray analysis press cell growth and sensitize HCC cells to cisplatin Total RNA was extracted from patients with cisplatin- in vitro and in vivo. The significant inhibitory effects resistant or cisplatin-sensitive HCC using the RNeasy indicate circRNA_101505 as a potential therapeutic target Mini Kit (Qiagen, GmBH, Hilden, Germany) according to for HCC treatment. Therefore, our findings provide sig- the manufacturer’s instructions. The adjacent tissues were nificant evidence to further elucidate the therapeutic use used as control. Patients with cisplatin-resistant HCC of circRNA in HCC. were defined as those with persistent disease more than two months, and those with recurrent disease more than Material and methods 2 months after completion of chemotherapy containing Human tissues collection cisplatin. Patients with cisplatin-sensitive HCC were This project was approved by the Ethic Committee of defined as those without local residual lesions or recur- The Third Xiangya Hospital of Central South University. rence at 2 months after completion of chemotherapy Written informed consent was obtained from each patient. containing cisplatin. Purified total RNA was quantified Hepatocellular carcinoma tissues (N = 120) and the adja- using the NanoDrop 2000 spectrophotometer. The total cent tissues (N = 78) were collected from The Third RNA was sent to Aksomics Co. Ltd. (Shanghai, China) to Xiangya Hospital of Central South University between analyze circRNA expression profiles. Differentially May 2008 and Nov 2018. Each sample was immediately expressed circRNAs were identified as fold change > 2 and flash-frozen in liquid nitrogen until RNA extraction. adjusted p < 0.05. Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 7 of 9 Fig. 5 The relationship of circRNA_101505, miR-103 and NOR1 in patients with HCC. a Scatter plots illustrating qRT-PCR analysis of expression fold change for miR-103 and NOR1 in cisplatin-resistant and cisplatin-sensitive HCC tissues. b circRNA_101505 expression was negatively correlated with miR-103. c circRNA_101505 expression was positively correlated with NOR1. d miR-103 expression was negatively correlated with NOR1. *p < 0.05 vs control Cell culture constructed and purchased from (GenomediTech, The cell lines used in the present study included Hep3B Shanghai, China). miR-103 inhibitors and mimics were and Huh7 cells were cultured in Roswell Park Memorial purchased from RiboBio (Guangzhou, China). Transfec- Institute (RPMI) 1640 medium (Gibco) supplemented tion was performed using Lipofectamine 3000 (Life- with 10% fetal bovine serum and maintained in a humi- Technologies, USA) according to the manufacturer’s dified atmosphere with 5% CO at 37 °C. instructions. The cisplatin-resistant Hep3B (Hep3B-R) and Huh7 (Huh7-R) cells was established in our lab. The Hep3B or Cell proliferation analysis Huh7 cells were treated with the initial concentration of A BrdU Cell Proliferation Assay Kit (CST (Shanghai) cisplatin (0.5 μg/L). The medium was changed once every Biological Reagents Company Limited, Shanghai, China) 3 days. Each dose was maintained for 2 weeks, and then was used to measure the cell proliferation rate according was doubled. to the manufacturer’s instructions. The cells were seeded To test the cell survival and proliferation rates, Hep3B- in a 96-well plate and incubated overnight. The cells then R and Huh7-R cells were exposed to cisplatin at a series of were transfected circRNA_101505, miR-103 or NOR1, concentrations (0, 2.5, 5, 10 and 20 μg/L) for 24 h or at with co-treatment of cisplatin and cells were incubated for 10 μg/L for 24 h. 24 h. Finally, 10 μM BrdU was added to the plate and cells were incubated for 4 h. The absorbance at 450 nm was Cell transfection read and recorded to calculate the relative proliferation circRNA_101505 overexpression plasmids (cir- rate. The experiment was done three times with triplicate cRNA_101505-OE) and NOR1 expressed plasmids were samples. Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 8 of 9 Cell apoptosis analysis Tumor xenograft in nude mice An Annexin V-FITC Apoptosis Detection Kit (CST Animal experiments were approved by the Ethical (Shanghai) Biological Reagents Company Limited, Committee for Animal Research of the Third Xiangya Shanghai, China) was used for analysis of apoptosis Hospital of Central South University. Ten nude mice (5 according to the manufacturer’s instructions. Briefly, cells mice per group, male, 2 months old) were purchased from with indicated treatment were resuspended in 236 μl Animal Center of Central South University. The Hep3B-R Annexin V-FITC binding buffer and then mixed well with cells transfected with circRNA_101505-OE was sub- 1 μl Annexin V-FITC and 13 μl Propidium Iodide for cutaneously injected into mice. The tumor sizes were 30 min incubation. The cells were analyzed by using a BD measured every week. The tumor volume was calculated FacScanto II flow cytometer (BD Biosciences, San Jose, using the formula: 0.5 × L × W where L and W are the CA). Unstained cells, and fluorescence minus one (FMO) long and short diameter of the tumor, respectively. controls were used for cytometry and gating set up. Immunohistochemistry Quantitative PCR analysis The expression of Ki67 in tumor tissues from nude mice Total RNA was extracted with Trizol reagent. The was analyzed by immunohistochemical analysis. Briefly, expression of circRNA_101505 and NOR1 was measured the tissues were fixed with 4% formaldehyde for 24 h, by TaqMan Fast Advanced Master Mix (Thermo Scien- embedded and cut into 4-μm-thick section. The sections tific, Shanghai, China) according to manufacturers’ were treated with 10 mmol/l sodium citrate buffer and instructions. Expression of β-actin was used as an endo- incubated with anti-Ki67 antibody (1: 200 dilution) over- genous control. QPCR was performed at the condition: night at 4 °C. The positive signaling was stained by using a Hold 50 °C for 2 min, 95.0 °C for 20 s, and 40 circles of Mouse and Rabbit Specific HRP/DAB (ABC) Detection 95.0 °C for 1 s and 60 °C for 20 s in 7900HT Fast Real- IHC kit (Abcam Trading (Shanghai) Company Ltd., Time PCR Instrument. The primers were used as fol- Shanghai, China), and counterstained with hematoxylin. lowing: circRNA_101505: forward, 5′-CCGAGTTCCTAA The relative integral optical density (IOD) of positive AAGCAGCC-3′; reverse, 5′-CCATCAGCAGTCCTAG signaling was obtained by ImageJ software. GTCC-3′. β-actin: forward, 5′-CACACTGTGCCCATC- TATGAGG-3′; reverse, 5′-TCGAAGTCTAGGGCGA- Statistical analysis CATAGC-3′. miR-103 expression was measured by Data were analyzed in GraphPad Prism software miRNA QPCR Master Mix (Agilent, Beijing, China) (GraphPad Software Inc., La Jolla, CA). Overall survival according to the manufacturer’s instructions. analysis was performed by Kaplan–Meier curves and log- rank test for significance. Student’s t test with two tails Luciferase reporter assay was used to assess the statistical significance in two The wild-type sequence and mutants in binding sites of groups and one-way ANOVA with post hoc Bonferroni circRNA_101505 was cloned downstream of FL reporter test were used in three or more groups. Correlations were vector. Hep3B cells were seeded and cultured in 96-well analyzed by Pearson correlation test. P < 0.05 was con- plates for 24 h. The cells were co-transfected with FL sidered statistically significant. reporter, Renilla luciferase reporter and miR-103 mimic for 48 h. Luciferase activity was measured using a Luci- Acknowledgements This work was supported by National Natural Science Foundation of China (No. ferase Reporter Assay Kit (BioVision Technologies, Inc, 81573091 and No. 81802668) and Natural Science Foundation of Hunan Exton, PA, USA). Relative luciferase activity was nor- Provincial China (No. 2018JJ3776 and No. 2017JJ3467). malized to Renilla activity. Authors Contributions RNA immunoprecipitation (RIP) F.L., X.N. and R.G. conceived the study and participated in the study design, A Magna RIP™ RNA-Binding Protein Immunoprecipi- performance, coordination and manuscript writing. Y.L., Y.F., R.H. and M.G. performed the research. All authors have read and approved the final tation Kit (Merck Life Science (Shanghai) Co., Ltd. manuscript. In Figure 1, Y.L. generated the data and assembled the figure. In Shanghai, China) was used in this assay as previously Figure 2, R.H. generated the data and labelled the image, Y.L. and X.N. describe (22234798). Briefly, Hep3B cells were fixed with assembled the figure. In Figure 3, Y.F. and R.H. generated the data and labelled the image, Y.L. and X.N. assembled the figure. In Figure 4, F.L. and R.G. formaldehyde, lysed with lysis buffer and sonicated. The generated the data and labelled the image, Y.L. assembled the figure. In Figure supernatant was collected and incubated with a 5, F.Y., F.L. and R.G. generated the data and labelled the image, F.L. and R.G. circRNA_101505-specific probes dynabeads (Invitrogen) assembled the figure. mixture overnight at 30 °C. On the next day, the dyna- beads was washed and incubated with 200 μl of lysis buffer. Finally, RNA was extracted from these complexes Competing interests and was used for qPCR. The authors declare no competing interests. Official journal of the Cell Death Differentiation Association Luo et al. Cell Death Discovery (2019) 5:121 Page 9 of 9 Publisher’s note 13. Su, M. et al. Circular RNAs in cancer: emerging functions in hallmarks, Springer Nature remains neutral with regard to jurisdictional claims in stemness, resistance and roles as potential biomarkers. Mol. Cancer 18,90 published maps and institutional affiliations. (2019). 14. Kun-Peng, Z.,Xiao-Long,M.&Chun-Lin,Z.Overexpressed circPVT1, a potential Received: 23 May 2019 Revised: 24 June 2019 Accepted: 26 June 2019 new circular RNA biomarker, contributes to doxorubicin and cisplatin resis- tance of osteosarcoma cells by regulating ABCB1. Int. J. Biol. Sci. 14, 321–330 (2018). 15. Huang, X. et al. Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression. Mol. Cancer 18,71 (2019). References 16. Chi, B. J. et al. Downregulation of hsa_circ_0000285 serves as a prognostic 1. Wang,M., Yu, F.& Li, P.CircularRNAs: characteristics, function andclinical biomarker for bladder cancer and is involved in cisplatin resistance. Neoplasma significance in hepatocellular carcinoma. Cancers 10,pii:E258(2018). 66,197–202 (2019). 2. Fu, L., Jiang, Z., Li, T., Hu, Y. & Guo, J. Circular RNAs in hepatocellular carcinoma: 17. Feng, W. et al. ncRNAs associated with drug resistance and the therapy of Functions and implications. Cancer Med. 7, 3101–3109 (2018). digestive system neoplasms. J. Cell Physiol.(2019) https://doi.org/10.1002/ 3. Qiu, L. P. et al. The emerging role of circular RNAs in hepatocellular carcinoma. jcp.28551. J. Cancer 9, 1548–1559 (2018). 18. Wan,L.etal. MicroRNA-103 confers the resistance to long-treatment of 4. Hu, J. et al. Progress and prospects of circular RNAs in Hepatocellular carci- adriamycin to human leukemia cells by regulation of COP1. J. Cell Biochem. noma: novel insights into their function. J. Cell Physiol. 233, 4408–4422 (2018). 119, 3843–3852 (2018). 5. Yao, R.,Zou,H.& Liao,W.Prospect ofcircular RNA in hepatocellular carcinoma: 19. Garofalo, M. et al. EGFR and MET receptor tyrosine kinase-altered microRNA a novel potential biomarker and therapeutic target. Front Oncol. 8, 332 (2018). expression induces tumorigenesis and gefitinib resistance in lung cancers. Nat. 6. Guo, J.,Duan, H.,Li, Y.,Yang, L. &Yuan, L. A novel circular RNA circ-ZNF652 Med. 18,74–82 (2011). promotes hepatocellular carcinoma metastasis through inducing snail- 20. Zhang, Z. et al. circARF3 alleviates mitophagy-mediated inflammation by mediated epithelial-mesenchymal transition by sponging miR-203/miR-502- 5p. Biochem. Biophys. Res. Commun. 513,812–819 (2019). targeting miR-103/TRAF3 in mouse adipose tissue. Mol. Ther. Nucleic Acids 14, 7. Xu, L. et al. CircSETD3 (Hsa_circ_0000567) acts as a sponge for microRNA-421 192–203 (2019). inhibiting hepatocellular carcinoma growth. J. Exp. Clin. Cancer Res. 38,98 21. Nie, X. et al. Cloning, expression, and mutation analysis of NOR1, a novel (2019). human gene down-regulated in HNE1 nasopharyngeal carcinoma cell line. J. 8. Matboli, M. et al. circRNAs (hsa_circ_00156, hsa_circ _000224, and hsa_circ Cancer Res. Clin. Oncol. 129,410–414 (2003). _000520) are novel potential biomarkers in hepatocellular carcinoma. J. Cell 22. Wang, W. et al. Oxidored-nitro domain containing protein 1 (NOR1) Biochem. 38, BSR20180915 (2018). expression suppresses slug/vimentin but not snail in nasopharyngeal 9. Ding,B., Lou, W.,Xu, L. &Fan,W.Non-codingRNA in drug resistance of carcinoma: Inhibition of EMT in vitro and in vivo in mice. Cancer Lett. 348, hepatocellular carcinoma. Biosci. Rep. 38, pii: BSR20180915 (2018). 109–118 (2014). 10. Zhu, H. et al. Molecular mechanisms of cisplatin resistance in cervical cancer. 23. Shan, Z. et al. Overexpression of oxidored-nitro domain containing protein 1 Drug Des. Devel. Ther. 10, 1885–1895 (2016). induces growth inhibition and apoptosis in human prostate cancer PC3 cells. 11. Ghosh,S.Cisplatin:the first metal based anticancer drug. Bioorg. Chem. 88, Oncol. Rep. 32,1939–1946 (2014). 102925 (2019). 24. Gui,R., Li,D., Qi, G., Suhad, A. & Nie,X. Inhibitionof Grb2-mediated activation of 12. Sun, C.Y., Zhang, Q. Y.,Zheng,G.J.&Feng, B.Phytochemicals: current strategy MAPK signal transduction suppresses NOR1/CB1954-induced cytotoxicity in to sensitize cancer cells to cisplatin. Biomed. Pharm. 110,518–527 (2019). the HepG2 cell line. Oncol. Lett. 4, 566–570 (2012). Official journal of the Cell Death Differentiation Association

Journal

Cell Death Discovery – Springer Journals

Published: Jul 29, 2019

There are no references for this article.