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. 2016 Nov 16:6:37233.
doi: 10.1038/srep37233.

Analysis of Microarray Data on Gene Expression and Methylation to Identify Long Non-coding RNAs in Non-small Cell Lung Cancer

Nannan Feng  1 , Travers Ching  2 , Yu Wang  3 , Ben Liu  3 , Hongyan Lin  1 , Oumin Shi  1 , Xiaohong Zhang  1 , Min Zheng  1 , Xin Zheng  1 , Ming Gao  3 , Zhi-Jie Zheng  1 , Herbert Yu  2 , Lana Garmire  2 , Biyun Qian  1
Affiliations

Analysis of Microarray Data on Gene Expression and Methylation to Identify Long Non-coding RNAs in Non-small Cell Lung Cancer

Nannan Feng et al. Sci Rep. .

Abstract

To identify what long non-coding RNAs (lncRNAs) are involved in non-small cell lung cancer (NSCLC), we analyzed microarray data on gene expression and methylation. Gene expression chip and HumanMethylation450BeadChip were used to interrogate genome-wide expression and methylation in tumor samples. Differential expression and methylation were analyzed through comparing tumors with adjacent non-tumor tissues. LncRNAs expressed differentially and correlated with coding genes and DNA methylation were validated in additional tumor samples using RT-qPCR and pyrosequencing. In vitro experiments were performed to evaluate lncRNA's effects on tumor cells. We identified 8,500 lncRNAs expressed differentially between tumor and non-tumor tissues, of which 1,504 were correlated with mRNA expression. Two of the lncRNAs, LOC146880 and ENST00000439577, were positively correlated with expression of two cancer-related genes, KPNA2 and RCC2, respectively. High expression of LOC146880 and ENST00000439577 were also associated with poor survival. Analysis of lncRNA expression in relation to DNA methylation showed that LOC146880 expression was down-regulated by DNA methylation in its promoter. Lowering the expression of LOC146880 or ENST00000439577 in tumor cells could inhibit cell proliferation, invasion and migration. Analysis of microarray data on gene expression and methylation allows us to identify two lncRNAs, LOC146880 and ENST00000439577, which may promote the progression of NSCLC.

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Figures

Figure 1
Figure 1. Bioinformatics analysis of lncRNA.
(a) PCA of all lncRNA expression results from the microarray analysis. (b) Heatmap results based on the top 3,690 lncRNAs significantly associated with tumor samples from the microarray analysis of gene expression. (c) IPA results based on the 1,345 cis-pairs of lncRNAs and mRNAs which were differentially expressed in NSCLC. (d) Distributions of the methylation loci that were significantly correlated with lncRNA expression and their associated lncRNAs on chromosome 17. X axis is the correlation coefficient; Y axis is the p value; Z axis is the location of CpG sites in reference to TSS.
Figure 2
Figure 2. Validation of LOC146880 and ENST00000439577 expression in tumor and adjacent non-tumor tissues of NSCLC.
(a) Higher expression of LOC146880 and KPNA2 in tumor than in adjacent non-tumor tissues. (b) A positive correlation between LOC146880 and KPNA2 expression. (c) High LOC146880 expression associated with poor overall survival (left: training set; right: validation set). (d) Lower methylation of cg12562461 in tumor than in adjacent non-tumor tissues. (e) Lower methylation of cg12562461 in high than in low LOC146880 expression tumors. (f) Higher expression of ENST00000439577 and RCC2 in tumor than in adjacent non-tumor tissues. (g) A positive correlation between ENST00000439577 and RCC2 expression. (h) High ENST00000439577 expression associated with poor overall survival (left: training set; right: validation set). Data are expressed as mean ± s.d.; *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
Figure 3. Functional analysis of LOC146880 expression in NSCLC cell lines.
(a) Declined expression of LOC146880 and KPNA2 after siRNA-LOC146880 transfection. Declined expression of KPNA2 proteins after siRNA-LOC146880 transfection. (b) Declined A549/PC9 cell proliferation after lowering the expression of LOC146880. (c) Declined A549/PC9 cell migration after lowering the expression of LOC146880. (d) Declined A549/PC9 cell invasion after lowering the expression of LOC147880. (e) Reduced cell cycle progression measured by the C6 flow cytometry after lowering the expression of LOC146880. (f) A promoter region of LOC146880 contains a CpG site which was predicted to be a transcription factor SP1 binding site. A LOC146880 fragment (−623/+123) was cloned into the pGL3-basic vector. The vector containing the LOC146880 (−623/+123) construct displayed 15-fold increases in promoter activities compared with the PGL3-basic vector. LOC146880 methylation is lower in tumors than in normal tissues. Data are expressed as mean ± s.d.; *P < 0.05, **P < 0.01. NC, Negative Control.
Figure 4
Figure 4. Functional analysis of ENST00000439577 expression in NSCLC cell lines A549 and H1299.
(a) Declined expression of ENST00000439577 and RCC2 after siRNA-ENST00000439577 transfection. (b) Reduced A549/H1299 cell proliferation after lowering the expression of ENST00000439577. (c) Reduced A549/H1299 cell migration after lowering the expression of ENST00000439577. (d) Reduced A549/H1299 cell invasion after lowering the expression of ENST00000439577. (e) Cell cycle analysis by the C6 flow cytometry after lowering the expression of ENST00000439577. Data are expressed as mean ± s.d.; *P < 0.05. NC, Negative Control.
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