J Breast Cancer.  2016 Dec;19(4):349-357. 10.4048/jbc.2016.19.4.349.

LncRNA Taurine-Upregulated Gene 1 Promotes Cell Proliferation by Inhibiting MicroRNA-9 in MCF-7 Cells

• Zhao XB
• Ren GS

• KMID: 2362921
• DOI: http://doi.org/10.4048/jbc.2016.19.4.349

Abstract

PURPOSE
This study was designed to investigate the role of taurine-upregulated gene 1 (TUG1) in MCF-7 breast cancer cells and the molecular mechanism involved in the regulation of microRNA-9 (miR-9).
METHODS
The expression of TUG1 in breast cancer tissues and cells was evaluated using quantitative reverse transcription polymerase chain reaction. Cell viability was examined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay; cell cycle progression and apoptosis were analyzed using flow cytometry. A dual luciferase reporter assay was used to detect the relationship between TUG1 and miR-9. The expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was measured by western blot.
RESULTS
Higher expression of TUG1 was observed in breast cancer tissues and cell lines than in the corresponding controls. TUG1 knockdown reduced proliferation, suppressed cell cycle progression, and promoted apoptosis of MCF-7 cells. The dual luciferase reporter assay showed that TUG1 could negatively regulate the expression of miR-9. MiR-9 inhibition abrogated the effect of TUG1 knockdown on the proliferation, cell cycle progression, and apoptosis of MCF-7 cells. TUG1 positively regulated the expression of MTHFD2 in breast cancer cells.
CONCLUSION
TUG1 knockdown was significantly associated with decreased cell proliferation and it promoted apoptosis of breast cancer cells through the regulation of miR-9.

Keyword

Apoptosis; Breast neoplasms; Cell proliferation; MicroRNA-9; TUG1 long noncoding RNA

MeSH Terms

Apoptosis
Blotting, Western
Breast Neoplasms
Cell Cycle
Cell Line
Cell Proliferation*
Cell Survival
Flow Cytometry
Luciferases
MCF-7 Cells*
Methylenetetrahydrofolate Dehydrogenase (NADP)
Polymerase Chain Reaction
Reverse Transcription
RNA, Long Noncoding*
Luciferases
Methylenetetrahydrofolate Dehydrogenase (NADP)
RNA, Long Noncoding

Figure

• 

  Figure 1 Expression of taurine-upregulated gene 1 (TUG1) in breast cancer tissues and cell lines. (A) Expression of TUG1 in 24 breast cancer tissues and paired normal tissues detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) (n=24, each). (B) Expression of TUG1 in several breast cancer cell lines BT474, MCF-7, MDA-MB-231, T47D and normal mammary epithelial cell line 76N using RT-qPCR (n=6, each). Data represent mean±SD. *p<0.05 vs. the corresponding control.

• 

  Figure 2 Effects of taurine-upregulated gene 1 (TUG1) on cell proliferation, cell cycle and cell apoptosis in MCF-7 cells. (A) The interference efficiency of si-TUG1, si-TUG1 2, and si-TUG1 3 on the expression of TUG1. (B) MTT assay showed that si-TUG1, si-TUG1 2, and si-TUG1 3 significantly reduced proliferation rate of MCF-7 cells. (C, D) si-TUG1 suppressed cell cycle progression in MCF-7 cells analyzed by flow cytometry. The bar chart represented the percentage of cells in G1–G0, S, or G2–M phase, as indicated. (E, F) TUG1 knockdown induced cell apoptosis of MCF-7 cells. Data represent mean±SD (n=6, each). si-NC=control si-RNA; OD=optaical density. *p<0.05 vs. si-NC.

• 

  Figure 3 Taurine-upregulated gene 1 (TUG1) regulated the expression of miR-9. (A) Binding site was predicted between TUG1 and microRNA-9 (miR-9) according to starBase v2.0 database. (B) Effects of TUG1 knockdown on the expression of miR-9. (C) Effects of TUG1 overexpression on the expression of miR-9. (D) The expression of TUG1 in MCF-7 cells transfected with miR-9 mimic or inhibitor. (E) Luciferase activity of reporters containing TUG1-WT or TUG1-mut sequence in MCF-7 cells transfected with miR-9 mimic or control. (F) Correlation analysis was performed to detect the relationship between TUG1 and miR-9 in breast cancer tissues. Data represent mean±SD (n=6, each). si-NC=control si-RNA; NC=negative control. *p<0.05 versus the corresponding control.

• 

  Figure 4 Effects of microRNA-9 (miR-9) inhibitor on the function of taurine-upregulated gene 1 (TUG1) on cell proliferation, cell cycle and cell apoptosis. MCF-7 cells were transfected with si-NC, si-TUG1, si-TUG1+miR-9 inhibitor and miR-9 inhibitor. (A) The expression of miR-9 in these cells. (B) MTT assay was performed to examine these cells' proliferation. (C, D) Cell cycle was detected in these cells. (E, F) Cell apoptosis was detected by flow cytometry. Data represent mean±SD (n=6, each). si-NC=control si-RNA. *p<0.05 vs. si-NC; †p<0.05 vs. si-TUG1; ‡p<0.05 vs. miR-9 inhibitor.

• 

  Figure 5 Effect of taurine-upregulated gene 1 (TUG1) on the expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in breast cancer. (A) The mRNA level of MTHFD2 was detected in MCF-7 cells transfected with si-NC or si-TUG1 or si-TUG1+ microRNA-9 (miR-9) inhibitor (n=6, each). (B) The protein level of MTHFD2 was decreased in MCF-7 cells transfected with si-NC or si-TUG1 or si-TUG1+ miR-9 inhibitor (n=6, each). (C) TUG1 was positively correlated with MTHFD2 expression in breast cancer tissues (n=24, each). Data represent mean±SD. si-NC=control si-RNA; GRPDH=glyceraldehyde-3-phosphate dehydrogenase. *p<0.05 vs. si-NC; †p<0.05 vs. si-TUG1.

• 

  Figure 6 Effect of taurine-upregulated gene 1 (TUG1) on the tumor growth in vivo. (A) The tumor picture from mouse model of MCF-7 cells transfected with shRNA-TUG1 (n=3, each) or shRNA-NC (n=3, each). (B) The tumor volume in mouse model of MCF-7 cells transfected with shRNA-TUG1 (n=5, each) or shRNA-NC (n=5, each). Data represent mean±SD. *p<0.05 vs. shRNA-NC.

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