Knockdown of lncRNA NEAT1 expression inhibits cell migration, invasion and EMT by regulating the miR‑24‑3p/LRG1 axis in retinoblastoma cells

Luan,Lan; Hu,Qiang; Wang,Yan; Lu,Lu; Ling,Jiaojiao

doi:10.3892/etm.2021.9798

Knockdown of lncRNA NEAT1 expression inhibits cell migration, invasion and EMT by regulating the miR‑24‑3p/LRG1 axis in retinoblastoma cells

Authors:
- Lan Luan
- Qiang Hu
- Yan Wang
- Lu Lu
- Jiaojiao Ling
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Affiliations: Department of Ophthalmology, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China
Published online on: February 18, 2021 https://doi.org/10.3892/etm.2021.9798
Article Number: 367
Copyright: © Luan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Retinoblastoma (RB) is the most common primary intraocular cancer type that occurs during retinal development in childhood. Previous studies have reported that long non‑coding RNAs (lncRNAs) are involved in the development of RB. Therefore, the aim of the present study was to investigate the effects and underlying regulatory mechanisms of nuclear paraspeckle assembly transcript 1 (NEAT1) in RB. The expression levels of NEAT1, microRNA (miR)‑24‑3p and leucine‑rich‑α‑2‑glycoprotein (LRG1) were detected using reverse transcription‑quantitative PCR (RT‑qPCR). Moreover, the protein expression levels of LRG1, matrix metalloproteinase 9, N‑cadherin and E‑cadherin were detected via western blotting. Furthermore, cell migration and invasion abilities were evaluated via Transwell assays. The targeting relationships between miR‑24‑3p and NEAT1 or LRG1 were predicted using online software and confirmed via dual‑luciferase reporter assay. In the present study, NEAT1 and LRG1 were upregulated, and miR‑24‑3p was downregulated in RB tissues and cells compared with the corresponding healthy tissues and cells. Moreover, miR‑24‑3p was identified as a target of NEAT and LRG1 was demonstrated to be a direct target gene of miR‑24‑3p. Knockdown of NEAT1 or LRG1 significantly suppressed RB cell migration and invasion ability, while the effects were reversed by an miR‑24‑3p inhibitor. In addition, the downregulation of LRG1 caused by miR‑24‑3p was restored following the overexpression of NEAT1 in RB cells. It was also demonstrated that NEAT1 knockdown inhibited the epithelial‑to‑mesenchymal transition (EMT) pathway by inhibiting the expression of LRG via targeting miR‑24‑3p. In conclusion, the present results suggest that silencing of NEAT1 suppresses cell migration, invasion and the EMT process by downregulating LRG1 expression via sponging miR‑24‑3p in RB, thus indicating that NEAT1 may be a potential candidate for RB treatment.

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1	Dimaras H, Kimani K, Dimba EA, Gronsdahl P, White A, Chan HS and Gallie BL: Retinoblastoma. Lancet. 379:1436–1446. 2012.PubMed/NCBI View Article : Google Scholar
2	Kivelä T: The epidemiological challenge of the most frequent eye cancer: Retinoblastoma, an issue of birth and death. Br J Ophthalmol. 93:1129–1131. 2009.PubMed/NCBI View Article : Google Scholar
3	Cimino PJ, Robirds DH, Tripp SR, Pfeifer JD, Abel HJ and Duncavage EJ: Retinoblastoma gene mutations detected by whole exome sequencing of merkel cell carcinoma. Mod Pathol. 27:1073–1087. 2014.PubMed/NCBI View Article : Google Scholar
4	Khan AA, Bukhari MH and Mehboob R: Association of retinoblastoma with clinical and histopathological risk factors. Nat Sci. 5:437–444. 2013.
5	Abramson DH, Shields CL, Munier FL and Chantada GL: Treatment of retinoblastoma in 2015: Agreement and disagreement. JAMA Ophthalmol. 133:1341–1347. 2015.PubMed/NCBI View Article : Google Scholar
6	Peng WX, Koirala P and Mo YY: LncRNA-mediated regulation of cell signaling in cancer. Oncogene. 36:5661–5667. 2017.PubMed/NCBI View Article : Google Scholar
7	Fan Q, Yang L, Zhang X, Peng X, Wei S, Su D, Zhai Z, Hua X and Li H: The emerging role of exosome-derived non-coding RNAs in cancer biology. Cancer Lett. 414:107–115. 2018.PubMed/NCBI View Article : Google Scholar
8	Gutschner T and Diederichs S: The hallmarks of cancer: A long non-coding RNA point of view. RNA Biol. 9:703–719. 2012.PubMed/NCBI View Article : Google Scholar
9	Yang Y and Peng XW: The silencing of long non-coding RNA ANRIL suppresses invasion, and promotes apoptosis of retinoblastoma cells through the ATM-E2F1 signaling pathway. Biosci Rep. 38(BSR20180558)2018.PubMed/NCBI View Article : Google Scholar
10	Zhang H, Zhong J, Bian Z, Fang X, Peng Y and Hu Y: Long non-coding RNA CCAT1 promotes human retinoblastoma SO-RB50 and Y79 cells through negative regulation of miR-218-5p. Biomed Pharmacother. 87:683–691. 2017.PubMed/NCBI View Article : Google Scholar
11	Zhang A, Shang W, Nie Q, Li T and Li S: Long non-coding RNA H19 suppresses retinoblastoma progression via counteracting miR-17-92 cluster. J Cell Biochem. 119:3497–3509. 2018.PubMed/NCBI View Article : Google Scholar
12	Yu X, Li Z, Zheng H, Chan MT and Wu WK: NEAT 1: A novel cancer-related long non-coding RNA. Cell Prolif. 50(e12329)2017.PubMed/NCBI View Article : Google Scholar
13	Li X, Wang S, Li Z, Long X, Guo Z, Zhang G, Zu J, Chen Y and Wen L: The lncRNA NEAT1 facilitates cell growth and invasion via the miR-211/HMGA2 axis in breast cancer. Int J Biol Macromol. 105:346–353. 2017.PubMed/NCBI View Article : Google Scholar
14	Guo H, Yang S, Zhao S, Li L, Yan M and Fan M: LncRNA NEAT1 regulates cervical carcinoma proliferation and invasion by targeting AKT/PI3K. Eur Rev Med Pharmacol Sci. 22:4090–4097. 2018.PubMed/NCBI View Article : Google Scholar
15	Lü J, Qian J, Chen F, Tang X, Li C and Cardoso WV: Differential expression of components of the microRNA machinery during mouse organogenesis. Biochem Biophys Res Commun. 334:319–323. 2005.PubMed/NCBI View Article : Google Scholar
16	Zhao JJ, Yang J, Lin J, Yao N, Zhu Y, Zheng J, Xu J, Cheng JQ, Lin JY and Ma X: Identification of miRNAs associated with tumorigenesis of retinoblastoma by miRNA microarray analysis. Childs Nerv Syst. 25:13–20. 2009.PubMed/NCBI View Article : Google Scholar
17	To KH, Pajovic S, Gallie BL and Thériault BL: Regulation of p14ARF expression by miR-24: A potential mechanism compromising the p53 response during retinoblastoma development. BMC Cancer. 12(69)2012.PubMed/NCBI View Article : Google Scholar
18	Yu F, Pang G and Zhao G: ANRIL acts as onco-lncRNA by regulation of microRNA-24/c-Myc, MEK/ERK and Wnt/β-catenin pathway in retinoblastoma. Int J Biol Macromol. 128:583–592. 2019.PubMed/NCBI View Article : Google Scholar
19	Takahashi N, Takahashi Y and Putnam FW: Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich alpha 2-glycoprotein of human serum. Proc Natl Acad Sci USA. 82:1906–1910. 1985.PubMed/NCBI View Article : Google Scholar
20	Serada S, Fujimoto M, Terabe F, Iijima H, Shinzaki S, Matsuzaki S, Ohkawara T, Nezu R, Nakajima S, Kobayashi T, et al: Serum leucine-rich alpha-2 glycoprotein is a disease activity biomarker in ulcerative colitis. Inflamm Bowel Dis. 18:2169–2179. 2012.PubMed/NCBI View Article : Google Scholar
21	Wang X, Abraham S, McKenzie JAG, Jeffs N, Swire M, Tripathi VB, Luhmann UFO, Lange CAK, Zhai Z, Arthur HM, et al: LRG1 promotes angiogenesis by modulating endothelial TGF-β signalling. Nature. 499:306–311. 2013.PubMed/NCBI View Article : Google Scholar
22	Nakajima M, Miyajima M, Ogino I, Watanabe M, Miyata H, Karagiozov KL, Arai H, Hagiwara Y, Segawa T, Kobayashi K and Hashimoto Y: Leucine-rich α-2-glycoprotein is a marker for idiopathic normal pressure hydrocephalus. Acta Neurochir (Wien). 153:1339–1346. 2011.PubMed/NCBI View Article : Google Scholar
23	Lindén M, Lind SB, Mayrhofer C, Segersten U, Wester K, Lyutvinskiy Y, Zubarev R, Malmström PU and Pettersson U: Proteomic analysis of urinary biomarker candidates for nonmuscle invasive bladder cancer. Proteomics. 12:135–144. 2012.PubMed/NCBI View Article : Google Scholar
24	Andersen JD, Boylan KL, Jemmerson R, Geller MA, Misemer B, Harrington KM, Weivoda S, Witthuhn BA, Argenta P, Vogel RI and Skubitz AP: Leucine-rich alpha-2-glycoprotein-1 is upregulated in sera and tumors of ovarian cancer patients. J Ovarian Res. 3(21)2010.PubMed/NCBI View Article : Google Scholar
25	Amer R, Tiosano L and Pe'er J: Leucine-rich α-2-glycoprotein-1 (LRG-1) expression in retinoblastoma. Invest Ophthalmol Vis Sci. 59:685–692. 2018.PubMed/NCBI View Article : Google Scholar
26	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.PubMed/NCBI View Article : Google Scholar
27	Yilmaz M and Christofori G: EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev. 28:15–33. 2009.PubMed/NCBI View Article : Google Scholar
28	Yang J and Weinberg RA: Epithelial-mesenchymal transition: At the crossroads of development and tumor metastasis. Dev Cell. 14:818–829. 2008.PubMed/NCBI View Article : Google Scholar
29	Wang JX, Yang Y and Li K: Long noncoding RNA DANCR aggravates retinoblastoma through miR-34c and miR-613 by targeting MMP-9. J Cell Physiol. 233:6986–6995. 2018.PubMed/NCBI View Article : Google Scholar
30	Hu C, Liu S, Han M, Wang Y and Xu C: Knockdown of lncRNA XIST inhibits retinoblastoma progression by modulating the miR-124/STAT3 axis. Biomed Pharmacother. 107:547–554. 2018.PubMed/NCBI View Article : Google Scholar
31	Liu F, Chen N, Gong Y, Xiao R, Wang W and Pan Z: The long non-coding RNA NEAT1 enhances epithelial-to-mesenchymal transition and chemoresistance via the miR-34a/c-Met axis in renal cell carcinoma. Oncotarget. 8:62927–62938. 2017.PubMed/NCBI View Article : Google Scholar
32	Ji S, Wang S, Zhao X and Lv L: Long noncoding RNA NEAT1 regulates the development of osteosarcoma through sponging miR-34a-5p to mediate HOXA13 expression as a competitive endogenous RNA. Mol Genet Genomic Med. 7(e673)2019.PubMed/NCBI View Article : Google Scholar
33	Wang L, Yang D, Tian R and Zhang H: NEAT1 promotes retinoblastoma progression via modulating miR-124. J Cell Biochem. 120:15585–15593. 2019.PubMed/NCBI View Article : Google Scholar
34	Wang P, Ning S, Zhang Y, Li R, Ye J, Zhao Z, Zhi H, Wang T, Guo Z and Li X: Identification of lncRNA-associated competing triplets reveals global patterns and prognostic markers for cancer. Nucleic Acids Res. 43:3478–3489. 2015.PubMed/NCBI View Article : Google Scholar
35	Song L, Yang J, Duan P, Xu J, Luo X, Luo F, Zhang Z, Hou T, Liu B and Zhou Q: MicroRNA-24 inhibits osteosarcoma cell proliferation both in vitro and in vivo by targeting LPAATβ. Arch Biochem Biophys. 535:128–135. 2013.PubMed/NCBI View Article : Google Scholar
36	Duan Y, Hu L, Liu B, Yu B, Li J, Yan M, Yu Y, Li C, Su L, Zhu Z, et al: Tumor suppressor miR-24 restrains gastric cancer progression by downregulating RegIV. Mol Cancer. 13(127)2014.PubMed/NCBI View Article : Google Scholar
37	Zhang S, Zhang C, Liu W, Zheng W, Zhang Y, Wang S, Huang D, Liu X and Bai Z: MicroRNA-24 upregulation inhibits proliferation, metastasis and induces apoptosis in bladder cancer cells by targeting CARMA3. Int J Oncol. 47:1351–1360. 2015.PubMed/NCBI View Article : Google Scholar
38	Bates RC and Mercurio AM: The epithelial-mesenchymal tansition (EMT) and colorectal cancer progression. Cancer Biol Ther. 4:371–376. 2005.PubMed/NCBI View Article : Google Scholar
39	Li L and Li W: Epithelial-mesenchymal transition in human cancer: Comprehensive reprogramming of metabolism, epigenetics, and differentiation. Pharmacol Ther. 150:33–46. 2015.PubMed/NCBI View Article : Google Scholar
40	Li X, Wang S, Li Z, Long X, Guo Z, Zhang G, Zu J, Chen Y and Wen L: NEAT1 induces epithelial-mesenchymal transition and 5-FU resistance through the miR-129/ZEB2 axis in breast cancer. FEBS Lett. 591(570)2017.PubMed/NCBI View Article : Google Scholar
41	Lu Y, Li T, Wei G, Liu L, Chen Q, Xu L, Zhang K, Zeng D and Liao R: The long non-coding RNA NEAT1 regulates epithelial to mesenchymal transition and radioresistance in through miR-204/ZEB1 axis in nasopharyngeal carcinoma. Tumour Biol. 37:11733–11741. 2016.PubMed/NCBI View Article : Google Scholar