Functional impact of the long non‑coding RNA MEG3 deletion by CRISPR/Cas9 in the human triple negative metastatic Hs578T cancer cell line

Deocesano‑Pereira,Carlos; Machado,Raquel  Arminda Carvalho; de Jesus‑Ferreira,Henrique  Cesar; Marchini,Thiago; Pereira,Tulio  Felipe; Carreira,Ana  Claudia Oliveira; Sogayar,Mari   Cleide

doi:10.3892/ol.2019.10969

Functional impact of the long non‑coding RNA MEG3 deletion by CRISPR/Cas9 in the human triple negative metastatic Hs578T cancer cell line

Authors:
- Carlos Deocesano‑Pereira
- Raquel Arminda Carvalho Machado
- Henrique Cesar de Jesus‑Ferreira
- Thiago Marchini
- Tulio Felipe Pereira
- Ana Claudia Oliveira Carreira
- Mari Cleide Sogayar
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Affiliations: Cell and Molecular Therapy Center, School of Medicine, University of São Paulo, São Paulo 05360‑130 SP, Brazil
Published online on: October 8, 2019 https://doi.org/10.3892/ol.2019.10969
Pages: 5941-5951
Copyright: © Deocesano‑Pereira et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Long non‑coding RNAs (lncRNAs) serve critical roles in regulating cellular homeostasis, and their deregulated expression/activity is associated with neoplastic transformation. The maternally expressed gene 3 (MEG3) has been extensively described as a tumor suppressor gene in different types of cancer, including breast cancer. Interestingly, using a panel of seven different breast cancer cell lines, the present study revealed that MEG3 is highly expressed in the triple negative metastatic human Hs578T breast cancer cell line, which is refractory to different therapeutic approaches. Therefore, the present study aimed to investigate the phenotypic impact of MEG3 deletion in this cell line. Using the CRISPR/Cas9 system, complete knockout (KO) of MEG3 was achieved. Deletion was confirmed by genomic PCR and reverse transcription‑quantitative PCR. The MEG3_KO cell population displaying the highest efficiency of genomic editing was selected for phenotypic in vitro assays, including wound scratch and Transwell assays, flow cytometry and immunofluorescence. The results demonstrated that MEG3 deletion increased cell proliferation, anchorage‑independent cell growth and cell motility, which was consistent with its well‑known tumor suppressor function. However, the present study revealed that MEG3_KO also lead to decreased cell invasiveness ability, supporting previous evidence that MEG3 modulates epithelial‑to‑mesenchymal inducing factors. The present study demonstrated that deletion of MEG3 promoted an increase in transforming growth factor β and N‑cadherin protein levels and significant reduction in matrix metallopeptidase 2, zinc‑finger E‑box binding homeobox 1 and collagen type III α1 chain gene expression levels. Additionally, MEG3_KO cells displayed significant resistance to doxorubicin treatment, demonstrating the role of this lncRNA in cancer cell survival by regulating apoptosis. The present study highlighted the utility of CRISPR/Cas9 for anticancer studies of intergenic lncRNAs and demonstrated that, although Hs578T cells express MEG3 at high levels, these cells display mechanisms to escape the growth suppression effects of this lncRNA. Notably, the detailed pathological mechanisms of MEG3 concerning tumor metastasis remain to be elucidated prior to applying MEG3 expression/activation in future therapeutic approaches for breast cancer treatment.

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1	Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, Huarte M, Zuk O, Carey BW, Cassady JP, et al: Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature. 458:223–227. 2009. View Article : Google Scholar : PubMed/NCBI
2	Qureshi IA and Mehler MF: Emerging roles of non-coding RNAs in brain evolution, development, plasticity and disease. Nat Rev Neurosci. 13:528–541. 2012. View Article : Google Scholar : PubMed/NCBI
3	Esteller M: Non-coding RNAs in human disease. Nat Rev Genet. 12:861–874. 2011. View Article : Google Scholar : PubMed/NCBI
4	Calore F, Lovat F and Garofalo M: Non-coding RNAs and cancer. Int J Mol Sci. 14:17085–17110. 2013. View Article : Google Scholar : PubMed/NCBI
5	Rinn JL and Chang HY: Genome regulation by long noncoding RNAs. Annu Rev Biochem. 81:145–166. 2012. View Article : Google Scholar : PubMed/NCBI
6	Zhao Y, Li H, Fang S, Kang Y, Wu W, Hao Y, Li Z, Bu D, Sun N, Zhang MQ and Chen R: NONCODE 2016: An informative and valuable data source of long non-coding RNAs. Nucleic Acids Res. 44(D1): D203–D208. 2016. View Article : Google Scholar : PubMed/NCBI
7	Li S, Li B, Zheng Y, Li M, Shi L and Pu X: Exploring functions of long noncoding RNAs across multiple cancers through co-expression network. Sci Rep. 7:7542017. View Article : Google Scholar : PubMed/NCBI
8	Fatica A and Bozzoni I: Long non-coding RNAs: New players in cell differentiation and development. Nat Rev Genet. 15:7–21. 2014. View Article : Google Scholar : PubMed/NCBI
9	Long Y, Wang X, Youmans DT and Cech TR: How do lncRNAs regulate transcription? Sci Adv. 3:eaao21102017. View Article : Google Scholar : PubMed/NCBI
10	Zhang X, Zhou Y, Mehta KR, Danila DC, Scolavino S, Johnson SR and Klibanski A: A pituitary-derived MEG3 isoform functions as a growth suppressor in tumor cells. J Clin Endocrinol Metab. 88:5119–5126. 2003. View Article : Google Scholar : PubMed/NCBI
11	Gibb EA, Brown CJ and Lam WL: The functional role of long non-coding RNA in human carcinomas. Mol Cancer. 10:382011. View Article : Google Scholar : PubMed/NCBI
12	Zhang J, Yao T, Wang Y, Yu J, Liu Y and Lin Z: Long noncoding RNA MEG3 is downregulated in cervical cancer and affects cell proliferation and apoptosis by regulating miR-21. Cancer Biol Ther. 17:104–113. 2016. View Article : Google Scholar : PubMed/NCBI
13	Wang P, Ren Z and Sun P: Overexpression of the long non-coding RNA MEG3 impairs in vitro glioma cell proliferation. J Cell Biochem. 113:1868–1874. 2012. View Article : Google Scholar : PubMed/NCBI
14	Luo G, Wang M, Wu X, Tao D, Xiao X, Wang L, Min F, Zeng F and Jiang G: Long non-coding RNA MEG3 inhibits cell proliferation and induces apoptosis in prostate cancer. Cell Physiol Biochem. 37:2209–2220. 2015. View Article : Google Scholar : PubMed/NCBI
15	Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA and Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 68:394–424. 2018. View Article : Google Scholar : PubMed/NCBI
16	Siegel RL, Miller KD and Jemal A: Cancer statistics, 2018. CA Cancer J Clin. 68:7–30. 2018. View Article : Google Scholar : PubMed/NCBI
17	Dai X, Li T, Bai Z, Yang Y, Liu X, Zhan J and Shi B: Breast cancer intrinsic subtype classification, clinical use and future trends. Am J Cancer Res. 5:2929–2943. 2015.PubMed/NCBI
18	Abramson VG, Lehmann BD, Ballinger TJ and Pietenpol JA: Subtyping of triple-negative breast cancer: Implications for therapy. Cancer. 121:8–16. 2015. View Article : Google Scholar : PubMed/NCBI
19	Zhang JJ, Guo SH and Jia BQ: Down-regulation of long non-coding RNA MEG3 serves as an unfavorable risk factor for survival of patients with breast cancer. Eur Rev Med Pharmacol Sci. 20:5143–5147. 2016.PubMed/NCBI
20	Tian T, Wang M, Lin S, Guo Y, Dai Z, Liu K, Yang P, Dai C, Zhu Y, Zheng Y, et al: The impact of lncRNA dysregulation on clinicopathology and survival of breast cancer: A systematic review and meta-analysis. Mol Ther Nucleic Acids. 12:359–369. 2018. View Article : Google Scholar : PubMed/NCBI
21	Cui X, Yi Q, Jing X, Huang Y, Tian J, Long C, Xiang Z, Liu J, Zhang C, Tan B, et al: Mining prognostic significance of MEG3 in human breast cancer using bioinformatics analysis. Cell Physiol Biochem. 50:41–51. 2018. View Article : Google Scholar : PubMed/NCBI
22	Zhang CY, Yu MS, Li X, Zhang Z, Han CR and Yan B: Overexpression of long non-coding RNA MEG3 suppresses breast cancer cell proliferation, invasion, and angiogenesis through AKT pathway. Tumour Biol. 39:10104283177013112017.PubMed/NCBI
23	Sun L, Li Y and Yang B: Downregulated long non-coding RNA MEG3 in breast cancer regulates proliferation, migration and invasion by depending on p53's transcriptional activity. Biochem Biophys Res Commun. 478:323–329. 2016. View Article : Google Scholar : PubMed/NCBI
24	Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA and Zhang F: Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 8:2281–2308. 2013. View Article : Google Scholar : PubMed/NCBI
25	Sánchez-Rivera FJ and Jacks T: Applications of the CRISPR-Cas9 system in cancer biology. Nat Rev Cancer. 15:387–395. 2015. View Article : Google Scholar : PubMed/NCBI
26	Wang H, La Russa M and Qi LS: CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem. 85:227–264. 2016. View Article : Google Scholar : PubMed/NCBI
27	Liu SJ, Horlbeck MA, Cho SW, Birk HS, Malatesta M, He D, Attenello FJ, Villalta JE, Cho MY, Chen Y, et al: CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science. 355(pii): aah71112017. View Article : Google Scholar : PubMed/NCBI
28	Chen W, Zhang G, Li J, Zhang X, Huang S, Xiang S, Hu X and Liu C: CRISPRlnc: A manually curated database of validated sgRNAs for lncRNAs. Nucleic Acids Res. 47:D63–D68. 2019. View Article : Google Scholar : PubMed/NCBI
29	Goyal A, Myacheva K, Groß M, Klingenberg M, Duran Arqué B and Diederichs S: Challenges of CRISPR/Cas9 applications for long non-coding RNA genes. Nucleic Acids Res. 45:e122017.PubMed/NCBI
30	Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29:e452001. View Article : Google Scholar : PubMed/NCBI
31	Zhou Y, Zhang X and Klibanski A: MEG3 noncoding RNA: A tumor suppressor. J Mol Endocrinol. 48:R45–R53. 2012. View Article : Google Scholar : PubMed/NCBI
32	Olson MF and Sahai E: The actin cytoskeleton in cancer cell motility. Clin Exp Metastasis. 26:273–287. 2009. View Article : Google Scholar : PubMed/NCBI
33	Terashima M, Tange S, Ishimura A and Suzuki T: MEG3 long noncoding RNA contributes to the epigenetic regulation of epithelial-mesenchymal transition in lung cancer cell lines. J Biol Chem. 292:82–99. 2017. View Article : Google Scholar : PubMed/NCBI
34	Xia Y, He Z, Liu B, Wang P and Chen Y: Downregulation of Meg3 enhances cisplatin resistance of lung cancer cells through activation of the WNT/β-catenin signaling pathway. Mol Med Rep. 12:4530–4537. 2015. View Article : Google Scholar : PubMed/NCBI
35	Liu J, Wan L, Lu K, Sun M, Pan X, Zhang P, Lu B, Liu G and Wang Z: The long noncoding RNA MEG3 contributes to cisplatin resistance of human lung adenocarcinoma. PLoS One. 10:e01145862015. View Article : Google Scholar : PubMed/NCBI
36	Zhao H, Wang X, Feng X, Li X, Pan L, Liu J, Wang F, Yuan Z, Yang L, Yu J, et al: Long non-coding RNA MEG3 regulates proliferation, apoptosis and autophagy and is associated with prognosis in glioma. J Neurooncol. 140:281–288. 2018. View Article : Google Scholar : PubMed/NCBI
37	Shi Y, Lv C, Shi L and Tu G: MEG3 inhibits proliferation and invasion and promotes apoptosis of human osteosarcoma cells. Oncol Lett. 15:1917–1923. 2018.PubMed/NCBI
38	Miyoshi N, Wagatsuma H, Wakana S, Shiroishi T, Nomura M, Aisaka K, Kohda T, Surani MA, Kaneko-Ishino T and Ishino F: Identification of an imprinted gene, Meg3/Gtl2 and its human homologue MEG3, first mapped on mouse distal chromosome 12 and human chromosome 14q. Genes Cells. 5:211–220. 2000. View Article : Google Scholar : PubMed/NCBI
39	Zhou Y, Cheunsuchon P, Nakayama Y, Lawlor MW, Zhong Y, Rice KA, Zhang L, Zhang X, Gordon FE, Lidov HG, et al: Activation of paternally expressed genes and perinatal death caused by deletion of the Gtl2 gene. Development. 137:2643–2652. 2010. View Article : Google Scholar : PubMed/NCBI
40	Zhou Y, Zhong Y, Wang Y, Zhang X, Batista DL, Gejman R, Ansell PJ, Zhao J, Weng C and Klibanski A: Activation of p53 by MEG3 non-coding RNA. J Biol Chem. 282:24731–24742. 2007. View Article : Google Scholar : PubMed/NCBI
41	Zhang X, Rice K, Wang Y, Chen W, Zhong Y, Nakayama Y, Zhou Y and Klibanski A: Maternally expressed gene 3 (MEG3) noncoding ribonucleic acid: Isoform structure, expression, and functions. Endocrinology. 151:939–947. 2010. View Article : Google Scholar : PubMed/NCBI
42	Tang Y, He Y, Zhang P, Wang J, Fan C, Yang L, Xiong F, Zhang S, Gong Z, Nie S, et al: LncRNAs regulate the cytoskeleton and related Rho/ROCK signaling in cancer metastasis. Mol Cancer. 17:772018. View Article : Google Scholar : PubMed/NCBI
43	Wang C, Yan G, Zhang Y, Jia X and Bu P: Long non-coding RNA MEG3 suppresses migration and invasion of thyroid carcinoma by targeting of Rac1. Neoplasma. 62:541–549. 2015. View Article : Google Scholar : PubMed/NCBI
44	Parri M and Chiarugi P: Rac and Rho GTPases in cancer cell motility control. Cell Commun Signal. 8:232010. View Article : Google Scholar : PubMed/NCBI
45	Mitra R, Chen X, Greenawalt EJ, Maulik U, Jiang W, Zhao Z and Eischen CM: Decoding critical long non-coding RNA in ovarian cancer epithelial-to-mesenchymal transition. Nat Commun. 8:16042017. View Article : Google Scholar : PubMed/NCBI
46	Mondal T, Subhash S, Vaid R, Enroth S, Uday S, Reinius B, Mitra S, Mohammed A, James AR, Hoberg E, et al: MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures. Nat Commun. 6:77432015. View Article : Google Scholar : PubMed/NCBI
47	Housman G, Byler S, Heerboth S, Lapinska K, Longacre M, Snyder N and Sarkar S: Drug resistance in cancer: An overview. Cancers (Basel). 6:1769–1792. 2014. View Article : Google Scholar : PubMed/NCBI
48	Li ZY, Yang L, Liu XJ, Wang XZ, Pan YX and Luo JM: The long noncoding RNA MEG3 and its target miR-147 regulate JAK/STAT pathway in advanced chronic myeloid leukemia. EBioMedicine. 34:61–75. 2018. View Article : Google Scholar : PubMed/NCBI