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. 2019 Jan;47(1):481-493.
doi: 10.1177/0300060518816592. Epub 2018 Dec 16.

Long intergenic noncoding RNA-p21 inhibits apoptosis by decreasing PUMA expression in non-small cell lung cancer

Tao Yang  1 , Wenjun Zhang  1 , Li Wang  2 , Chunyan Xiao  1 , Bingling Guo  1 , Yi Gong  1 , Xiping Liang  1 , Dehong Huang  1 , Qiying Li  1 , Yingyu Nan  1 , Ying Xiang  1 , Jianghe Shao  1
Affiliations

Long intergenic noncoding RNA-p21 inhibits apoptosis by decreasing PUMA expression in non-small cell lung cancer

Tao Yang et al. J Int Med Res. 2019 Jan.

Abstract

Objective: Long noncoding RNAs (lncRNAs) are important mediators in tumor progression. Long intergenic noncoding RNA-p21 (lincRNA-p21) participates in multiple biological processes. This study explored the role of lincRNA-p21 in human non-small cell lung cancer (NSCLC) progression and potential regulatory mechanisms.

Methods: LincRNA-p21 expression in NSCLC tissues and cell lines (A549, H1299, H1650, and NCI-H2087) was determined by quantitative real-time PCR. LincRNA-p21 overexpressing and sh-lincRNA-p21 lentiviral were respectively transfected into H1299 and A549 cells. Flow cytometry was used to measure apoptosis. Microarray analysis and RNA pull-down assay were used to predict the target genes of lincRNA-p21. Finally, PUMA siRNA and overexpressing PUMA were transfected into NSCLC cells, and the extent of cell apoptosis was measured. The protein expression levels of the relative genes were confirmed by western blot analysis.

Results: LincRNA-p21 was significantly upregulated in NSCLC tissues and cells. The upregulation of lincRNA-p21 considerably inhibited cell apoptosis while the downregulation of lincRNA-p21 showed the opposite effect. PUMA was a direct target gene of lincRNA-p21 and was negatively correlated with lincRNA-p21 in NSCLC specimens. The anti-apoptotic effect of lincRNA-p21 can be effectively attenuated by the upregulation of PUMA.

Conclusion: LincRNA-p21 is aberrantly upregulated in NSCLC and inhibits cell apoptosis by decreasing PUMA expression.

Keywords: Long noncoding RNAs; PUMA; apoptosis; lincRNA-p21; non-small cell lung cancer; p53.

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Figures

Figure 1.
Figure 1.
LincRNA-p21 level in NSCLC tissues and cell lines, and lincRNA-p21 overexpressing or knockdown cells. a. LincRNA-p21 is upregulated in non-small cell lung cancer tissues. LincRNA-p21 expression levels in non-small cell lung cancer tissues and adjacent normal lung tissues examined by real-time PCR. n = 31. A paired Student’s t-test was performed with SPSS Statistics for Windows, Version 17.0, to analyze real-time PCR data. A significant increase of lincRNA-p21 was observed in cancer tissues compared with adjacent normal lung tissues, P < 0.001. b. Real-time PCR analysis of lincRNA-p21 expression levels in four NSCLC cell lines (A549, H1299, H1650, and NCI-H2087) and the normal bronchial epithelial cell line 16HBE. Levels represent means ± SEM from at least three independent experiments. c. Relative mRNA expression of lincRNA-p21 in A549 cells infected with the control lentivirus and lincRNA-p21 overexpressing lentivirus examined by real-time PCR, *P < 0.05. Data are expressed as means ± SEM from at least three independent experiments. d. Representative real-time PCR data showing lentivirus-mediated overexpression of lincRNA-p21 in H1299 cells, **P < 0.01
Figure 2.
Figure 2.
LincRNA-p21 regulates apoptosis of non-small cell lung cancer cells. a. Representative FACS result showing that lincRNA-p21 knockdown enhanced A549 cell apoptosis by FITC-Annexin V/PI staining. b. Histogram showing that lncRNA-p21 knockdown increased the percentage of apoptotic A549 cells, **P< 0.01. c. Representative FACS result showing that lincRNA-p21 overexpression inhibited H1299 cell apoptosis by FITC-Annexin V/PI staining. d. Histogram showing that lncRNA-p21 overexpression decreased the percentage of apoptotic H1299 cells, *P< 0.05
Figure 3.
Figure 3.
PUMA is identified as a target of lincRNA-p21. a. Expression profile in A549 cells after knockdown of lincRNA-p21. b. Expression profile in H1299 cells after lincRNA-p21 was overexpressed. c. Venn diagram showing overlapping differentially expressed genes in A549 and H1299 gene expression profiles. d. RT-qPCR assay showing the relative mRNA level of PUMA in A549 cells after lincRNA-p21 pull-down. e, f. Expression of PUMA mRNA in sh-lincRNA-p21-infected A549 cells or lincRNA-p21-infected H1299 cells as determined by real-time PCR, *P < 0.05, **P < 0.01. g. Protein expression of PUMA in sh-lincRNA-p21-infected A549 cells or lincRNA-p21-infected H1299 cells as determined by western blotting. h. Correlation between mRNA levels of PUMA and lincRNA-p21 in non-small cell lung cancer specimens
Figure 4.
Figure 4.
PUMA attenuates the anti-apoptotic effect of lincRNA-p21. a. Protein expression of PUMA in sh-linRNA-p21 A549 cells transfected with control and PUMA siRNAs as determined by western blotting. b. Histogram showing the protein expression of PUMA in sh-linRNA-p21 A549 cells transfected with control and PUMA siRNAs; **P < 0.01, ##P < 0.01. c. Representative FACS result showing the extent of apoptosis in sh-linRNA-p21 A549 cells transfected with control and PUMA siRNAs as measured by FITC-Annexin V/PI staining. d. Histogram showing the percentage of apoptotic sh-linRNA-p21 A549 cells transfected with control and PUMA siRNAs; **P < 0.01, #P < 0.05. e. Protein expression of PUMA in linRNA-p21 H1299 cells transfected with control and PUMA overexpressing plasmid as determined by western blotting. f. Histogram showing the protein expression of PUMA in sh-linRNA-p21 H1299 cells transfected with control and PUMA overexpressing plasmids; **P < 0.01, #P < 0.05. g. Representative FACS result showing the extent of apoptosis in linRNA-p21 H1299 cells transfected with control and PUMA overexpressing plasmid as measured by FITC-Annexin V/PI staining. h. Histogram showing the percentage of apoptotic cells in linRNA-p21 H1299 cells transfected with control and PUMA overexpressing plasmids; **P < 0.01, #P < 0.05
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