LncRNA TUG1 is upregulated and promotes cell proliferation in osteosarcoma

Abstract

Objective: To examine the expression and function of long non-coding RNA taurine up-regulated 1 (TUG1) in human osteosarcoma cells.

Methods: Real-time quantitive PCR was used to detect the transcription level of TUG1 in a series of osteosarcoma cell lines. Knockdown of TUG1 in U2OS cells was carried out by transient transfection of siRNAs. MTT assay was performed to access the cell growth rates. Afterwards, RNA and protein of these cells were extracted to analyze the transfection efficient as well as the expression of other molecules.

Results: Compared to the normal cell line, TUG1 exhibited a significant upregulation in osteosarcoma cells. Phenotyping analysis showed the growth-promotion activity of TUG1, since knockdown of TUG1 resulted in declined proliferation. We also found that AKT phosphorylation was impaired after TUG1 was inhibited, suggesting that the AKT pathway was involved in the regulation of TUG1 in U2OS cells.

Conclusion: Our data provided evidence that TUG1 was upregulated and acted as a possible oncogene via positively regulating cell proliferation in osteosarcoma cells.

Keywords: AKT; TUG1; osteosarcoma; proliferation.

Figure 1

LncRNA TUG1 is upregulated in osteosarcoma cell line. QRT-PCR analysis performed to detect expression of LncRNA TUG1 expression in human normal osteoblastic cell line hF081.19 and human osteosarcoma cell lines. LncRNA TUG1 expression levels were calculated by the 2-^∆CT method and normalized to GAPDH. ∆Ct is the difference in Ct values between LncRNA and GAPDH. The plot shows the TUG1 expression was elevated.

Figure 2

TUG1 promotes tumor proliferation in U2OS cells. (A) U2OS cells were transfected with control siRNA or lncRNA TUG1 siRNA. In 48h, the cells were harvested for examining knockdown effect. (B) U2OS cells were transfected with control siRNA or lncRNA TUG1 siRNA. In 24h, cells were trypsinized and seeded into four 96-well plates at a density of 3×10³ cells for MTT assay as described under Materials and Methods. Data are expressed as the mean ± s.d. of the experiments performed in triplicate. *P < 0.05, **P < 0.01.

Figure 3

Knockdown of TUG1 reduces survival markers in vitro. U2OS cells were transfected with control siRNA or lncRNA TUG1 siRNA. Forty-eight hours later, cells were harvested for western blotting. Protein level of PCNA, p-Akt (S473) and t-Akt were tested. β-actin was used as the loading control in western blotting. The results showed that inhibited expression of TUG1 suppressed cell proliferation marker PCNA, and also suppressed cell survival marker p-Akt.