A tetracycline‑inducible CRISPR/Cas9 system, targeting two long non‑coding RNAs, suppresses the malignant behavior of bladder cancer cells

Peng,Lu; Pan,Peng; Chen,Jinbu; Yu,Xueyuan; Wu,Jun; Chen,Yong

doi:10.3892/ol.2018.9157

A tetracycline‑inducible CRISPR/Cas9 system, targeting two long non‑coding RNAs, suppresses the malignant behavior of bladder cancer cells

Authors:
- Lu Peng
- Peng Pan
- Jinbu Chen
- Xueyuan Yu
- Jun Wu
- Yong Chen
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Affiliations: Department of Clinical Laboratory, Affiliated Brain Hospital of Nanjing Medical University, Nanjing, Jiangsu 210000, P.R. China, Reproductive Medicine Center, Nanjing General Hospital, Nanjing, Jiangsu 210000, P.R. China
Published online on: July 17, 2018 https://doi.org/10.3892/ol.2018.9157
Pages: 4309-4316
Copyright: © Peng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) technology has been applied in varied biological studies, including cancer studies. However, stable mRNA expression of Cas9 has potential risks in future gene therapy. Therefore, in the present study, a tetracycline‑inducible switch was used to control the mRNA expression of Cas9. Long non‑coding RNAs (lncRNAs) may be important functional regulators in tumor development, including in bladder cancer. RNA was designed to simultaneously target two lncRNAs, PVT1 and ANRIL, which are considered to be bladder cancer oncogenes. The mRNA expression of Cas9 was controlled by doxycycline. Reverse transcription‑quantitative polymerase chain reaction revealed that the expression of PVT1 and ANRIL was significantly inhibited by the tetracycline‑inducible CRISPR/Cas9 system. Functional assays demonstrated that this system could inhibit proliferation, induce apoptosis and suppress cell migration. Therefore, the tetracycline‑inducible CRISPR/Cas9 system was demonstrated to repress the malignant behavior of bladder cancer cells by controlling the expression of Cas9 and simultaneously targeting two oncogenic lncRNAs.

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1	Kaufman DS, Shipley WU and Feldman AS: Bladder cancer. Lancet. 374:239–249. 2009. View Article : Google Scholar : PubMed/NCBI
2	Marta GN, Hanna SA, Gadia R, Correa SF, Silva JL and Carvalho A: The role of radiotherapy in urinary bladder cancer: Current status. Int Braz J Urol. 38:144–153. 2012. View Article : Google Scholar : PubMed/NCBI
3	Racioppi M, Agostino DD, Totaro A, Pinto F, Sacco E, D'Addessi A, Marangi F, Palermo G and Bassi PF: Value of current chemotherapy and surgery in advanced and metastatic bladder cancer. Urol Int. 88:249–258. 2012. View Article : Google Scholar : PubMed/NCBI
4	Amit D and Hochberg A: Development of targeted therapy for bladder cancer mediated by a double promoter plasmid expressing diphtheria toxin under the control of H19 and IGF2-P4 regulatory sequences. J Transl Med. 8:1342010. View Article : Google Scholar : PubMed/NCBI
5	Droop J, Szarvas T, Schulz WA, Niedworok AC, Niegisch G, Scheckenbach K and Hoffmann MJ: Diagnostic and prognostic value of long noncoding RNAs as biomarkers in urothelial carcinoma. PloS One. 12:e01762872017. View Article : Google Scholar : PubMed/NCBI
6	Berrondo C, Flax J, Kucherov V, Siebert A, Osinski T, Rosenberg A, Fucile C, Richheimer S and Beckham CJ: Expression of the long non-coding RNA HOTAIR correlates with disease progression in bladder cancer and is contained in bladder cancer patient urinary exosomes. PloS One. 11:01472362016. View Article : Google Scholar
7	Heubach J, Monsior J, Deenen R, Niegisch G, Szarvas T, Niedworok C, Schulz WA and Hoffmann MJ: The long noncoding RNA HOTAIR has tissue and cell type-dependent effects on HOX gene expression and phenotype of urothelial cancer cells. Mol Cancer. 14:1082015. View Article : Google Scholar : PubMed/NCBI
8	Song J, Wu X, Liu F, Li M, Sun Y, Wang Y, Wang C, Zhu K, Jia X, Wang B and Ma X: Long non-coding RNA PVT1 promotes glycolysis and tumor progression by regulating miR-497/HK2 axis in osteosarcoma. Biochem Biophys Res Commun. 490:217–224. 2017. View Article : Google Scholar : PubMed/NCBI
9	Zhuang C, Li J, Liu Y, Chen M, Yuan J, Fu X, Zhan Y, Liu L, Lin J, Zhou Q, et al: Tetracycline-inducible shRNA targeting long non-coding RNA PVT1 inhibits cell growth and induces apoptosis in bladder cancer cells. Oncotarget. 6:41194–41203. 2015. View Article : Google Scholar : PubMed/NCBI
10	Li T, Meng XL and Yang WQ: Long noncoding RNA PVT1 acts as a ‘sponge’ to inhibit microRNA-152 in gastric cancer cells. Dig Dis Sci. 62:3021–3028. 2017. View Article : Google Scholar : PubMed/NCBI
11	Lillycrop K, Murray R, Cheong C, Teh AL, Clarke-Harris R, Barton S, Costello P, Garratt E, Cook E, Titcombe P, et al: ANRIL promoter DNA methylation: A perinatal marker for later adiposity. EBioMedicine. 19:60–72. 2017. View Article : Google Scholar : PubMed/NCBI
12	Wei X, Wang C, Ma C, Sun W, Li H and Cai Z: Retraction note: Long noncoding RNA ANRIL is activated by hypoxia-inducible factor-1α and promotes osteosarcoma cell invasion and suppresses cell apoptosis upon hypoxia. Cancer Cell Int. 17:602017. View Article : Google Scholar : PubMed/NCBI
13	Zhu H, Li X, Song Y, Zhang P, Xiao Y and Xing Y: Long non-coding RNA ANRIL is up-regulated in bladder cancer and regulates bladder cancer cell proliferation and apoptosis through the intrinsic pathway. Biochem Biophys Res Commun. 467:223–228. 2015. View Article : Google Scholar : PubMed/NCBI
14	Clement F, Grockowiak E, Zylbersztejn F, Fossard G, Gobert S and Maguer-Satta V: Stem cell manipulation, gene therapy and the risk of cancer stem cell emergence. Stem Cell Investig. 4:672017. View Article : Google Scholar : PubMed/NCBI
15	Zhang F, Wen Y and Guo X: CRISPR/Cas9 for genome editing: Progress, implications and challenges. Hum Mol Genet. 23:R40–46. 2014. View Article : Google Scholar : PubMed/NCBI
16	Jinek M, Chylinski K, Fonfara I, Hauer MJ, Doudna A and Charpentier E: A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337:816–821. 2012. View Article : Google Scholar : PubMed/NCBI
17	Chen X, Janssen JM, Liu J, Maggio I, 't Jong AEJ, Mikkers HMM and Goncalves MAFV: In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting. Nat Commun. 8:6572017. View Article : Google Scholar : PubMed/NCBI
18	Hruscha A, Krawitz P, Rechenberg A, Heinrich V, Hecht J, Haass C and Schmid B: Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development. 140:4982–4987. 2013. View Article : Google Scholar : PubMed/NCBI
19	Shen B, Zhang J, Wu H, Wang J, Ma J, Li Z, Zhang X, Zhang P and Huang X: Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23:720–723. 2013. View Article : Google Scholar : PubMed/NCBI
20	Chen Y, Zeng S, Hu R, Wang X, Huang W, Liu J, Wang L, Liu G, Cao Y and Zhang Y: Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish. PloS One. 12:e01825282017. View Article : Google Scholar : PubMed/NCBI
21	Huang X, Zhuang C, Zhuang C, Xiong T, Li Y and Gui Y: An enhanced hTERT promoter-driven CRISPR/Cas9 system selectively inhibits the progression of bladder cancer cells. Mol BioSyst. 13:1713–1721. 2017. View Article : Google Scholar : PubMed/NCBI
22	Sanchez-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
23	Vilaboa N and Voellmy R: Regulatable gene expression systems for gene therapy. Curr Gene Ther. 6:421–438. 2006. View Article : Google Scholar : PubMed/NCBI
24	Goverdhana S, Puntel M, Xiong W, Zirger JM, Barcia C, Curtin JF, Soffer EB, Mondkar S, King GD, Hu J, et al: Regulatable gene expression systems for gene therapy applications: Progress and future challenges. Mol Ther. 12:189–211. 2005. View Article : Google Scholar : PubMed/NCBI
25	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
26	Zhuang C, Huang X, Zhuang C, Luo X, Zhang X, Cai Z and Gui Y: Synthetic regulatory RNAs selectively suppress the progression of bladder cancer. J Exp Clin Cancer Res. 36:1512017. View Article : Google Scholar : PubMed/NCBI
27	Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA and Zhang F: Multiplex genome engineering using CRISPR/Cas systems. Science. 339:819–823. 2013. View Article : Google Scholar : PubMed/NCBI
28	Jinek M, East A, Cheng A, Lin S, Ma E and Doudna J: RNA-programmed genome editing in human cells. Elife. 2:e004712013. View Article : Google Scholar : PubMed/NCBI
29	Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP and Lim WA: Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 152:1173–1183. 2013. View Article : Google Scholar : PubMed/NCBI
30	Ebina H, Misawa N, Kanemura Y and Koyanagi Y: Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep. 3:25102013. View Article : Google Scholar : PubMed/NCBI
31	Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK and Sander JD: High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 31:822–826. 2013. View Article : Google Scholar : PubMed/NCBI
32	Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L and Church GM: CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 31:833–838. 2013. View Article : Google Scholar : PubMed/NCBI
33	Li J, Li Z, Zheng W, Li X, Wang Z, Cui Y and Jiang X: LncRNA-ATB: An indispensable cancer-related long noncoding RNA. Cell Prolif. 50:2017. View Article : Google Scholar