Yonsei Med J.  2017 Nov;58(6):1092-1100. 10.3349/ymj.2017.58.6.1092.

Knockdown of Long Non-Coding RNA NEAT1 Inhibits Proliferation and Invasion and Induces Apoptosis of Osteosarcoma by Inhibiting miR-194 Expression

Affiliations
  • 1Department of Orthopedics, Zhoukou Central Hospital, Zhoukou, China. wangheping112@yeah.net
  • 2Department of Surgery, Zhoukou Central Hospital, Zhoukou, China.

Abstract

PURPOSE
Long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) has been implicated as an oncogene in the development and progression of osteosarcoma. This study aims to explore the mechanism of NEAT1 in osteosarcoma.
MATERIALS AND METHODS
Expressions of NEAT1 and miR-194 in osteosarcoma tissues and cells were detected by quantitative real-time PCR. The effects of NEAT1 knockdown or miR-194 overexpression on cell proliferation, invasion, and apoptosis were determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT) assay, transwell invasive assay, and flow cytometry analysis, respectively. Luciferase reporter assay was performed to observe the possible interaction between NEAT1 and miR-194.
RESULTS
NEAT1 was upregulated and miR-194 was downregulated in osteosarcoma tissues and cells. Knockdown of NEAT1 or overexpression of miR-194 suppressed proliferation and invasion and induced apoptosis of osteosarcoma cells in vitro. Luciferase reporter assay validated that NEAT1 could interact with miR-194 and negatively modulated its expression. Furthermore, inhibition of miR-194 reversed the suppression of proliferation and invasion and the promotion of apoptosis induced by NEAT1 depletion in osteosarcoma cells.
CONCLUSION
Knockdown of NEAT1 suppressed proliferation and invasion and induced apoptosis in osteosarcoma cells by inhibiting miR-194 expression.

Keyword

lncRNA; tumorigenesis; osteosarcoma; miR-194

MeSH Terms

Apoptosis/*genetics
Bone Neoplasms/genetics
Cell Proliferation
Disease Progression
Down-Regulation
*Gene Expression Regulation, Neoplastic
Humans
MicroRNAs/*genetics/metabolism
Osteosarcoma/*genetics/pathology
RNA, Long Noncoding/*genetics
Real-Time Polymerase Chain Reaction
Up-Regulation
MicroRNAs
RNA, Long Noncoding

Figure

  • Fig. 1 Expression of NEAT1 and miR-194 in osteosarcoma tissues and cells. (A and B) The expression levels of NEAT1 and miR-194 in osteosarcoma tissue samples (n=15) and normal tissue samples (n=15) by qRT-PCR analysis. (C and D) qRT-PCR analysis of NEAT1 and miR-194 in MG63 and U2OS cells. *p<0.05, †p<0.01, ‡p<0.001. NEAT1, nuclear paraspeckle assembly transcript 1; qRT-PCR, quantitative real-time PCR.

  • Fig. 2 Knockdown of NEAT1 inhibits proliferation and invasion and promotes apoptosis of MG63 and U2OS cells. MG63 and U2OS cells were transfected with si-NEAT1 or si-con. (A) qRT-PCR analysis was performed to detect NEAT1 levels in MG63 and U2OS cells. (B) MTT assay was performed to detect viability of MG63 and U2OS cells at days 1, 2, 3, and 4 after transfection. (C and D) Transwell invasive assay was carried out to assess invasive ability of MG63 and U2OS cells 24 h post transfection. (E and F) Flow cytometry analysis was conducted to determine the apoptotic rate of MG63 and U2OS cells 24 h after transfection. †p<0.01, ‡p<0.001 vs. si-con. NEAT1, nuclear paraspeckle assembly transcript 1; qRT-PCR, quantitative real-time PCR; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide; PI, propidium iodide; FITC, fluorescein isothiocyanate.

  • Fig. 3 miR-194 overexpression suppressed proliferation and invasion and induced apoptosis of MG63 and U2OS cells. MG63 and U2OS cells were transfected with miR-194 mimic or miR-control. (A) qRT-PCR analysis was performed to detect the miR-194 expression in MG63 and U2OS cells. (B) MTT assay of viability at days 1, 2, 3 and 4 in MG63 and U2OS cells. (C and D) Transwell invasive assay of invasive capacity 24 h post transfection in MG63 and U2OS cells. (E and F) Flow cytometry analysis of apoptosis of 24 h post transfection in MG63 and U2OS cells. †p<0.01, ‡p<0.001 vs. miR-con. qRT-PCR, quantitative real-time PCR; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide.

  • Fig. 4 NEAT1 inhibited miR-194 expression in MG63 and U2OS cells. (A) The binding sequences of miR-194 in NEAT1 were marked. (B) Luciferase reporter assay was performed in MG63 and U2OS cells co-transfected with Wt- or Mut-NEAT1 and miR-194 mimic or miR-con. (C) qRT-PCR analysis of miR-194 expression in MG63 and U2OS cells transfected with si-NEAT1 or pcDNA-NEAT1. †p<0.01, ‡p<0.001 vs. controls. Wt-NEAT1, wild type-nuclear paraspeckle assembly transcript 1; Mut-NEAT1, mutant-nuclear paraspeckle assembly transcript 1; NC, blank control; qRT-PCR, quantitative real-time PCR.

  • Fig. 5 miR-194 inhibition overturned suppression of proliferation and invasion and the promotion of apoptosis elicited by NEAT1 knockdown in MG63 and U2OS cells. MG63 and U2OS cells were transfected with si-NEAT1 or co-transfected with si-NEAT1 and anti-miR-194. (A and B) MTT assay was used to test the viability of transfected MG63 and U2OS cells. (C and D) Transwell invasive assay was applied to measure the invasive ability of transfected MG63 and U2OS cells. (E and F) Flow cytometry analysis was conducted to observe the apoptotic rate of transfected MG63 and U2OS cells. †p<0.01, ‡p<0.001 vs. controls. NEAT1, nuclear paraspeckle assembly transcript 1; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide.


Reference

1. Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res. 2009; 152:3–13.
Article
2. Stiller CA. International patterns of cancer incidence in adolescents. Cancer Treat Rev. 2007; 33:631–645.
Article
3. Yang J, Zhang W. New molecular insights into osteosarcoma targeted therapy. Curr Opin Oncol. 2013; 25:398–406.
Article
4. Luetke A, Meyers PA, Lewis I, Juergens H. Osteosarcoma treatment - where do we stand? A state of the art review. Cancer Treat Rev. 2014; 40:523–532.
Article
5. Chen L, Wang X, Wang H, Li Y, Yan W, Han L, et al. miR-137 is frequently down-regulated in glioblastoma and is a negative regulator of Cox-2. Eur J Cancer. 2012; 48:3104–3111.
Article
6. Duan Z, Choy E, Harmon D, Liu X, Susa M, Mankin H, et al. MicroRNA-199a-3p is downregulated in human osteosarcoma and regulates cell proliferation and migration. Mol Cancer Ther. 2011; 10:1337–1345.
Article
7. Liu LH, Li H, Li JP, Zhong H, Zhang HC, Chen J, et al. miR-125b suppresses the proliferation and migration of osteosarcoma cells through down-regulation of STAT3. Biochem Biophys Res Commun. 2011; 416:31–38.
Article
8. Hong Q, Fang J, Pang Y, Zheng J. Prognostic value of the microRNA-29 family in patients with primary osteosarcomas. Med Oncol. 2014; 31:37.
Article
9. Meng Z, Fu X, Chen X, Zeng S, Tian Y, Jove R, et al. miR-194 is a marker of hepatic epithelial cells and suppresses metastasis of liver cancer cells in mice. Hepatology. 2010; 52:2148–2157.
Article
10. Dong P, Kaneuchi M, Watari H, Hamada J, Sudo S, Ju J, et al. MicroRNA-194 inhibits epithelial to mesenchymal transition of endometrial cancer cells by targeting oncogene BMI-1. Mol Cancer. 2011; 10:99.
Article
11. Song Y, Zhao F, Wang Z, Liu Z, Chiang Y, Xu Y, et al. Inverse association between miR-194 expression and tumor invasion in gastric cancer. Ann Surg Oncol. 2012; 19:Suppl 3. S509–S517.
Article
12. Han K, Zhao T, Chen X, Bian N, Yang T, Ma Q, et al. microRNA-194 suppresses osteosarcoma cell proliferation and metastasis in vitro and in vivo by targeting CDH2 and IGF1R. Int J Oncol. 2014; 45:1437–1449.
Article
13. Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nat Rev Genet. 2009; 10:155–159.
Article
14. Cheetham SW, Gruhl F, Mattick JS, Dinger ME. Long noncoding RNAs and the genetics of cancer. Br J Cancer. 2013; 108:2419–2425.
Article
15. Li W, Xie P, Ruan WH. Overexpression of lncRNA UCA1 promotes osteosarcoma progression and correlates with poor prognosis. J Bone Oncol. 2016; 5:80–85.
Article
16. Cong M, Li J, Jing R, Li Z. Long non-coding RNA tumor suppressor candidate 7 functions as a tumor suppressor and inhibits proliferation in osteosarcoma. Tumour Biol. 2016; 37:9441–9450.
Article
17. Yun-Bo F, Xiao-Po L, Xiao-Li L, Guo-Long C, Pei Z, Fa-Ming T. LncRNA TUG1 is upregulated and promotes cell proliferation in osteosarcoma. Open Med (Wars). 2016; 11:163–167.
Article
18. Yin Z, Ding H, He E, Chen J, Li M. Overexpression of long non-coding RNA MFI2 promotes cell proliferation and suppresses apoptosis in human osteosarcoma. Oncol Rep. 2016; 36:2033–2040.
Article
19. Choudhry H, Albukhari A, Morotti M, Haider S, Moralli D, Smythies J, et al. Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival. Oncogene. 2015; 34:4482–4490.
Article
20. Zhao H, Zhao Y, Tao J, Ma C, Zhang J, Xu H, et al. Up-regulated expression of lncRNA NEAT1 promotes progression of osteosarcoma by regulating the activity of Wnt/β-catenin pathway. Int J Clin Exp Pathol. 2016; 9:11466–11472.
21. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008; 3:1101–1108.
Article
22. Dong Y, Liang G, Yuan B, Yang C, Gao R, Zhou X. MALAT1 promotes the proliferation and metastasis of osteosarcoma cells by activating the PI3K/Akt pathway. Tumour Biol. 2015; 36:1477–1486.
Article
23. Li Z, Zhao L, Wang Q. Overexpression of long non-coding RNA HOTTIP increases chemoresistance of osteosarcoma cell by activating the Wnt/β-catenin pathway. Am J Transl Res. 2016; 8:2385–2393.
24. Sun XH, Yang LB, Geng XL, Wang R, Zhang ZC. Increased expression of lncRNA HULC indicates a poor prognosis and promotes cell metastasis in osteosarcoma. Int J Clin Exp Pathol. 2015; 8:2994–3000.
25. Zhao H, Hou W, Tao J, Zhao Y, Wan G, Ma C, et al. Upregulation of lncRNA HNF1A-AS1 promotes cell proliferation and metastasis in osteosarcoma through activation of the Wnt/β-catenin signaling pathway. Am J Transl Res. 2016; 8:3503–3512.
26. Clemson CM, Hutchinson JN, Sara SA, Ensminger AW, Fox AH, Chess A, et al. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol Cell. 2009; 33:717–726.
Article
27. Jiang P, Wu X, Wang X, Huang W, Feng Q. NEAT1 upregulates EGCG-induced CTR1 to enhance cisplatin sensitivity in lung cancer cells. Oncotarget. 2016; 7:43337–43351.
Article
28. Wang P, Wu T, Zhou H, Jin Q, He G, Yu H, et al. Long noncoding RNA NEAT1 promotes laryngeal squamous cell cancer through regulating miR-107/CDK6 pathway. J Exp Clin Cancer Res. 2016; 35:22.
Article
29. Chen X, Kong J, Ma Z, Gao S, Feng X. Up regulation of the long noncoding RNA NEAT1 promotes esophageal squamous cell carcinoma cell progression and correlates with poor prognosis. Am J Cancer Res. 2015; 5:2808–2815.
30. Stahlhut C, Slack FJ. MicroRNAs and the cancer phenotype: profiling, signatures and clinical implications. Genome Med. 2013; 5:111.
Article
31. Zhou Q, Chen F, Zhao J, Li B, Liang Y, Pan W, et al. Long non-coding RNA PVT1 promotes osteosarcoma development by acting as a molecular sponge to regulate miR-195. Oncotarget. 2016; 7:82620–82633.
Article
32. Xie CH, Cao YM, Huang Y, Shi QW, Guo JH, Fan ZW, et al. Long non-coding RNA TUG1 contributes to tumorigenesis of human osteosarcoma by sponging miR-9-5p and regulating POU2F1 expression. Tumour Biol. 2016; 37:15031–15041.
Article
33. Wu X, Liu T, Fang O, Leach LJ, Hu X, Luo Z. miR-194 suppresses metastasis of non-small cell lung cancer through regulating expression of BMP1 and p27(kip1). Oncogene. 2014; 33:1506–1514.
Article
34. Zhang M, Zhuang Q, Cui L. MiR-194 inhibits cell proliferation and invasion via repression of RAP2B in bladder cancer. Biomed Pharmacother. 2016; 80:268–275.
Article
Full Text Links
  • YMJ
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr