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. 2016 Nov 28:11:2951-2964.
doi: 10.2147/COPD.S109570. eCollection 2016.

"V体育官网" Screening of long non-coding RNA and TUG1 inhibits proliferation with TGF-β induction in patients with COPD

Wenxiang Tang  1 , Zhenyu Shen  2 , Jiang Guo  2 , Shenghua Sun  1
Affiliations

Screening of long non-coding RNA and TUG1 inhibits proliferation with TGF-β induction in patients with COPD

V体育官网 - Wenxiang Tang et al. Int J Chron Obstruct Pulmon Dis. .

"VSports手机版" Abstract

Objective: To evaluate differentially expressed long noncoding RNAs (lncRNAs) and the potential role of lncRNA TUG1 in patients with chronic obstructive pulmonary disease (COPD) VSports手机版. .

Methods: Total RNA was extracted from both COPD and non-COPD lung tissues, and microarray analysis was performed with 25,628 lncRNA probes and 20,106 mRNA probes. In addition, five up-regulated and five down-regulated lncRNAs were selected for identification using quantitative real-time polymerase chain reaction. COPD cell model was established by transforming growth factor β (TGF-β) treatment. Cell Counting Kit-8 assay was used to detect BEAS-2B and HFL1 cell proliferation after TUG-siRNA transfection with TGF-β treatment. In addition, the expression levels of α-SMA and fibronectin proteins were determined using Western blot in BEAS-2B and HFL1 cells after TUG-siRNA transfection with TGF-β treatment. V体育安卓版.

Results: There were 8,376 (32. 7%) differentially expressed lncRNAs and 5,094 (25. 3%) differentially expressed mRNAs in COPD lung tissues compared with non-COPD lung tissues V体育ios版. Five of the analyzed lncRNAs (BC038205, BC130595, TUG1, MEG3, and LOC646329) were markedly increased, while five lncRNAs (LOC729178, PLAC2, LOC339529, LINC00229, and SNHG5) were significantly decreased in COPD lung tissues compared with non-COPD lung tissues (n=20) (***P<0. 001). Knockdown of lncRNA TUG1 promotes BEAS-2B and HFL1 cell proliferation after TGF-β treatment through inhibiting the expression levels of α-SMA and fibronectin. .

Conclusion: Abundant, differentially expressed lncRNAs and mRNAs were identified by microarray analysis and these might play a partial or key role in the diagnosis of patients with COPD. LncRNA TUG1 may become a very important class of biomarker and may act as a potential diagnostic and therapeutic target for patients with COPD. VSports最新版本.

Keywords: COPD; TGF-β; lncRNA TUG1; long noncoding RNA; microarray analysis. V体育平台登录.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
The expression of lncRNAs and mRNAs in COPD lung tissues. (A) The percentage of differentially expressed lncRNAs in COPD lung tissues compared with non-COPD lung tissues. (B) The percentage of differentially expressed mRNAs in COPD lung tissues relative to non-COPD lung tissues. (C) The hierarchical clustering of differentially expressed lncRNAs, which are displayed on a scale from green (low) to red (high), between COPD and non-COPD lung tissues. (D) Heat map of distinguishable mRNA expression amid lung tissues samples; red shows relatively high expression and green shows relatively low expression. Abbreviation: lncRNAs, long noncoding RNAs.
Figure 2
Figure 2
KEGG pathway analysis of the differentially expressed mRNAs. (A) Pathway analysis demonstrates the significant pathways of differentially down-regulated mRNAs in model vs normal group. The P-value (EASE-score, Fisher’s P-value, or hypergeometric P-value) denotes the significance of the pathway correlated to the conditions. The lower the P-value, more significant is the pathway (the recommended P-value cutoff is 0.05). (B) Differentially up-regulated mRNAs in model vs normal group. Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; Sig, significant.
Figure 3
Figure 3
Gene ontology (GO) enrichment analysis of the differentially down-expressed mRNAs. (A) GO biological process classification for biological process of down-regulated mRNAs. (B) GO biological process classification for cellular components of differentially down-expressed mRNAs. (C) GO biological process classification for molecular function of differentially down-expressed mRNAs. (D) GO analysis indicates the top 10 counts of enrichment score in the biological process of down-regulated mRNAs. (E) GO analysis indicates the top 10 counts of enrichment score in cellular components of down-regulated mRNAs. (F) GO analysis indicates the top 10 counts of enrichment score in molecular function of down-regulated mRNAs. Abbreviation: Sig, significant.
Figure 4
Figure 4
Gene ontology (GO) enrichment analysis of the differentially up-expressed mRNAs. (A) GO biological process classification for biological process of up-regulated mRNAs. (B) GO biological process classification for cellular components of differentially up-expressed mRNAs. (C) GO biological process classification for molecular function of differentially up-expressed mRNAs. (D) GO analysis indicates the top 10 counts of enrichment score in biological process of up-regulated mRNAs. (E) GO analysis indicates the top 10 counts of enrichment score in cellular components of up-regulated mRNAs. (F) GO analysis indicates the top 10 counts of enrichment score in molecular function of up-regulated mRNAs. Abbreviation: Sig, significant.
Figure 5
Figure 5
Ten differentially expressed lncRNAs are identified. (A) The relative expression levels of 10 differentially up-regulated lncRNAs in COPD lung tissues compared with non-COPD lung tissues (n=20) were measured by qRT-PCR. (B) The relative expression levels of 10 differentially down-regulated lncRNAs beween COPD and non-COPD lung tissues (n=20) were detected by qRT-PCR. GAPDH served as the control for RNA loading (*P<0.05; **P<0.01; ***P<0.001). Abbreviations: lncRNA, long noncoding RNA; qRT-PCR, quantitative real-time polymerase chain reaction.
Figure 6
Figure 6
Silencing of TUG1 affects the proliferation ability of BEAS-2B and HFL1 cells after TGF-β pretreatment. (A) The relative expression levels of lncRNAs TUG1 in BEAS-2B cells were measured by qRT-PCR (**P<0.01). (B) The relative expression levels of lncRNAs TUG1 in HFL1 cells were measured by qRT-PCR (**P<0.01). (C) The capacity of proliferation was detected with CCK-8. About 50 μM of NC siRNAs or TUG1 siRNAs (si-TUG1) were transfected into BEAS-2B cells for 48 h. Silencing of TUG1 expression by siRNA promoted the capacity to proliferation in BEAS-2B cells. TGF-β (2 ng/mL) was treated for 48 h. TGF-β inhibited the capacity to proliferate. #P<0.05 as compared with BEAS-2B cells treated with 50 μM NC siRNAs and TGF-β (2 ng/mL); *P<0.05 as compared with BEAS-2B cells. (D) The capacity to proliferate was also detected using CCK-8 assay. Silencing of TUG1 expression by siRNA promoted the capacity of proliferation. TGF-β inhibited the capacity of proliferation in HFL1 cells. #P<0.05 as compared with HFL1 cells treated with 50 μM NC siRNAs and TGF-β (2 ng/mL); *P<0.05 as compared with HFL1 cells. Abbreviations: TGF-β, transforming growth factor β; lncRNA, long noncoding RNA; qRT-PCR, quantitative real-time polymerase chain reaction; CCK, Cell Counting Kit; NC, negative control; OD, optical density.
Figure 7
Figure 7
Silencing of TUG1 suppresses the expression levels of α-SMA and fibronectin proteins in BEAS-2B and HFL1 cells. (A) Silencing of TUG1 expression by siRNA inhibited the expression levels of α-SMA and fibronectin proteins, while TGF-β promoted the expression levels of α-SMA and fibronectin proteins. About 50 μM negative control (NC) siRNAs or TUG1 siRNAs (si-TUG1) and 2 ng/mL TGF-β were transfected into BEAS-2B cells for 48 h. The expression levels of α-SMA and fibronectin proteins were detected by Western blot in BEAS-2B cells. (B) Gray statistical analysis of the expression levels of α-SMA and fibronectin proteins in BEAS-2B cells. #P<0.05 as compared with BEAS-2B cells treated with 50 μM NC siRNAs and TGF-β (2 ng/mL); *P<0.05 as compared with BEAS-2B cells. **P<0.01. (C) The expression levels of α-SMA and fibronectin proteins were detected by Western blot in HFL1 cells treated as mentioned earlier. GAPDH was used for internal control. (D) Gray statistical analysis of the expression levels of α-SMA and fibronectin proteins in HFL1 cells. #P<0.05 as compared with HFL1 cells treated with 50 μM NC siRNAs and TGF-β (2 ng/mL); *P<0.05 as compared with HFL1 cells. ***P<0.001.
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