Long noncoding RNA TUG1 is a diagnostic factor in lung adenocarcinoma and suppresses apoptosis via epigenetic silencing of BAX

Abstract

Lung cancer is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel diagnostic markers and therapeutic targets. Increasing evidences have indicated that long non-coding RNAs (lncRNAs) play an important role in initiation and progression of lung cancer. However, the role of lncRNA Taurine upregulated 1 (TUG1) in lung adenocarcinoma (LAD) progression is not well known. In this study, we determined the diagnostic value of TUG1 in LAD patients, and further uncovered the underlying functional mechanism. Our results showed that TUG1 was significantly upregulated in LAD cells and serum samples. Receiver operator characteristic (ROC) analysis suggested a relatively higher area under the curve (AUC) of TUG1 (0.756) contrast to cyfra21-1 (0.619). In addition, high TUG1 level was associated with enhanced tumor size, degree of differentiation, lymph node metastases, distant metastasis and TNM stage. Cell functional assays showed that knockdown of TUG1 suppressed LAD cell viability and promoted cell apoptosis. We then sought to reveal the underlying regulatory mechanism, and the pro-apoptotic protein BAX was then identified as the downstream target of TUG1. Gain and loss functional assays showed that inhibition of BAX reversed the induced apoptosis by TUG1 knockdown. Finally, RNA immunoprecipitation and Chromatin immunoprecipitation revealed that TUG1 suppressed BAX expression through physically interacting with EZH2. In conclusion, lncRNA TUG1 is a promising diagnostic marker for LAD patients and suppression of TUG1 levels could be a future direction to promote the prognosis of LAD patients.

Keywords: BAX; EZH2; diagnosis; lncRNA TUG1; lung adenocarcinoma.

Figure 1. The investigation of lncRNA TUG1 as a diagnostic marker for LAD patients

(A) RT-qPCR showed that TUG1 was dramatically upregualted in 60 serum samples of LAD patients compared to healthy individuals. (B) TUG1 was upregulated in most LAD cell lines in contrast to normal human bronchial epithelial cell line 16HBE. (C–D) The ROC curve analysis of TUG1 (C) and cyfra21-1 (D) in 60 serum samples from LAD patients and 60 from healthy population. (E) Spearmen correlation test suggested a positive correlation between of TUG1 and cyfra21-1 expression in 60 serum samples. ^*P < 0.05; ^**P < 0.01

Figure 2. The effect of TUG1 on cell viability and apoptosis in LAD cell lines

(A) LncRNA TUG1 was silenced by specific siRNAs. (B) Knockdown of TUG1 inhibited cell viability of SPC-A1 and H1299 cells. (C) FACS apoptosis assay showed that cells transfected with si-TUG1-3 significantly promoted cell apoptosis in LAD. (D) TUNEL assay revealed that knockdown of TUG1 promoted nuclear apoptosis of SPC-A1 cells. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 3. BAX was identified as the downstream target of TUG1 in LAD cells

(A) Spearman correlation analysis indicated a significantly negative correlation between TUG1 and BAX mRNA expression in 60 serum samples of LAD patients. (B) Both mRNA and protein levels of BAX were upregulated by TUG1 knockdown. (C) BAX was silenced by specific siRNA. (D) CCK8 assay showed that transfection of si-BAX promoted cell viability in both SPC-A1 and H1299 cells. (E) TUNEL assay indicated that co-transfection with si-BAX reversed the si-TUG1-3 induced promotion of apoptosis in SPC-A1 cells. ^*P < P < 0.05; ^**P < 0.01

Figure 4. TUG1 directly interacted with EZH2 in LAD cells

(A) RT-qPCR showed that EZH2 mRNA was upregulated in serum samples of LAD patients. (B–C) EZH2 mRNA (B) and protein (C) was also upregulated in LAD cell lines when control cells. (D) RIP experiments were performed using the EZH2 antibody to immunoprecipitate RNA and two primers to detect lncRNA TUG1, and a relatively higher enrichment of TUG1 was found compared with IgG control. (E) RIP experiments showed that no enrichment of β-actin or lncRNA control was confirmed in SPC-A1 cells. (F) RNA pulldown assay was then performed to validate the the direct interaction between the two molecules. ^**P < 0.01; ^***P < 0.001

Figure 5. TUG1 suppressed BAX expression through interacting with EZH2

(A) EZH2 mRNA was silenced by specific siRNA. (B–C) BAX mRNA (B) and protein (C) was upregulated by EZH2 knockdown. (D) TUG1 was overexpressed by pTUG1 transfection. (E–H) ChIP analysis of SPC-A1 cells treated with p-TUG1 was conducted on BAX promoter (primer a-c) and GAPDH promoter (primer d-e) regions using the indicated antibodies. Enrichment was determined relative to input controls. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.