Yonsei Med J.  2018 Mar;59(2):226-235. 10.3349/ymj.2018.59.2.226.

Long Non-Coding RNA TUG1 Promotes Proliferation and Inhibits Apoptosis of Osteosarcoma Cells by Sponging miR-132-3p and Upregulating SOX4 Expression

• Li G
• Liu K
• Du X

• KMID: 2418785
• DOI: http://doi.org/10.3349/ymj.2018.59.2.226

Abstract

PURPOSE
Long non-coding RNA taurine upregulated gene 1 (TUG1) is reported to be a vital regulator of the progression of various cancers. This study aimed to explore the exact roles and molecular mechanisms of TUG1 in osteosarcoma (OS) development.
MATERIALS AND METHODS
Real-time quantitative PCR was applied to detect the expressions of TUG1 and microRNA-132-3p (miR-132-3p) in OS tissues and cells. Western blot was performed to measure protein levels of sex determining region Y-box 4 (SOX4). Cell viability was assessed using XTT assay. Cell apoptosis was evaluated using flow cytometry and caspase-3 activity detection assays. Bioinformatics analysis and luciferase reporter experiments were employed to confirm relationships among TUG1, miR-132-3p, and SOX4.
RESULTS
TUG1 was highly expressed in human OS tissues, OS cell lines, and primary OS cells. TUG1 knockdown hindered proliferation and induced apoptosis in human OS cell lines and primary OS cells. Moreover, TUG1 inhibited miR-132-3p expression by direct interaction, and introduction of miR-132-3p inhibitor partly abrogated the effect of TUG1 knockdown on the proliferation and apoptosis of OS cells. Furthermore, SOX4 was validated as a target of miR-132-3p. Further functional analyses revealed that miR-132-3p inhibited proliferation and induced apoptosis of OS cells, while this effect was greatly abated following SOX4 overexpression. Moreover, TUG1 knockdown suppressed proliferation and promoted apoptosis by upregulating miR-132-3p and downregulating SOX4 in primary OS cells.
CONCLUSION
TUG1 facilitated proliferation and suppressed apoptosis by regulating the miR-132-3p/SOX4 axis in human OS cell lines and primary OS cells. This finding provides a potential target for OS therapy.

Keyword

Osteosarcoma; TUG1; miR-132-3p; SOX4

MeSH Terms

Apoptosis/*genetics
Biomarkers, Tumor
Bone Neoplasms/genetics/metabolism/*pathology
Cell Line, Tumor
Cell Proliferation
Gene Expression Regulation, Neoplastic/genetics
Gene Knockdown Techniques
Humans
MicroRNAs/*genetics/metabolism
Osteosarcoma/genetics/metabolism/*pathology
RNA, Long Noncoding/*genetics/metabolism
Real-Time Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
SOXC Transcription Factors/genetics/*metabolism
Transcriptional Activation
Tumor Cells, Cultured
Up-Regulation
Biomarkers, Tumor
MicroRNAs
RNA, Long Noncoding
SOXC Transcription Factors

Figure

• 

  Fig. 1 TUG1 expression is up-regulated in OS tissues and cells, and TUG1 depletion hampers proliferation and induces apoptosis of OS cells. (A-E) Real-time quantitative PCR assays were employed to measure the expression of TUG1 in tissues and cells. (A) Expression of TUG1 in 22 cases of OS tissues with the expression in correspondingly adjacent normal tissues as a normalization standard. (B) Overall expression analysis of TUG1 in 22 pairs of OS tissues and adjacent normal tissues with β-actin as an internal control. (C) TUG1 expression in human normal osteoblastic cell line FOB1.19 and OS cell lines (U2OS, MG-63, Saos-2, 143B) with β-actin as an internal control. (D and E) The transfection efficiency of pcDNA3.1-TUG1 in MG-63 cells (D) and the knockdown efficiency of TUG1 siRNA in U2OS cells (E). (F and G) The effect of TUG1 overexpression (F) and knockdown (G) on OS cell proliferation was assessed by XTT assays at the indicated time point (0, 24, 48, and 72 h) after transfection. (H and I) The effect of TUG1 knockdown on U2OS cell apoptosis was evaluated by flow cytometry (H) and caspase-3 activity detection (I) after transfection 48 h. *p<0.05. TUG1, taurine upregulated gene 1; OS, osteosarcoma.

• 

  Fig. 2 si-TUG1 exerts its anti-proliferation and pro-apoptosis effect by targeting miR-132-3p in OS cells. (A) The putative binding sites between TUG1 and miR-132-3p and the mutation sites of TUG1 in the MUT-TUG1 reporter. (B) The effect of miR-132-3p on luciferase activity of WT-TUG1 or MUT-TUG1 reporter. (C) The effect of TUG1 overexpression and knockdown on miR-132-3p expression in MG-63 and U2OS cells. (D) The effect of miR-132-3p on TUG1-mediated pro-proliferation effect in MG-63 cells. (E-G) The influence of miR-132-3p inhibitor on si-TUG1-mediated anti-proliferation (E) and pro-apoptosis (F and G) effect in U2OS cells. *p<0.05. TUG1, taurine upregulated gene 1; OS, osteosarcoma; WT-TUG1, wild-type TUG1; MUT-TUG1, mutant TUG1.

• 

  Fig. 3 SOX4 is a target of miR-132-3p. (A) The putative binding sites between SOX4-3′UTR and miR-132-3p and the mutation sites of SOX4-3′UTR in the MUT-SOX4-3′UTR reporter. (B) The effect of miR-132-3p on luciferase activity of WT-SOX4-3′UTR or MUT-SOX4-3′UTR reporter. (C) SOX4 protein expression in miR-132-3p-transfected MG-63 cells and anti-miR-132-3p-transfected U2OS cells. The lower histogram presented the gray values. *p<0.05. SOX4, sex determining region Y-box 4; WT, wild-type; MUT, mutant.

• 

  Fig. 4 MiR-132-3p inhibits proliferation and induces apoptosis of MG-63 and U2OS cells partly by targeting SOX4. (A, C, D) MG-63 cells were transfected with miR-NC, miR-132-3p, miR-132-3p+pcDNA3.1vector, or miR-132-3p+pcDNA3.1-SOX4, followed by the detection of cell proliferation (A), apoptosis rate (C) and caspase-3 activity (D). (B) U2OS cells were transfected with anti-miR-NC, anti-miR-132-3p inhibitor, anti-miR-132-3p+si-NC scramble control, or anti-miR-132-3p+si-SOX4, followed by cell proliferation by XTT assays. *p<0.05. SOX4, sex determining region Y-box 4.

• 

  Fig. 5 TUG1 acts as a ceRNA of miR-132-3p to facilitate SOX4 expression in OS cells. (A) MG-63 cells were transfected with pcDNA3.1 vector, pcDNA3.1-TUG1, pcDNA3.1-TUG1+miR-NC, or pcDNA3.1-TUG1+miR-132-3p. (B) U2OS cells were transfected with si-NC, si-TUG1, si-TUG+anti-miR-NC, si-TUG1+anti-miR-132-3p. After transfection 48 h, SOX4 protein levels in MG-63 (A) and U2OS (B) cells were detected by western blot assays. *p<0.05. TUG1, taurine upregulated gene 1; SOX4, sex determining region Y-box 4.

• 

  Fig. 6 Knockdown of TUG1 suppresses proliferation and promotes apoptosis by regulating miR-132-3p/SOX4 axis in primary OS cells. (A) RT-qPCR assays were performed to measure TUG1 expression in FOB1.19 and primary OS cells. (B) Primary OS cells were transfected with pcDNA3.1 empty vector, pcDNA3.1-TUG1, si-NC, or si-TUG1 for 48 h, followed by the detection of TUG1 level using RT-qPCR assays. (C and D) Primary OS cells were transfected with pcDNA3.1 empty vector, pcDNA3.1-TUG1, si-NC, or si-TUG1. At the indicated time points (0, 24, 48, 72 h) after transfection, cell viability was determined using XTT assays. (E and F) The effects of TUG1 knockdown on apoptosis rate and caspase-3 activity were measured in primary OS cells at 48 h following transfection. (G and H) Primary OS cells were transfected with pcDNA3.1 empty vector, pcDNA3.1-TUG1, si-NC, or si-TUG1 for 48 h, followed by the detection of miR-132-3p and SOX4 expressions. *p<0.05. TUG1, taurine upregulated gene 1; SOX4, sex determining region Y-box 4; OS, osteosarcoma; RT-qPCR, real-time quantitative PCR.

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