Abstract
BackgroundAbnormal expression of numerous long non-coding RNAs (lncRNAs) has been reported in esophageal squamous cell carcinoma (ESCC) recently, but the great majority of their roles and mechanisms remain largely unclear. We aim to identify the critical ESCC-associated lncRNAs and elucidate the functions and mechanisms in detail.MethodsMicroarrays were used to analyze the differentially expressed lncRNAs in ESCC tissues. qRT-PCR was used to verify the result of microarrays. The effects of the most up-regulated lncRNA, cancer susceptibility candidate 9(CASC9), on cell growth, proliferation and cell cycle were investigated by in vivo and in vitro assays. Microarrays and recovery tests were used to discover the regulatory targets of CASC9. RNA FISH and subcellular fractionation assays were used to detect the subcellular location of CASC9. Finally, the mechanism of CASC9 regulating PDCD4 was explored by RIP, RNA-protein pull down and ChIP assays.ResultsESCC tissue microarrays showed that CASC9 was the most up-regulated lncRNA. qRT-PCR analysis indicated that CASC9 expression was positively associated with tumor size and TNM stage, and predicted poor overall survival of ESCC patients. Knockdown of CASC9 inhibited ESCC cell growth in vitro and tumorigenesis in nude mice. Furthermore interfering CASC9 decreased cell proliferation and blocked cell cycle G1/S transition. CASC9-associated microarrays indicated that PDCD4 might be the target of CASC9. Consistent with this, PDCD4 expression was negatively associated with CASC9 expression in ESCC tissues and predicted good prognosis. Manipulating CASC9 expression in ESCC cells altered both PDCD4 mRNA and protein levels and cell cycle arrest caused by CASC9 knockdown could be rescued by suppressing PDCD4 expression. CASC9 located both in the nucleus and cytoplasm. Mechanistically, enhancer of zeste homolog2 (EZH2) could bind to both CASC9 and PDCD4 promoter region. Interfering CASC9 reduced the enrichment of EZH2 and H3K27me3 in the PDCD4 promoter region.ConclusionsOur study firstly demonstrates that lncRNA CASC9 functions as an oncogene by negatively regulating PDCD4 expression through recruiting EZH2 and subsequently altering H3K27me3 level. Our study implicates lncRNA CASC9 as a valuable biomarker for ESCC diagnosis and prognosis.
Highlights
Abnormal expression of numerous long non-coding RNAs has been reported in esophageal squamous cell carcinoma (ESCC) recently, but the great majority of their roles and mechanisms remain largely unclear
Results long non-coding RNAs (lncRNAs) expression profile in ESCC To identify the lncRNAs involved in ESCC progression, we selected 5 pairs of ESCC and adjacent normal tissues to conduct lncRNAs expression profiling, which included 24,011 probes in total
Further analysis revealed that the deregulated lncRNAs in ESCC included many well-known tumor-associated lncRNAs, such as H19, TUG1 and SOX2OT, which have been reported to contribute to tumorigenicity in other cancers [20,21,22]
Summary
Abnormal expression of numerous long non-coding RNAs (lncRNAs) has been reported in esophageal squamous cell carcinoma (ESCC) recently, but the great majority of their roles and mechanisms remain largely unclear. Li et al has studied the lncRNA expression profile of ESCC by microarray including 119 pairs of tumor and normal tissues and found that a three-lncRNA signature (including the lncRNAs ENST00000435885.1, XLOC_013014 and ENS T00000547963.1) is significantly associated with the overall survival of ESCC patients [15]. Tong and his colleagues have identified lncRNA POU3F3 could help to improve the efficiency of early ESCC screening [16]
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