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. 2016 Sep 15:6:33535.
doi: 10.1038/srep33535.

Analysis of long non-coding RNA expression profiles in pancreatic ductal adenocarcinoma

Xue-Liang Fu  1 , De-Jun Liu  1 , Ting-Ting Yan  2 , Jian-Yu Yang  1 , Min-Wei Yang  1 , Jiao Li  1 , Yan-Miao Huo  1 , Wei Liu  1 , Jun-Feng Zhang  1 , Jie Hong  2 , Rong Hua  1 , Hao-Yan Chen  2 , Yong-Wei Sun  1
Affiliations

Analysis of long non-coding RNA expression profiles in pancreatic ductal adenocarcinoma

Xue-Liang Fu et al. Sci Rep. .

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive and lethal malignancies. Long non-coding RNAs (lncRNAs) are a novel class of non-protein-coding transcripts that have been implicated in cancer biogenesis and prognosis. By repurposing microarray probes, we herein analysed the lncRNA expression profiles in two public PDAC microarray datasets and identified 34 dysregulated lncRNAs in PDAC. In addition, the expression of 6 selected lncRNAs was confirmed in Ren Ji cohort and pancreatic cell lines, and their association with 80 PDAC patients' clinicopathological features and prognosis was investigated. Results indicated that AFAP1-AS1, UCA1 and ENSG00000218510 might be involved in PDAC progression and significantly associated with overall survival of PDAC. UCA1 and ENSG00000218510 expression status may serve as independent prognostic biomarkers for overall survival of PDAC. Gene set enrichment analysis (GSEA) analysis suggested that high AFAP1-AS1, UCA1 and low ENSG00000218510 expression were correlated with several tumorigenesis related pathways. Functional experiments demonstrated that AFAP1-AS1 and UCA1 were required for efficient invasion and/or proliferation promotion in PDAC cell lines, while ENSG00000218510 acted the opposite. Our findings provide novel information on lncRNAs expression profiles which might be beneficial to the precise diagnosis, subcategorization and ultimately, the individualized therapy of PDAC.

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Figures

Figure 1
Figure 1. Flowchart of the study overview.
LncRNA expression profiles were retrieved from Affymetrix HG-U133 Plus 2.0 microarray through a lncRNA-mining approach. LncRNA expression analyses were performed in the training dataset (GSE16515) first and then validated in the testing dataset (GSE15471). The 6 selected lncRNAs were then verified in Ren Ji cohort and pancreatic cell lines.
Figure 2
Figure 2. Distinctive lncRNA expressions between PDAC tissues and normal pancreas.
(A) One-way hierarchical clustering of the 39 lncRNA probe sets (corresponding to 34 lncRNAs) identified as significantly different between PDAC tissues and normal pancreas in the training dataset (GSE16515). (B) Validation of the 39 probe signatures in the testing dataset (GSE15471). Each column represents one sample and each row represents one lncRNA probe set. Gene expression levels are indicated as follows: red, high expression (+3.0); green, low expression (−3.0). The bar colors in the dendrogram represent the sample types as indicated: blue, tumor; yellow, normal. (C,D) The distribution of all 2448 lncRNA probe sets (corresponding to 1970 lncRNAs) and the acquired 39 distinctive probe sets expression differentials between the experimental dataset GSE16515 and the validation dataset GSE15471.
Figure 3
Figure 3. Validation of candidate lncRNAs in PDAC patients and cell lines by qRT-PCR analysis.
(A–F) Analysis of CRNDE, NR_036488, ENSG00000244649, AFAP1-AS1, UCA1 and ENSG00000218510 relative expression in human PDAC and their matched normal pancreas samples, Ren Ji cohort, n = 80, paired sample t-test. (G–L) Relative expression level of CRNDE, NR_036488, ENSG00000244649, AFAP1-AS1, UCA1 and ENSG00000218510 in 8 PDAC cell lines compared to the nonmalignant HPDE6-C7 cell line. *P < 0.05 (Student’s t-test), ns: not significant. The qRT-PCR expression data were all shown as mean ± SD and normalised by GAPDH. GAPDH: glyceraldehyde-phosphate dehydrogenase; NP: normal pancreas.
Figure 4
Figure 4. The potential value of candidate lncRNAs expression in predicting PDAC and patient prognosis.
(A–C) Kaplan-Meier analysis of overall survival in Ren Ji cohort. Patients were scored as low and high expression group using the median value as cutoff according to lncRNAs expression. Results showed that patients with higher AFAP1-AS1, UCA1 and lower ENSG00000218510 expression have a poorer overall survival after surgery than their corresponding counterparts in PDAC. P-values were calculated by log-rank test. (D) ROC curve analyses of CRNDE, NR_036488, ENSG00000244649, AFAP1-AS1, UCA1 and ENSG00000218510 for prediction of PDAC using qRT-PCR-based expression level in Ren Ji cohort. AUC: the area under curve.
Figure 5
Figure 5. LncRNA UCA1 and ENSG00000218510 regulate the proliferation and/or invasion abililty of PDAC cell lines.
(A) UCA1 and ENSG00000218510 knockdown efficiency was confirmed by qRT-PCR in MIA PaCa-2 and Capan-2 cell line, respectively. (B) The effect of UCA1 and ENSG00000218510 knockdown on PDAC cell line proliferation was determined by CCK-8 assays. *P < 0.05, Student’s t-test. Data are represented as the mean ± SD. (C) Representative images of transwell assay after UCA1 and ENSG00000218510 knockdown in MIA PaCa-2 and Capan-2, respectively.
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