RESUMEN
BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown. RESULTS: We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3'-end. Consequently, the secondary structures were changed, the RNA-protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3'-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs. CONCLUSIONS: Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study.
Asunto(s)
Técnicas de Transferencia de Gen , Lentivirus/genética , ARN Largo no Codificante , Lentivirus/metabolismo , ARN Largo no Codificante/química , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Terminación de la Transcripción GenéticaRESUMEN
Several studies have shown that long non-coding RNAs (lncRNAs) may play an essential role in Epithelial-Mesenchymal Transition (EMT), which is an important step in tumor metastasis; however, little is known about the global change of lncRNA transcriptome during EMT. To investigate how lncRNA transcriptome alterations contribute to EMT progression regulation, we deep-sequenced the whole-transcriptome of MCF10A as the cells underwent TGF-ß-induced EMT. RESULTS: Deep-sequencing results showed that the long RNA transcriptome of MCF10A had undergone global changes as early as 8h after treatment with TGF-ß. The expression of 3403 known and novel lncRNAs, and 570 known and novel circRNAs were altered during EMT. To identify the key lncRNA-regulator, we constructed the co-expression network and found all junction nodes in the network are lncRNAs. One junction node, RP6-65G23.5, was further verified as a key regulator of EMT. Intriguingly, we identified 216 clusters containing lncRNAs which were located in "gene desert" regions. The expressions of all lncRNAs in these clusters changed concurrently during EMT, strongly suggesting that these clusters might play important roles in EMT. Our study reveals a global reprogramming of lncRNAs transcriptome during EMT and provides clues for the future study of the molecular mechanism of EMT.
Asunto(s)
Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal/genética , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Largo no Codificante/biosíntesis , Neoplasias de la Mama/patología , Línea Celular Tumoral , Reprogramación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis de la Neoplasia , ARN Largo no Codificante/genética , Transcriptoma/genéticaRESUMEN
Rationale: The forkhead box A1 (FOXA1) is a crucial transcription factor in initiation and development of breast, lung and prostate cancer. Previous studies about the FOXA1 transcriptional network were mainly focused on protein-coding genes. Its regulatory network of long non-coding RNAs (lncRNAs) and their role in FOXA1 oncogenic activity remains unknown. Methods: The Cancer Genome Atlas (TCGA) data, RNA-seq and ChIP-seq data were used to analyze FOXA1 regulated lncRNAs. RT-qPCR was used to detect the expression of DSCAM-AS1, RT-qPCR and Western blotting were used to determine the expression of FOXA1, estrogen receptor α (ERα) and Y box binding protein 1 (YBX1). RNA pull-down and RIP-qPCR were employed to investigate the interaction between DSCAM-AS1 and YBX1. The effect of DSCAM-AS1 on malignant phenotypes was examined through in vitro and in vivo assays. Results: In this study, we conducted a global analysis of FOXA1 regulated lncRNAs. For detailed analysis, we chose lncRNA DSCAM-AS1, which is specifically expressed in lung adenocarcinoma, breast and prostate cancer. The expression level of DSCAM-AS1 is regulated by two super-enhancers (SEs) driven by FOXA1. High expression levels of DSCAM-AS1 was associated with poor prognosis. Knockout experiments showed DSCAM-AS1 was essential for the growth of xenograft tumors. Moreover, we demonstrated DSCAM-AS1 can regulate the expression of the master transcriptional factor FOXA1. In breast cancer, DSCAM-AS1 was also found to regulate ERα. Mechanistically, DSCAM-AS1 interacts with YBX1 and influences the recruitment of YBX1 in the promoter regions of FOXA1 and ERα. Conclusion: Our study demonstrated that lncRNA DSCAM-AS1 was transcriptionally activated by super-enhancers driven by FOXA1 and exhibited lineage-specific expression pattern. DSCAM-AS1 can promote cancer progression by interacting with YBX1 and regulating expression of FOXA1 and ERα.
Asunto(s)
Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/metabolismo , ARN Largo no Codificante/genética , Proteína 1 de Unión a la Caja Y/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/mortalidad , Adenocarcinoma del Pulmón/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Secuenciación de Inmunoprecipitación de Cromatina , Biología Computacional , Conjuntos de Datos como Asunto , Progresión de la Enfermedad , Elementos de Facilitación Genéticos/genética , Receptor alfa de Estrógeno/genética , Retroalimentación Fisiológica , Femenino , Técnicas de Inactivación de Genes , Células HEK293 , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Pronóstico , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/patología , ARN Largo no Codificante/metabolismo , RNA-Seq , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Estrogen receptor (ER) plays important roles in cell growth, development and tumorigenesis. Although ER-regulated genes have been extensively investigated, little is known about roles of ER-regulated lncRNAs in breast cancer. Here, we conducted genome-wide study of ER-regulated lncRNAs by using RNA-seq, ChIP-seq and TCGA data. A total of identified 114 ER-regulated lncRNAs were identified, many of them were overexpressed in ER+ breast cancer and co-expressed with some key regulators. Silencing one of most prominent lncRNA, AP000439.3, resulted in inhibition of cell cycle progression and proliferation. Further study revealed AP000439.3 can regulate expression of CCND1 through enhancing estrogen receptor induction of CCND1. This finding revealed lncRNAs may serve as important effectors of ER in regulation of gene expression and cell phenotype in breast cancer.