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Title: Epigenetic regulation mediated by antisense non-coding RNAs and its impact on oncogenic pathways: the HMGA2/RPSAP52 locus
Author: Oliveira Mateos, Cristina
Director/Tutor: Guil Domènech, Sònia
Esteller, Manel
Keywords: Epigenètica
Regulació genètica
Genetic regulation
Issue Date: 16-Jun-2020
Publisher: Universitat de Barcelona
Abstract: [eng] The vast majority of the human genome is transcribed giving rise to non-coding RNAs. Antisense transcripts are one of the most abundant types of long non-coding RNAs and many of them have regulatory roles in the transcription of the nearby genes. Specifically, we study gene pairs with divergent transcription and a GC skew in the promoter region that allows the formation of an R-loop by the antisense. The HMGA2/RPSAP52 pair was selected for further investigation due to the aberrant expression of HMGA2 in a multitude of cancers. In this case, the R-loop formed by RPSAP52 reduces chromatin compaction, which has a positive impact on HMGA2 transcription. Both genes are overexpressed in some human cancers, with a positive correlation in breast cancer patients and cell lines; and hypermethylation of their promoter correlates with their repression. On the other hand, RPSAP52 enrichment in the cytoplasm, its polyadenylation and its association with polysomes indicate a possible function in translation. In this sense, we described the binding of RPSAP52 to IGF2BP2 protein, a transcriptional target of HMGA2 that regulates the translation of mRNAs such as IGF1R and RAS. RPSAP52 depletion resulted in an increase of let-7 expression that correlated with low levels of the proteins IGF2BP2, IGF1R and RAS, whose mRNAs are let-7 targets. LIN28B is not expressed in MCF10A cells and LIN28A does not change with RPSAP52 depletion. Thus, it is not possible to explain these results by alterations in the levels of LIN28 proteins, the main negative regulators of let-7 biogenesis. The phenotypic impact of RPSAP52 depletion in breast cancer cell lines includes a decrease in cell proliferation, migration and clonogenicity. Also, the protein levels of the stemness markers NANOG and OCT4 are reduced in these cells. Similarly, the weight and volume of subcutaneous tumors is lower in immunosuppressed mice injected with RPSAP52-depleted cells. Given the role played by the HMGA2-IGF2BP2-NRAS axis in embryonic rhabdomyosarcoma, the study was extended to sarcomas using A673 cell line as a model. As seen in MCF10A, an increase in let-7 expression was observed in RPSAP52-depleted clones, and the interaction between IGF2BP2 and RPSAP52 was confirmed. In this case, IGF2BP2 and RAS proteins remain unchanged, but the pathway is affected downstream with the decrease of p-ERK. It is noteworthy to mention the reduction of LIN28B protein in the clones, which is abundantly expressed in A673 cells. The reduction in tumor formation was the consequence of RPSAP52 depletion in vivo. Importantly, we described the binding of IGF2BP2 to LIN28B mRNA, and confirmed the interaction using iCLIP-Seq. RPSAP52 knockdown caused a specific loss of IGF2BP2 affinity for particular mRNAs, influencing the translational efficiency of HMGA2 and LIN28B. Moreover, RPSAP52 mediates IGF2BP2 recruitment to polysomes. This could be the mechanism by which RPSAP52 controls the expression of LIN28B and, therefore, let-7 levels. An expression array was performed to study the genome-wide impact of RPSAP52 silencing. The increase of some tumor suppressor genes was detected in the clones, as well as the decrease of genes usually overexpressed in cancer. In support of the influence that RPSAP52 has in tumorigenicity, high expression levels imply a worse survival rate in sarcoma patients. Our findings provide new knowledge about NATs-mediated regulatory mechanisms and highlight their impact on cancer-related genes and on tumor progression itself. According to our results, RPSAP52 regulates HMGA2 expression through the formation of an R-loop and IGF2BP2 function through the binding to this protein. We also demonstrated that LIN28B mRNA constitutes a new target of IGF2BP2. Thus, RPSAP52 affects LIN28B/let-7 balance and promotes tumorigenesis. In conclusion, our work establishes RPSAP52 as a master regulator with oncogenic properties.
[spa] La gran mayoría del genoma humano se transcribe, dando lugar en muchos casos a RNAs no codificantes. Los transcritos antisentido son uno de los tipos más abundantes de RNAs no codificantes largos y muchos poseen importantes funciones en la regulación de los genes cercanos. Este es el caso de RPSAP52, transcrito antisentido del gen codificante HMGA2. La expresión de ambos genes es elevada en varios cánceres humanos y correlaciona positivamente como consecuencia de la regulación que RPSAP52 ejerce sobre HMGA2. RPSAP52 forma un R-loop en la región promotora de los dos genes, lo que modifica la conformación de la cromatina y favorece la transcripción de HMGA2. RPSAP52 desempeña funciones adicionales en el citoplasma gracias a su unión a la proteína IGF2BP2, cuya transcripción es regulada por HMGA2. IGF2BP2 promueve la traducción de genes relacionados con importantes rutas proliferativas, y su interacción con RPSAP52 afecta su unión a algunos RNAs mensajeros, así como su reclutamiento a polisomas. En este trabajo demostramos que LIN28B, uno de los principales reguladores negativos de la maduración del miRNA let-7, es uno de sus targets. De este modo, RPSAP52 aumenta la traducción de LIN28B y reduce los niveles del supresor tumoral let-7. La regulación mediada por RPSAP52 tiene un importante impacto en rutas génicas relacionadas con el cáncer. Su depleción afecta negativamente las características tumorigénicas de las células in vitro y disminuye la progresión tumoral in vivo. Además, RPSAP52 puede ser considerado como un biomarcador en sarcomas, ya que sus altos niveles se asocian a un peor pronóstico. En resumen, el presente trabajo propone un modelo regulador mediado por RPSAP52 con dos niveles diferentes de acción. Este transcrito antisentido promueve la activación transcripcional de HMGA2 y, a su vez, regula la función de la proteína IGF2BP2. Dado que HMGA2 e IGF2BP2 están en la misma vía proliferativa, RPSAP52 refuerza la función de HMGA2 tanto sobre IGF2BP2 como sobre sus efectores posteriores, lo que afecta la progresión del cáncer. Debido a los importantes roles desempeñados por RPSAP52 y a sus propiedades oncogénicas, podría ser una potencial diana terapéutica para el desarrollo de nuevos tratamientos contra el cáncer.
Appears in Collections:Tesis Doctorals - Facultat - Medicina

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