Abstract
Here, we show that AU-1, spirostanol saponin isolated from Agavaceae plants, causes a transient increase in cyclin-dependent kinase inhibitor (CDKI) p21/Cip1 through the upregulation of miRNAs, miR-34 and miR-21. AU-1 stimulated p21/Cip1 expression without exerting cytotoxicity against different types of carcinoma cell lines. In renal adenocarcinoma ACHN cells, AU-1 transiently elevated the expression level of p21/Cip1 protein without marked increases in p21/Cip1 mRNA levels. Rapid and transient increases in miR-34 and miR-21, both of which are known to upregulate p21/Cip1, were observed in AU-1-treated cells. Inhibitor for miR-34 and for miR-21 significantly blocked the AU-1-caused increase in p21/Cip1, indicating that elevation of p21/Cip1 protein by AU-1 is dependent on these microRNAs. We further clarified that NAD-dependent deacetylase SIRT1, a direct target of miR-34, is decreased by the treatment with AU-1. Furthermore, we found that SIRT1-knockdown increases p21/Cip1 protein levels in an miR-21-dependent manner. On the other hand, ectopic expression of p21/Cip1 resulted in the lowered expression of miR-34 and miR-21, suggesting that reciprocal regulation exists between p21/Cip1 and these miRNAs. We propose that the following feedback network composed of miR-34/SIRT1/miR-21/p21 is triggered by the treatment with AU-1: in cells treated with AU-1, transient elevation of miR-34 leads to the downregulation of SIRT1, thereby miR-21 is freed from SIRT1-dependent suppression. Then, elevated miR-21 upregulates p21/Cip1 protein, followed by the suppression of miR-34 expression.
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Fujino T, Takeuchi A, Maruko-Ohtake A, Ohtake Y, Satoh J, Kobayashi T, Tanaka T, Ito H, Sakamaki R, Kashimura R, Ando K, Nishimaki-Mogami T, Ohkubo Y, Kitamura N, Sato R, Kikugawa K, Hayakawa M (2012) Critical role of farnesoid X receptor for hepatocellular carcinoma cell proliferation. J Biochem 152:577–586
Fujino T, Maruko-Ohtake A, Ohtake Y, Kobayashi T, Ando K, Takeuchi A, Ohkubo Y, Hayakawa M (2015) Farnesoid X receptor knockdown provides significant growth inhibition in hepatocellular carcinoma cells while it does not interfere with the proliferation of primary human hepatocyte-derived cells. J Toxicol Sci 40:501–508
Fujino T, Kuroda M, Matsuo Y, Kubo S, Tamura C, Sakamoto N, Mimaki Y, Hayakawa M (2015) Cardenolide glycosides from the seeds of Digitalis purpurea exhibit carcinoma-specific cytotoxicity toward renal adenocarcinoma and hepatocellular carcinoma cells. Biosci Biotechnol Biochem 79:177–184
Yokosuka A, Jitsuno M, Yui S, Yamazaki M, Mimaki Y (2009) Steroidal glycosides from Agave utahensis and their cytotoxic activity. J Nat Prod 72:1399–1404
Wilczynska A, Bushell M (2015) The complexity of miRNA-mediated repression. Cell Death Differ 22:22–33
Di Leva G, Garofalo M, Croce CM (2014) MicroRNAs in cancer. Annu Rev Pathol 9:287–314
Dellago H, Preschitz-Kammerhofer B, Terlecki-Zaniewicz L, Schreiner C, Fortschegger K, Chang MW, Hackl M, Monteforte R, Kühnel H, Schosserer M, Gruber F, Tschachler E, Scheideler M, Grillari-Voglauer R, Grillari J, Wieser M (2013) High levels of oncomiR-21 contribute to the senescence-induced growth arrest in normal human cells and its knock-down increases the replicative lifespan. Aging Cell 12:446–458
Zhao J, Lammers P, Torrance CJ, Bader AG (2013) TP53-independent function of miR-34a via HDAC1 and p21(CIP1/WAF1). Mol Ther 21:1678–1686
Wang Y, Zheng X, Zhang Z, Zhou J, Zhao G, Yang J, Xia L, Wang R, Cai X, Hu H, Zhu C, Nie Y, Wu K, Zhang D, Fan D (2012) MicroRNA-149 inhibits proliferation and cell cycle progression through the targeting of ZBTB2 in human gastric cancer. PLoS One 7:e41693
Jenkins RH, Davies LC, Taylor PR, Akiyama H, Cumbes B, Beltrami C, Carrington CP, Phillips AO, Bowen T, Fraser DJ (2014) miR-192 induces G2/M growth arrest in aristolochic acid nephropathy. Am J Pathol 184:996–1009
Yamakuchi M, Ferlito M, Lowenstein CJ (2008) miR-34a repression of SIRT1 regulates apoptosis. Proc Natl Acad Sci U S A 105:13421–13426
Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, Pandita TK, Guarente L, Weinberg RA (2001) hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 107:149–159
el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75:817–825
Dey N, Ghosh-Choudhury N, Kasinath BS, Choudhury GG (2012) TGFβ-stimulated microRNA-21 utilizes PTEN to orchestrate AKT/mTORC1 signaling for mesangial cell hypertrophy and matrix expansion. PLoS One 7:e42316
Bera A, Das F, Ghosh-Choudhury N, Kasinath BS, Abboud HE, Choudhury GG (2014) microRNA-21-induced dissociation of PDCD4 from rictor contributes to Akt-IKKβ-mTORC1 axis to regulate renal cancer cell invasion. Exp Cell Res 328:99–117
Acknowledgments
We thank Ken Ando, Toshiyuki Oshima, and Harutaka Ichikawa for their helpful advice and discussions. This work was supported, in part, by a grant from the Japan Private School Promotion Foundation.
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Fujino, T., Yokosuka, A., Higurashi, H. et al. AU-1 from Agavaceae plants causes transient increase in p21/Cip1 expression in renal adenocarcinoma ACHN cells in an miR-34-dependent manner. J Nat Med 71, 36–43 (2017). https://doi.org/10.1007/s11418-016-1024-y
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DOI: https://doi.org/10.1007/s11418-016-1024-y