Quercetin protects PC-12 cells against hypoxia injury by down-regulation of miR-122

Document Type : Original Article


Department of Children Rehabilitation, Women & Children’s Health Care Hospital of Linyi, Linyi 276016, Shandong, China


Objective(s): Impairment of nerve cells of brain induced by hypoxia results in energy-deprivation and dysfunction, which accompanies with neurons apoptosis. Improving function of nerve cells is important for treating cerebral anoxia. This study aimed to investigate the role of Quercetin (Quer) in hypoxia-induced injury of pheochromocytoma (PC-12) cells.
Materials and Methods: PC-12 cells were cultured under anoxic condition for induction of hypoxia injury and/or treatment with Quer, transfection with pre-miR-122, anti-miR-122 or their negative controls. After Quer treatment, viability, migration, and cell apoptosis of PC-12 cells were analyzed by CCK-8 assay, transwell assay and flowcytometry analysis, respectively. Cell proliferation-associated proteins and cell apoptosis-associated proteins were analyzed by Western blot. Relative miR-122 expression in Quer-treated cells or transfection efficacy of miR-122 was analyzed by qRT-PCR. Finally, main components in AMP-activated protein kinase (AMPK) and Wnt/β-catenin signaling pathways were analyzed by Western blot.
Results: Quer alleviated hypoxia-induced injury in PC-12 cells by increasing viability, promoting cell proliferation, enhancing migration and repressing apoptosis. Also, miR-122 was down-regulated in Quer-treated cells. miR-122 overexpression decreased cell viability and migration, and increased cell apoptosis in hypoxia- treated PC-12 cells, but miR-122 silencing led to the opposite results. We also found that AMPK and Wnt/β-catenin signaling pathways were activated by Quer in hypoxia-induced injury, but were inactivated in hypoxia-induced cells by overexpression of miR-122.
Conclusion: Quer could repress hypoxia-induced injury in PC-12 cells by activating AMPK and Wnt/β-catenin signaling pathways via down-regulation of miR-122.


Main Subjects

1. Aly H, Elmahdy H, El-Dib M, Rowisha M, Awny M, El-Gohary T, et al. Melatonin use for neuroprotection in perinatal asphyxia: a randomized controlled pilot study. J Perinatol 2015; 35:186-191.
2. Shankaran S. Neonatal encephalopathy: treatment with hypothermia. J Neurotrauma 2009; 26:437-443.
3. Lin EP, Miles L, Hughes EA, McCann JC, Vorhees CV, McAuliffe JJ, et al. A combination of mild hypothermia and sevoflurane affords long-term protection in a modified neonatal mouse model of cerebral hypoxia-ischemia. Anesth Analg 2014; 119:1158-1173.
4. van Handel M, Swaab H, de Vries LS, Jongmans MJ. Long-term cognitive and behavioral consequences of neonatal encephalopathy following perinatal asphyxia: a review. Eur J Pediatr 2007; 166:645-654.
5. Zhang P, Cheng G, Chen L, Zhou W, Sun J. Cerebral hypoxia-ischemia increases toll-like receptor 2 and 4 expression in the hippocampus of neonatal rats. Brain Dev 2015; 37:747-752.
6. Long FY, Shi MQ, Zhou HJ, Liu DL, Sang N, Du JR. Klotho upregulation contributes to the neuroprotection of ligustilide against cerebral ischemic injury in mice. Eur J Pharmacol 2018; 820:198-205.
7. Zhou L, Bondy SC, Jian L, Wen P, Yang F, Luo H, et al. Tanshinone IIA attenuates the cerebral ischemic injury-induced increase in levels of GFAP and of caspases-3 and -8. Neurosci 2015; 288:105-111.
8. Blasina F, Vaamonde L, Silvera F, Tedesco AC, Dajas F. Intravenous nanosomes of quercetin improve brain function and hemodynamic instability after severe hypoxia in newborn piglets. Neurochem Int 2015; 89:149-156.
9. Chiş IC, Baltaru D, Dumitrovici A, Coseriu A, Radu BC, Moldovan R, et al. Quercetin ameliorate oxidative/nitrosative stress in the brain of rats exposed to intermittent hypobaric hypoxia. Rev Virtual Qui 2016; 8:369-383.
10. Lee HN, Shin SA, Choo GS, Kim HJ, Park YS, Kim BS, et al. Antiinflammatory effect of quercetin and galangin in LPS stimulated RAW264.7 macrophages and DNCB induced atopic dermatitis animal models. Int J Mol Med 2018; 41:888-898.
11. Li X, Zhou N, Wang J, Liu Z, Wang X, Zhang Q, et al. Quercetin suppresses breast cancer stem cells (CD44(+)/CD24(-)) by inhibiting the PI3K/Akt/mTOR-signaling pathway. Life Sci 2018; 196:56-62.
12. Kamada C, Mukai R, Kondo A, Sato S, Terao J. Effect of quercetin and its metabolite on caveolin-1 expression induced by oxidized LDL and lysophosphatidylcholine in endothelial cells. J Clin Biochem Nutr 2016; 58:193-201.
13. Marunaka Y, Marunaka R, Sun H, Yamamoto T, Kanamura N, Inui T, et al. Actions of quercetin, a polyphenol, on blood pressure. Molecules 2017; 22:E209.
14. Singh S, Kushwah V, Agrawal AK, Jain S. Insulin- and quercetin-loaded liquid crystalline nanoparticles: implications on oral bioavailability, antidiabetic and antioxidant efficacy. Nanomedicine (Lond) 2018; 13:521-537.
15. Lei X, Chao H, Zhang Z, Lv J, Li S, Wei H, et al. Neuroprotective effects of quercetin in a mouse model of brain ischemic/reperfusion injury via anti-apoptotic mechanisms based on the Akt pathway. Mol Med Rep 2015;12:3688-3696.
16. Li X, Wang H. Neuroprotection by quercetin via mitochondrial function adaptation in traumatic brain injury: PGC-1alpha pathway as a potential mechanism. J Cell Mol Med  2018; 22:883-891.
17. Pandey AK, Shukla SC, Bhattacharya P, Patnaik R. A possible therapeutic potential of quercetin through inhibition of mu-calpain in hypoxia induced neuronal injury: a molecular dynamics simulation study. Neural Regen Res 2016; 11:1247-1253.
18. Qu X, Qi D, Dong F, Wang B, Guo R, Luo M, et al. Quercetin improves hypoxia-ischemia induced cognitive deficits via promoting remyelination in neonatal rat. Brain Res 2014; 17:31-40.
19. Sonkoly E, Pivarcsi A. microRNAs in inflammation. Int Rev Immunol 2009; 28:535-561.
20. Hsu S, Wang B, Kota J, Yu J, Costinean S, Kutay H, et al. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest 2012; 122:2871-2883.
21. Boesch-Saadatmandi C, Wagner AE, Wolffram S, Rimbach G. Effect of quercetin on inflammatory gene expression in mice liver in vivo – role of redox factor 1, miRNA-122 and miRNA-125b. Pharmacol Res 2012; 65:523-530.
22. Csak T, Bala S, Lippai D, Satishchandran A, Catalano D, Kodys K, et al. microRNA-122 regulates hypoxia-inducible factor-1 and vimentin in hepatocytes and correlates with fibrosis in diet-induced steatohepatitis. Liver Int 2015; 35:532-541.
23. Thatipamula S, Al Rahim M, Zhang J, Hossain MA. Genetic deletion of neuronal pentraxin 1 expression prevents brain injury in a neonatal mouse model of cerebral hypoxia-ischemia. Neurobiol Dis 2015; 75:15-30.
24. Kalenderian E, Pegus C, Francis C, Goodwin N, Jacques HS, Lasa D. Cardiovascular disease urban intervention: baseline activities and findings. J Community Health 2009; 34:282-287.
25. Bova RJ, Quinn DI, Nankervis JS, Cole IE, Sheridan BF, Jensen MJ, et al. Cyclin D1 and p16INK4A expression predict reduced survival in carcinoma of the anterior tongue. Clin Cancer Res 1999; 5:2810-2819.
26. Chen X, Tian Y, Yao L, Zhang J, Liu Y. Hypoxia stimulates proliferation of rat neural stem cells with influence on the expression of cyclin D1 and c-Jun N-terminal protein kinase signaling pathway in vitro. Neurosci 2009; 165:705-714.
27. Bindra RS, Vasselli JR, Stearman R, Linehan WM, Klausner RD. VHL-mediated hypoxia regulation of cyclin D1 in renal carcinoma cells. Cancer Res 2002; 62:3014-3019.
28. Emmi P, Peppi K, Kirsi-Maria H, Risto B, Arja JV. The prognostic significance and value of cyclin D1, CDK4 and p16 in human breast cancer. Breast Cancer Res 2013; 15:R5.
29. Fu M, Wang C, Li Z, Sakamaki T, Pestell RG. Cyclin D1: normal and abnormal functions. Endocrinol 2005; 145:5439-5447.
30. Wen W, Ding J, Sun W, Wu K, Ning B, Gong W, et al. Suppression of cyclin D1 by hypoxia-inducible factor-1 via direct mechanism inhibits the proliferation and 5-fluorouracil-induced apoptosis of A549 cells. Cancer Res 2010; 70:2010-2019.
31. Bae S, Jeong HJ, Cha HJ, Kim K, Choi YM, An IS, et al. The hypoxia-mimetic agent cobalt chloride induces cell cycle arrest and alters gene expression in U266 multiple myeloma cells. Int J Mol Med 2012; 30:1180-1186.
32. Zygmunt A, Tedesco VC, Udho E, Krucher NA. Hypoxia stimulates p16 expression and association with cdk4. Exp Cell Res 2002; 278:53-60.
33. Conrad PW, Rust RT, Han J, Millhorn DE, Beitnerjohnson D. Selective activation of p38alpha and p38gamma by hypoxia. Role in regulation of cyclin D1 by hypoxia in PC12 cells. J Biol Chem 1999; 274:23570-23576.
34. Blasina F, Vaamonde L, Silvera F, Tedesco AC, Dajas F. Intravenous nanosomes of quercetin improve brain function and hemodynamic instability after severe hypoxia in newborn piglets. Neurochem Int 2015; 89:149-156.
35. Liu P, Zou D, Yi L, Chen M, Gao Y, Zhou R, et al. Quercetin ameliorates hypobaric hypoxia-induced memory impairment through mitochondrial and neuron function adaptation via the PGC-1α pathway. Restor Neurol Neurosci 2015; 33:143-157.
36. Alrasheed NM, Fadda L, Attia HA, Sharaf IA, Mohamed AM, Alrasheed NM. Original research paper. Pulmonary prophylactic impact of melatonin and/or quercetin: A novel therapy for inflammatory hypoxic stress in rats. Acta Pharm 2017; 67:125-135.
37. Tsai WC, Hsu PWC, Lai TC, Chau GY, Lin CW, Chen CM, et al. MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma. Hepatology 2009; 49:1571-1582.
38. Wang X, Lam EK, Zhang J, Jin H, Sung JJ. MicroRNA-122a functions as a novel tumor suppressor downstream of adenomatous polyposis coli in gastrointestinal cancers. Biochem Biophys Res Commun 2009; 387:376-380.
39. Liang W, Guo J, Li J, Bai C, Dong Y. Downregulation of miR-122 attenuates hypoxia/reoxygenation (H/R)-induced myocardial cell apoptosis by upregulating GATA-4. Biochem Biophys Res Commun 2016; 478:1416-1422.
40. Zhang Z, Li H, Chen S, Li Y, Cui Z, Ma J. Knockdown of microRNA-122 protects H9c2 cardiomyocytes from hypoxia-induced apoptosis and promotes autophagy. Med Sci Monit 2017; 23:4284-4290.
41. Xiao Q, Ye QF, Wang W, Fu BQ, Xia ZP, Liu ZZ, et al. Mild hypothermia pretreatment protects hepatocytes against ischemia reperfusion injury via down-regulating miR-122 and IGF-1R/AKT pathway. Cryobiology 2017; 75:100-105.
42. Rousset CI, Leiper FC, Kichev A, Gressens P, Carling D, Hagberg H, et al. A dual role for AMP-activated protein kinase (AMPK) during neonatal hypoxic–ischaemic brain injury in mice. J Neurochem 2015; 133:242-252.
43. Kim SG, Kim JR, Choi HC. Quercetin-induced AMP-activated protein kinase activation attenuates vasoconstriction through LKB1-AMPK signaling pathway. J Med Food 2018; 21:146-153.
44. Dhanya R, Arya AD, Nisha P, Jayamurthy P. Quercetin, a lead compound against type 2 diabetes ameliorates glucose uptake via AMPK pathway in skeletal muscle cell line. Front Pharmacol 2017; 8:336-345.
45. Fancy SPJ, Harrington EP, Baranzini SE, Silbereis JC, Shiow LR, Yuen TJ, et al. Parallel states of pathological Wnt signaling in neonatal brain injury and colon cancer. Nat Neurosci 2014; 17:506-512.
46. Jie X, Zhu X, Wu L, Rong Y, Yang Z, Wang Q, et al. MicroRNA‐122 suppresses cell proliferation and induces cell apoptosis in hepatocellular carcinoma by directly targeting Wnt/β‐catenin pathway. Liver Int 2012; 32:752-760.
47. Varela-Nallar L, Rojas-Abalos M, Abbott AC, Moya EA, Iturriaga R, Inestrosa NC. Chronic hypoxia induces the activation of the Wnt/β-catenin signaling pathway and stimulates hippocampal neurogenesis in wild-type and APPswe-PS1ΔE9 transgenic mice in vivo. Front Cell Neurosci 2014; 8:17-26.