MicroRNA‑204 may participate in the pathogenesis of hypoxic‑ischemic encephalopathy through targeting KLLN

Chen,Ronglin; Wang,Meixia; Fu,Shaopin; Cao,Feng; Duan,Pengkai; Lu,Jiefu

doi:10.3892/etm.2019.7936

MicroRNA‑204 may participate in the pathogenesis of hypoxic‑ischemic encephalopathy through targeting KLLN

Authors:
- Ronglin Chen
- Meixia Wang
- Shaopin Fu
- Feng Cao
- Pengkai Duan
- Jiefu Lu
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Affiliations: Department of Critical Care Medicine, Longgang District Central Hospital, Shenzhen, Guangdong 518116, P.R. China, Department of Intensive Care Unit, Affiliated General Hospital of Guangzhou Military Command of Southern Medical University, Guangzhou, Guangdong 510515, P.R. China
Published online on: August 26, 2019 https://doi.org/10.3892/etm.2019.7936
Pages: 3299-3306
Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Hypoxic‑ischemic encephalopathy (HIE) is a common neonatal disease that can lead to high neonatal mortality rates. Previous studies have indicated that microRNAs (miRs) may be involved in the pathogenesis of HIE; however, the specific mechanisms underlying their involvement require further investigation. The aim of the present study was to investigate the roles of miR‑204 and its target gene killin p53 regulated DNA replication inhibitor (KLLN) in HIE using rat HIE models. Brain injury was induced by surgery and incubation of hypoxic incubator brain using 10‑day‑old pup rats. On day 3, rats were sacrificed, and the infarct size of the brain was determined using a tetrazolium chloride assay. Terminal deoxynucleotidyl transferase UTP nick‑end labeling staining was performed to detect the cell death rate in the brain tissue. Following this, the brain tissues were collected, and reverse transcription‑quantitative polymerase chain reaction, western blot analysis and immunohistochemistry assays were performed to examine the expression levels of miR‑204 and KLLN. Furthermore, neurons were cultured and transfected with miR‑204 inhibitors or mimics, and the effect of miR‑204 on the proliferation and apoptosis of neurons was examined using MTT and flow cytometric assays. Finally, a dual‑luciferase reporter assay was performed to confirm whether KLLN is a direct target of miR‑204. The expression of miR‑204 was significantly downregulated and the expression of KLLN was significantly increased in the brain tissue of HIE rats (P<0.001). In addition, the transfection with miR‑204 inhibitors significantly decreased the proliferation rates and significantly increased the apoptosis rate of neurons; however, transfection with miR‑204 mimics prompted the opposite results. The dual‑luciferase reporter assay also confirmed that KLLN is a direct target of miR‑204. Taken together, the results of the present study demonstrated that miR‑204 was downregulated in HIE and that miR‑204 may serve important roles in the pathogenesis of HIE through targeting KLLN.

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1	Leroy-Terquem E, Vermersch AI, Dean P, Assaf Z, Boddaert N, Lapillonne A and Magny JF: Abnormal interhemispheric synchrony in neonatal hypoxic-ischemic encephalopathy: A retrospective pilot study. Neonatology. 112:359–364. 2017. View Article : Google Scholar : PubMed/NCBI
2	Yum SK, Seo YM, Kwun Y, Moon CJ, Youn YA and Sung IK: Therapeutic hypothermia in infants with hypoxic-ischemic encephalopathy and reversible persistent pulmonary hypertension: Short-term hospital outcomes. J Matern Fetal Neonatal Med. 31:3108–3114. 2018. View Article : Google Scholar : PubMed/NCBI
3	Jain SV, Pagano L, Gillam-Krakauer M, Slaughter JC, Pruthi S and Engelhardt B: Cerebral regional oxygen saturation trends in infants with hypoxic-ischemic encephalopathy. Early Human Dev. 113:55–61. 2017. View Article : Google Scholar
4	Ma Q and Zhang L: MiR210 in neonatal hypoxic-ischemic encephalopathy. Oncotarget. 8:38078–38079. 2017.PubMed/NCBI
5	Chen J, Cui C, Yang X, Xu J, Venkat P, Zacharek A, Yu P and Chopp M: MiR-126 affects brain-heart interaction after cerebral ischemic stroke. Transl Stroke Res. 8:374–385. 2017. View Article : Google Scholar : PubMed/NCBI
6	Wang J, Chen T and Shan G: miR-148b regulates proliferation and differentiation of neural stem cells via Wnt/β-catenin signaling in rat ischemic stroke model. Front Cell Neurosci. 11:3292017. View Article : Google Scholar : PubMed/NCBI
7	Wei H, Zhang JJ and Tang QL: MiR-638 inhibits cervical cancer metastasis through Wnt/beta-catenin signaling pathway and correlates with prognosis of cervical cancer patients. Eur Rev Med Pharmacol Sci. 21:5587–5593. 2017.PubMed/NCBI
8	Wu H, Liu HY, Liu WJ, Shi YL and Bao D: miR-377-5p inhibits lung cancer cell proliferation, invasion, and cell cycle progression by targeting AKT1 signaling. J Cell Biochem. Nov 28–2018.(Epub ahead of print).
9	Pan L, Tang Z, Pan L and Tang R: miR-3666 inhibits lung cancer cell proliferation, migration and invasion by targeting BPTF. Biochem Cell Biol. 97:415–422. 2019. View Article : Google Scholar : PubMed/NCBI
10	Mansoori B, Mohammadi A, Ghasabi M, Shirjang S, Dehghan R, Montazeri V, Holmskov U, Kazemi T, Duijf P, Gjerstorff M and Baradaran B: miR-142-3p as tumor suppressor miRNA in the regulation of tumorigenicity, invasion and migration of human breast cancer by targeting Bach-1 expression. J Cell Physiol. 234:9816–9825. 2019. View Article : Google Scholar : PubMed/NCBI
11	Yang L, Hou J, Cui XH, Suo LN and Lv YW: MiR-133b regulates the expression of CTGF in epithelial-mesenchymal transition of ovarian cancer. Eur Rev Med Pharmacol Sci. 21:5602–5609. 2017.PubMed/NCBI
12	Qiao Y, Peng C, Li J, Wu D and Wang X: Spinal cord ischemia-reperfusion causes damage of neurocyte by inhibiting RAP2C. Neurol Res. 39:877–884. 2017. View Article : Google Scholar : PubMed/NCBI
13	Peng T, Jia YJ, Wen QQ, Guan WJ, Zhao EY and Zhang BA: Expression of microRNA in neonatal rats with hypoxic-ischemic brain damage. Zhongguo Dang Dai Er Ke Za Zhi. 12:373–376. 2010.(In Chinese). PubMed/NCBI
14	Edwards AB, Feindel KW, Cross JL, Anderton RS, Clark VW, Knuckey NW and Meloni BP: Modification to the Rice-Vannucci perinatal hypoxic-ischaemic encephalopathy model in the P7 rat improves the reliability of cerebral infarct development after 48 hours. J Neurosci Methods. 288:62–71. 2017. View Article : Google Scholar : PubMed/NCBI
15	Gonzalez-Rodriguez PJ, Li Y, Martinez F and Zhang L: Dexamethasone protects neonatal hypoxic-ischemic brain injury via L-PGDS-dependent PGD2-DP1-pERK signaling pathway. PLoS One. 9:e1144702014. View Article : Google Scholar : PubMed/NCBI
16	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
17	Jin Q, He W, Chen L, Yang Y, Shi K and You Z: MicroRNA-101-3p inhibits proliferation in retinoblastoma cells by targeting EZH2 and HDAC9. Exp Ther Med. 16:1663–1670. 2018.PubMed/NCBI
18	Ma Q, Dasgupta C, Li Y, Huang L and Zhang L: MicroRNA-210 suppresses junction proteins and disrupts blood-brain barrier integrity in neonatal rat hypoxic-ischemic brain injury. Int J Mol Sci. 18:E13562017. View Article : Google Scholar : PubMed/NCBI
19	Wang L, Ke J, Li Y, Ma Q, Dasgupta C, Huang X, Zhang L and Xiao D: Inhibition of miRNA-210 reverses nicotine-induced brain hypoxic-ischemic injury in neonatal rats. Int J Biol Sci. 13:76–84. 2017. View Article : Google Scholar : PubMed/NCBI
20	Looney AM, Walsh BH, Moloney G, Grenham S, Fagan A, O'Keeffe GW, Clarke G, Cryan JF, Dinan TG, Boylan GB and Murray DM: Downregulation of umbilical cord blood levels of miR-374a in neonatal hypoxic ischemic encephalopathy. J Pediatr. 167:269–273.e2. 2015. View Article : Google Scholar : PubMed/NCBI
21	Ding X, Sun B, Huang J, Xu L, Pan J, Fang C, Tao Y, Hu S, Li R, Han X, et al: The role of miR-182 in regulating pineal CLOCK expression after hypoxia-ischemia brain injury in neonatal rats. Neurosci Lett. 591:75–80. 2015. View Article : Google Scholar : PubMed/NCBI
22	Fang C, Xie L, Liu C, Fu C, Ye W, Liu H and Zhang B: Tanshinone IIA improves hypoxic ischemic encephalopathy through TLR-4-mediated NF-κB signal pathway. Mol Med Rep. 18:1899–1908. 2018.PubMed/NCBI
23	Wang B, Armstrong JS, Lee JH, Bhalala U, Kulikowicz E, Zhang H, Reyes M, Moy N, Spicer D, Zhu J, et al: Rewarming from therapeutic hypothermia induces cortical neuron apoptosis in a swine model of neonatal hypoxic-ischemic encephalopathy. J Cereb Blood Flow Metab. 35:781–793. 2015. View Article : Google Scholar : PubMed/NCBI
24	Wang CM, Cheng BH, Xue QJ, Chen J and Bai B: MiR-1298 affects cell proliferation and apoptosis in C6 cells by targeting SET domain containing 7. Int J Immunopathol Pharmacol. 30:264–271. 2017. View Article : Google Scholar : PubMed/NCBI
25	Huang Q, Zheng Y, Ou Y, Xiong H, Yang H, Zhang Z, Chen S and Ye Y: miR-34a/Bcl-2 signaling pathway contributes to age-related hearing loss by modulating hair cell apoptosis. Neurosci Lett. 661:51–56. 2017. View Article : Google Scholar : PubMed/NCBI
26	Shu Y, Ren L, Xie B, Liang Z and Chen J: MiR-204 enhances mitochondrial apoptosis in doxorubicin-treated prostate cancer cells by targeting SIRT1/p53 pathway. Oncotarget. 8:97313–97322. 2017. View Article : Google Scholar : PubMed/NCBI
27	Zhao R, He H, Zhu Y, Wan J, Li Y, Gao S and Zhang C: MiR-204/14-3-3ζ axis regulates osteosarcoma cell proliferation through SATA3 pathway. Pharmazie. 72:593–598. 2017.PubMed/NCBI
28	Lin YC, Lin JF, Tsai TF, Chou KY, Chen HE and Hwang TI: Tumor suppressor miRNA-204-5p promotes apoptosis by targeting BCL2 in prostate cancer cells. Asian J Surg. 40:396–406. 2017. View Article : Google Scholar : PubMed/NCBI
29	Sankunny M and Eng C: KLLN-mediated DNA damage-induced apoptosis is associated with regulation of p53 phosphorylation and acetylation in breast cancer cells. Cell Death Dis. 4:312018.
30	Cho YJ and Liang P: Killin is a p53-regulated nuclear inhibitor of DNA synthesis. Proc Natl Acad Sci USA. 105:5396–5401. 2008. View Article : Google Scholar : PubMed/NCBI
31	Wang Y, He X, Yu Q and Eng C: Androgen receptor-induced tumor suppressor, KLLN, inhibits breast cancer growth and transcriptionally activates p53/p73-mediated apoptosis in breast carcinomas. Hum Mol Genet. 22:2263–2272. 2013. View Article : Google Scholar : PubMed/NCBI
32	Wang Y, Radhakrishnan D, He X, Peehl DM and Eng C: Transcription factor KLLN inhibits tumor growth by AR suppression, induces apoptosis by TP53/TP73 stimulation in prostate carcinomas, and correlates with cellular differentiation. J Clin Endocrinol Metab. 98:E586–E594. 2013. View Article : Google Scholar : PubMed/NCBI
33	Nizialek EA, Mester JL, Dhiman VK, Smiraglia DJ and Eng C: KLLN epigenotype-phenotype associations in Cowden syndrome. Eur J Hum Genet. 23:1538–1543. 2015. View Article : Google Scholar : PubMed/NCBI
34	Wang Y, Yu Q, He X, Romigh T, Altemus J and Eng C: Activation of AR sensitizes breast carcinomas to NVP-BEZ235's therapeutic effect mediated by PTEN and KLLN upregulation. Mol Cancer Ther. 13:517–527. 2014. View Article : Google Scholar : PubMed/NCBI
35	Hu K and Liang M: Upregulated microRNA-224 promotes ovarian cancer cell proliferation by targeting KLLN. In Vitro Cell Dev Biol Anim. 53:149–156. 2017. View Article : Google Scholar : PubMed/NCBI
36	Qiu R, Li W and Liu Y: MicroRNA-204 protects H9C2 cells against hypoxia/reoxygenation-induced injury through regulating SIRT1-mediated autophagy. Biomed Pharmacother. 100:15–19. 2018. View Article : Google Scholar : PubMed/NCBI