Long noncoding RNA TP53TG1 promotes pancreatic ductal adenocarcinoma development by acting as a molecular sponge of microRNA-96

Abstract

Long noncoding RNAs (lncRNAs) are emerging as key regulators in cancer initiation and progression. TP53TG1 is a recently identified lncRNA and several studies have shown that TP53TG1 may play the role of tumor suppressor gene or oncogene in different tumors. Nevertheless, the involvement of TP53TG1 in carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) has not been characterized. In our studies, we identified that TP53TG1 was highly expressed in PDAC and was a novel regulator of PDAC development. Knockdown of TP53TG1 inhibited proliferation, induced apoptosis, and decreased migration and invasion in PDAC cells, whereas enhanced expression of TP53TG1 had the opposite effects. Mechanistically, TP53TG1 could directly bind to microRNA (miR)-96 and effectively function as a sponge for miR-96, thus antagonizing the functions of miR-96 and leading to derepression of its endogenous target KRAS, which is a core oncogene in the initiation and maintenance of PDAC. Taken together, these observations imply that TP53TG1 contributes to the growth and progression of PDAC by acting as a competing endogenous RNA (ceRNA) to competitively bind to miR-96 and regulate KRAS expression, which highlights the importance of the complicated miRNA-lncRNA network in modulating the progression of PDAC.

Keywords: KRAS; ceRNA; lncRNA TP53TG1; miR-96; pancreatic ductal adenocarcinoma.

Figure 1

TP53TG1 is a novel long noncoding RNA and is upregulated in pancreatic ductal adenocarcinoma (PDAC). A, Genome browser representation of TP53TG1. Current gene annotations from our rapid amplification of cDNA ends (RACE) data and The University of California Santa Cruz (UCSC) genes are shown. B, The full sequence of TP53TG1 extending from 5′ and 3′ ends RACE technique is compared with the NCBI sequence (NR_015381.1). Red letters represent the 12 extra bases at the 5′ end of TP53TG1. C, TP53TG1 expression in normal pancreatic tissues and PDAC tissues, according to the array data (NCBI/GEO/GSE16515, GSE15471). Values are median with 95% CI. D, TP53TG1 expression in normal pancreatic tissues and PDAC tissues, as determined by RT‐qPCR

Figure 2

Downregulation of TP53TG1 inhibits pancreatic ductal adenocarcinoma cell proliferation. A, TP53TG1 expression was measured by RT‐qPCR following treatment of PANC‐1 and MIA PaCa‐2 cells with negative control (NC) or with TP53TG1 siRNAs. ***P < .001. B, CCK‐8 assay was carried out to determine the growth curves of si‐TP53TG1‐transfected PANC‐1 and MIA PaCa‐2 cells. **P < .01. C, Proliferation in PANC‐1 and MIA PaCa‐2 cells after TP53TG1 knockdown was detected through EdU‐incorporation assays. Proliferating cells were labeled with EdU. Scale bars, 100 μm. ***P < .001. D, Apoptosis was determined following treatment of PANC‐1 and MIA PaCa‐2 cells with NC or si‐TP53TG1 by flow cytometric analysis. *P < .05

Figure 3

Downregulation of TP53TG1 inhibits pancreatic ductal adenocarcinoma (PDAC) cell migration and invasion. A, Wound‐healing assays were carried out to determine the migratory ability of si‐TP53TG1‐transfected PANC‐1 and MIA PaCa‐2 cells. **P < .01, ***P < .001. B, Effect of TP53TG1 downregulation on the invasive abilities of PANC‐1 and MIA PaCa‐2 cells was measured using Transwell assays. ***P < .001. C, Colony formation assays were carried out to determine the anchorage‐independent growth abilities of PANC‐1 and MIA PaCa‐2 cells after TP53TG1 knockdown. **P < .001. NC, negative control

Figure 4

Enforced expression of TP53TG1 promotes the proliferation and invasion of pancreatic ductal adenocarcinoma (PDAC) cells. A, TP53TG1 expression in PANC‐1 and MIA PaCa‐2 cells transfected with pcDNA3.1‐TP53TG1 or empty pcDNA3.1 vector was measured by RT‐qPCR. ***P < .001. B, CCK‐8 assay was carried out to determine the growth curves of PANC‐1 and MIA PaCa‐2 cells after transfection with pcDNA3.1‐TP53TG1 or empty pcDNA3.1 vector. **P < .01, ***P < .001. C, Proliferation in PANC‐1 and MIA PaCa‐2 cells after TP53TG1 overexpression was detected through EdU‐incorporation assays. Proliferating cells were labeled with EdU. Scale bars, 100 μm. **P < .01. D, Effect of TP53TG1 upregulation on the anchorage‐independent growth capacity of PANC‐1 and MIA PaCa‐2 cells was determined using colony formation assays. **P < .01. E, Transwell assays were carried out to determine the invasive ability of PANC‐1 and MIA PaCa‐2 cells after upregulation of TP53TG1. ***P < .001

Figure 5

Long noncoding RNA (lncRNA) TP53TG1 is negatively regulated by microRNA (miR)‐96. A, Predicted binding sites for miR‐96 in TP53TG1 sequences. Numbers show the nucleotides relative to the transcriptional start site of TP53TG1. B, PANC‐1 and MIA PaCa‐2 cells were transfected with miR‐96 mimic and, 48 h later, total RNA was isolated and the expression of TP53TG1 was analyzed by RT‐qPCR. **P < .01, ***P < .001. C, Localization of TP53TG1 in PANC‐1 and MIA PaCa‐2 cells was determined by RNA‐in situ hybridization assays. The signal of TP53TG1 is stained blue. Scale bars, 50 μm. D, RT‐qPCR detection of the percentage of TP53TG1, miR‐96, GAPDH and MALAT1 in the cytoplasmic and nuclear fractions of PANC‐1 and MIA PaCa‐2 cells. GAPDH and MALAT1 serve as cytoplasmic and nuclear localization indicators, respectively

Figure 6

TP53TG1 acts as a competing endogenous RNA to regulate KRAS expression by sponging microRNA (miR)‐96. A, Bioinformatics analysis shows that there are putative binding sites for miR‐96 on both TP53TG1 and the 3′UTR of KRAS mRNA and mutation was generated in the complementary site for the seed region of miR‐96. B, Luciferase activity in PANC‐1 cells cotransfected with miR‐96 mimics and wild type (WT) or mutant (MUT) TP53TG1 or KRAS luciferase reporters was measured using a dual‐luciferase reporter gene assay system. ***P < .001. C, Upper panel: Immunoprecipitation of Ago2 protein by Ago2 or IgG antibody was detected by western blot assay. Lower panel: RIP followed by RT‐qPCR was used to detect miR‐96, TP53TG1 and KRAS endogenously associated with Ago2. Expression level of miR‐96, TP53TG1 and KRAS was normalized to that in the IgG antibody group. ***P < .001. D‐E, Expression of KRAS and miR‐96 in pancreatic ductal adenocarcinoma tissues and corresponding normal pancreatic tissues was detected by RT‐qPCR, and the associations between expression levels of KRAS, miR‐96 and TP53TG1 were determined by Pearson correlation analysis. F, Effect of miR‐96 mimic or TP53TG1‐siRNAs on the expression of KRAS in PANC‐1 cells was detected by western blot. G, Effect of cotransfected PANC‐1 cells with TP53TG1 expression plasmid and miR‐96 mimic on the expression of KRAS was detected by western blot