Abstract
Introduction: The current study aimed to construct a circNR4A1 loaded exosome (EXO) nano system for the treatment of oral squamous cell carcinoma (OSCC) and elucidate the related regulation mechanism in chemoresistance and tumor biology.
Methods: Exosomes were isolated from the HEK293 cells by the ultracentrifugation method. Then, the purified nanoproduction was characterized and identified by transmission electron microscopy, dynamic light scattering, and Western blot. The hydrophobic circNR4A1 was then loaded into exosomes by the coincubation method. The effects of circNR4A1 on chemotherapy and apoptosis were evaluated in three multiresistant OSCC cell lines.
Results: The range of size distribution of the exosomes was found to be 40-170 nm. Positive TSG101 and CD63 expressions were observed, and the expression of calnexin was negative. In the cisplatin group, circNR4A1 could sensitize 67% of cell lines, while circNR4A1-EXO could sensitize 100% of cell lines. In the 5FU group, circNR4A1 could only sensitize 33% of cell lines, while circNR4A1-EXO could sensitize 67% of cell lines. circNR4A1-EXO is involved in multiple signaling pathways, which can not only activate K-RAS/ERK and MDR-1 signaling pathways but activate DNMT-1 gene expression simultaneously.
Conclusion: circNR4A1-EXO can increase the sensitivity of OSCC to anticancer drugs, which may be due to the regulation of the K-RAS/ERK and p53 signaling pathway.
Keywords: circNR4A1, Oral squamous cell carcinoma, Chemotherapy, Drug resistance
Graphical Abstract
[1]
Feng, X.; Luo, Q.; Wang, H.; Zhang, H.; Chen, F. MicroRNA-22 suppresses cell proliferation, migration and invasion in oral squamous cell carcinoma by targeting NLRP3. J. Cell. Physiol., 2018, 233(9), 6705-6713.
[http://dx.doi.org/10.1002/jcp.26331] [PMID: 29319163]
[http://dx.doi.org/10.1002/jcp.26331] [PMID: 29319163]
[2]
Smirk, R.; Kyzas, P. Outcome of salvage procedures for recurrent oral and oropharyngeal cancer. Br. J. Oral Maxillofac. Surg., 2018, 56(9), 847-853.
[http://dx.doi.org/10.1016/j.bjoms.2018.09.008] [PMID: 30293806]
[http://dx.doi.org/10.1016/j.bjoms.2018.09.008] [PMID: 30293806]
[3]
Economopoulou, P.; Kotsantis, I.; Psyrri, A. The promise of immunotherapy in head and neck squamous cell carcinoma: Combinatorial immunotherapy approaches. ESMO Open, 2017, 1(6), e000122-e000131.
[http://dx.doi.org/10.1136/esmoopen-2016-000122] [PMID: 28848660]
[http://dx.doi.org/10.1136/esmoopen-2016-000122] [PMID: 28848660]
[4]
Li, Z.; Zhu, X.; Huang, S. Extracellular vesicle long non-coding RNAs and circular RNAs: Biology, functions and applications in cancer. Cancer Lett., 2020, 489, 111-120.
[http://dx.doi.org/10.1016/j.canlet.2020.06.006] [PMID: 32561417]
[http://dx.doi.org/10.1016/j.canlet.2020.06.006] [PMID: 32561417]
[5]
Rybak-Wolf, A.; Stottmeister, C.; Glažar, P.; Jens, M.; Pino, N.; Giusti, S.; Hanan, M.; Behm, M.; Bartok, O.; Ashwal-Fluss, R.; Herzog, M.; Schreyer, L.; Papavasileiou, P.; Ivanov, A.; Öhman, M.; Refojo, D.; Kadener, S.; Rajewsky, N. Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed. Mol. Cell, 2015, 58(5), 870-885.
[http://dx.doi.org/10.1016/j.molcel.2015.03.027] [PMID: 25921068]
[http://dx.doi.org/10.1016/j.molcel.2015.03.027] [PMID: 25921068]
[6]
Chen, B.; Huang, S. Circular RNA: An emerging non-coding RNA as a regulator and biomarker in cancer. Cancer Lett., 2018, 418, 41-50.
[http://dx.doi.org/10.1016/j.canlet.2018.01.011] [PMID: 29330104]
[http://dx.doi.org/10.1016/j.canlet.2018.01.011] [PMID: 29330104]
[7]
Chen, L.L. The expanding regulatory mechanisms and cellular functions of circular RNAs. Nat. Rev. Mol. Cell Biol., 2020, 21(8), 475-490.
[http://dx.doi.org/10.1038/s41580-020-0243-y] [PMID: 32366901]
[http://dx.doi.org/10.1038/s41580-020-0243-y] [PMID: 32366901]
[8]
Yao, Z.; Luo, J.; Hu, K.; Lin, J.; Huang, H.; Wang, Q.; Zhang, P.; Xiong, Z.; He, C.; Huang, Z.; Liu, B.; Yang, Y. ZKSCAN1 gene and its related circular RNA (circZKSCAN1) both inhibit hepatocellular carcinoma cell growth, migration, and invasion but through different signaling pathways. Mol. Oncol., 2017, 11(4), 422-437.
[http://dx.doi.org/10.1002/1878-0261.12045] [PMID: 28211215]
[http://dx.doi.org/10.1002/1878-0261.12045] [PMID: 28211215]
[9]
Jin, H.; Jin, X.; Zhang, H.; Wang, W. Circular RNA hsa-circ-0016347 promotes proliferation, invasion and metastasis of osteosarcoma cells. Oncotarget, 2017, 8(15), 25571-25581.
[http://dx.doi.org/10.18632/oncotarget.16104] [PMID: 28424426]
[http://dx.doi.org/10.18632/oncotarget.16104] [PMID: 28424426]
[10]
Feng, D.; Xu, Y.; Hu, J.; Zhang, S.; Li, M.; Xu, L. A novel circular RNA, hsa-circ-0000211, promotes lung adenocarcinoma migration and invasion through sponging of hsa-miR-622 and modulating HIF1-α expression. Biochem. Biophys. Res. Commun., 2020, 521(2), 395-401.
[http://dx.doi.org/10.1016/j.bbrc.2019.10.134] [PMID: 31668923]
[http://dx.doi.org/10.1016/j.bbrc.2019.10.134] [PMID: 31668923]
[11]
Mathivanan, S.; Ji, H.; Simpson, R.J. Exosomes: Extracellular organelles important in intercellular communication. J. Proteomics, 2010, 73(10), 1907-1920.
[http://dx.doi.org/10.1016/j.jprot.2010.06.006] [PMID: 20601276]
[http://dx.doi.org/10.1016/j.jprot.2010.06.006] [PMID: 20601276]
[12]
Sharma, A. Role of stem cell derived exosomes in tumor biology. Int. J. Cancer, 2018, 142(6), 1086-1092.
[http://dx.doi.org/10.1002/ijc.31089] [PMID: 28983919]
[http://dx.doi.org/10.1002/ijc.31089] [PMID: 28983919]
[13]
Didiot, M.C.; Hall, L.M.; Coles, A.H.; Haraszti, R.A.; Godinho, B.M.; Chase, K.; Sapp, E.; Ly, S.; Alterman, J.F.; Hassler, M.R.; Echeverria, D.; Raj, L.; Morrissey, D.V.; DiFiglia, M.; Aronin, N.; Khvorova, A. Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing. Mol. Ther., 2016, 24(10), 1836-1847.
[http://dx.doi.org/10.1038/mt.2016.126] [PMID: 27506293]
[http://dx.doi.org/10.1038/mt.2016.126] [PMID: 27506293]
[14]
Théry, C.; Amigorena, S.; Raposo, G.; Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr. Protoc. Cell Biol; , 2006, Chapter 3, p. (1)22.
[http://dx.doi.org/10.1002/0471143030.cb0322s30] [PMID: 18228490]
[http://dx.doi.org/10.1002/0471143030.cb0322s30] [PMID: 18228490]
[15]
Johnsen, K.; Gudbergsson, J.; Skov, M.; Pilgaard, L.; Moos, T.; Duroux, M. 2014. A comprehensive overview of exosomes as drug delivery vehicles—Endogenous nanocarriers for targeted cancer therapy. Biochimica et Biophysica Acta (BBA) -. Rev. Can., 1846, (1), 75-87.
[16]
Vader, P.; Mol, E.A.; Pasterkamp, G.; Schiffelers, R.M. Extracellular vesicles for drug delivery. Adv. Drug Deliv. Rev, 2016, 106(Pt A), 148-156.
[http://dx.doi.org/10.1016/j.addr.2016.02.006] [PMID: 26928656]
[http://dx.doi.org/10.1016/j.addr.2016.02.006] [PMID: 26928656]
[17]
Kim, M.S.; Haney, M.J.; Zhao, Y.; Mahajan, V.; Deygen, I.; Klyachko, N.L.; Inskoe, E.; Piroyan, A.; Sokolsky, M.; Okolie, O.; Hingtgen, S.D.; Kabanov, A.V.; Batrakova, E.V. Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine, 2016, 12(3), 655-664.
[http://dx.doi.org/10.1016/j.nano.2015.10.012] [PMID: 26586551]
[http://dx.doi.org/10.1016/j.nano.2015.10.012] [PMID: 26586551]
[18]
Kooijmans, S.A.A.; Stremersch, S.; Braeckmans, K.; de Smedt, S.C.; Hendrix, A.; Wood, M.J.A.; Schiffelers, R.M.; Raemdonck, K.; Vader, P. Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles. J. Control. Release, 2013, 172(1), 229-238.
[http://dx.doi.org/10.1016/j.jconrel.2013.08.014] [PMID: 23994516]
[http://dx.doi.org/10.1016/j.jconrel.2013.08.014] [PMID: 23994516]
[19]
Steinbichler, T.B.; Dudás, J.; Skvortsov, S.; Ganswindt, U.; Riechelmann, H.; Skvortsova, I.I. Therapy resistance mediated by exosomes. Mol. Cancer, 2019, 18(1), 58-69.
[http://dx.doi.org/10.1186/s12943-019-0970-x] [PMID: 30925921]
[http://dx.doi.org/10.1186/s12943-019-0970-x] [PMID: 30925921]
[20]
Bach, D.H.; Hong, J.Y.; Park, H.J.; Lee, S.K. The role of exosomes and miRNAs in drug-resistance of cancer cells. Int. J. Cancer, 2017, 141(2), 220-230.
[http://dx.doi.org/10.1002/ijc.30669] [PMID: 28240776]
[http://dx.doi.org/10.1002/ijc.30669] [PMID: 28240776]
[21]
Lin, B.; Kolluri, S.K.; Lin, F.; Liu, W.; Han, Y.H.; Cao, X.; Dawson, M.I.; Reed, J.C.; Zhang, X.K. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell, 2004, 116(4), 527-540.
[http://dx.doi.org/10.1016/S0092-8674(04)00162-X] [PMID: 14980220]
[http://dx.doi.org/10.1016/S0092-8674(04)00162-X] [PMID: 14980220]
[22]
Lindner, K.; Borchardt, C.; Schöpp, M.; Bürgers, A.; Stock, C.; Hussey, D.J.; Haier, J.; Hummel, R. Proton pump inhibitors (PPIs) impact on tumour cell survival, metastatic potential and chemotherapy resistance, and affect expression of resistance-relevant miRNAs in esophageal cancer. J. Exp. Clin. Cancer Res., 2014, 33(1), 73.
[http://dx.doi.org/10.1186/s13046-014-0073-x] [PMID: 25175076]
[http://dx.doi.org/10.1186/s13046-014-0073-x] [PMID: 25175076]
[23]
Patop, I.L.; Kadener, S. circRNAs in Cancer. Curr. Opin. Genet. Dev., 2018, 48, 121-127.
[http://dx.doi.org/10.1016/j.gde.2017.11.007] [PMID: 29245064]
[http://dx.doi.org/10.1016/j.gde.2017.11.007] [PMID: 29245064]
[24]
Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc., 2008, 3(6), 1101-1108.
[http://dx.doi.org/10.1038/nprot.2008.73] [PMID: 18546601]
[http://dx.doi.org/10.1038/nprot.2008.73] [PMID: 18546601]
[25]
Zhang, B.; Wang, Z.; Shen, Y.; Yang, H. Silencing circular RNA hsa_circ_009755 promotes growth and metastasis of oral squamous cell carcinoma. Genomics, 2020, 112(6), 5275-5281.
[http://dx.doi.org/10.1016/j.ygeno.2020.09.035] [PMID: 32956844]
[http://dx.doi.org/10.1016/j.ygeno.2020.09.035] [PMID: 32956844]
[26]
Fan, H.Y.; Jiang, J.; Tang, Y.J.; Liang, X.H.; Tang, Y.L. CircRNAs: A new chapter in oral squamous cell carcinoma biology. OncoTargets Ther., 2020, 13, 9071-9083.
[http://dx.doi.org/10.2147/OTT.S263655] [PMID: 32982296]
[http://dx.doi.org/10.2147/OTT.S263655] [PMID: 32982296]
[27]
Liu, Y.Y.; Zhang, L.Y.; Du, W.Z. Circular RNA circ-PVT1 contributes to paclitaxel resistance of gastric cancer cells through the regulation of ZEB1 expression by sponging miR-124-3p. Biosci. Rep., 2019, 39(12), e1-e11.
[http://dx.doi.org/10.1042/BSR20193045] [PMID: 31793989]
[http://dx.doi.org/10.1042/BSR20193045] [PMID: 31793989]
[28]
Shang, Q.; Yang, Z.; Jia, R.; Ge, S. The novel roles of circRNAs in human cancer. Mol. Cancer, 2019, 18(1), 6.
[http://dx.doi.org/10.1186/s12943-018-0934-6] [PMID: 30626395]
[http://dx.doi.org/10.1186/s12943-018-0934-6] [PMID: 30626395]
[29]
Gao, D.; Zhang, X.; Liu, B.; Meng, D.; Fang, K.; Guo, Z.; Li, L. Screening circular RNA related to chemotherapeutic resistance in breast cancer. Epigenomics, 2017, 9(9), 1175-1188.
[http://dx.doi.org/10.2217/epi-2017-0055] [PMID: 28803498]
[http://dx.doi.org/10.2217/epi-2017-0055] [PMID: 28803498]
[30]
Mincheva-Nilsson, L.; Baranov, V.; Nagaeva, O.; Dehlin, E. Isolation and Characterization of Exosomes from Cultures of Tissue Explants and Cell Lines. Curr. Protoc. Immunol, 2016, 115(1), 42.1-21.
[http://dx.doi.org/10.1002/cpim.17] [PMID: 27801511]
[http://dx.doi.org/10.1002/cpim.17] [PMID: 27801511]
[31]
O’Loughlin, A.J.; Mäger, I.; de Jong, O.G.; Varela, M.A.; Schiffelers, R.M.; El Andaloussi, S.; Wood, M.J.A.; Vader, P. Functional delivery of lipid-conjugated siRNA by extracellular vesicles. Mol. Ther., 2017, 25(7), 1580-1587.
[http://dx.doi.org/10.1016/j.ymthe.2017.03.021] [PMID: 28392161]
[http://dx.doi.org/10.1016/j.ymthe.2017.03.021] [PMID: 28392161]
[32]
Xia, L.; Wu, L.; Bao, J.; Li, Q.; Chen, X.; Xia, H.; Xia, R. Circular RNA circ-CBFB promotes proliferation and inhibits apoptosis in chronic lymphocytic leukemia through regulating miR-607/FZD3/Wnt/β-catenin pathway. Biochem. Biophys. Res. Commun., 2018, 503(1), 385-390.
[http://dx.doi.org/10.1016/j.bbrc.2018.06.045] [PMID: 29902450]
[http://dx.doi.org/10.1016/j.bbrc.2018.06.045] [PMID: 29902450]
[33]
Xu, G.; Chen, Y.; Fu, M.; Zang, X.; Cang, M.; Niu, Y.; Zhang, W.; Zhang, Y.; Mao, Z.; Shao, M.; Qian, H.; Xu, W.; Cai, H.; Jiang, P.; Zhang, X. Circular RNA CCDC66 promotes gastric cancer progression by regulating c-Myc and TGF-β signaling pathways. J. Cancer, 2020, 11(10), 2759-2768.
[http://dx.doi.org/10.7150/jca.37718] [PMID: 32226494]
[http://dx.doi.org/10.7150/jca.37718] [PMID: 32226494]
[34]
Han, W.; Wang, L.; Zhang, L.; Wang, Y.; Li, Y. Circular RNA circ-RAD23B promotes cell growth and invasion by miR-593-3p/CCND2 and miR-653-5p/TIAM1 pathways in non-small cell lung cancer. Biochem. Biophys. Res. Commun., 2019, 510(3), 462-466.
[http://dx.doi.org/10.1016/j.bbrc.2019.01.131] [PMID: 30722989]
[http://dx.doi.org/10.1016/j.bbrc.2019.01.131] [PMID: 30722989]
[35]
Chen, N.; Zhao, G.; Yan, X.; Lv, Z.; Yin, H.; Zhang, S.; Song, W.; Li, X.; Li, L.; Du, Z.; Jia, L.; Zhou, L.; Li, W.; Hoffman, A.R.; Hu, J.F.; Cui, J. A novel FLI1 exonic circular RNA promotes metastasis in breast cancer by coordinately regulating TET1 and DNMT1. Genome Biol., 2018, 19(1), 218.
[http://dx.doi.org/10.1186/s13059-018-1594-y] [PMID: 30537986]
[http://dx.doi.org/10.1186/s13059-018-1594-y] [PMID: 30537986]
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