Next Generation Sequencing of piRNAs in 3 prostate cancer tissues and 3 paired prostate tissues
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ABSTRACT: iRNAs have been demonstrated to play crucial roles in cancer development by directing the post-transcriptional regulation of target gene expression. We analyzed the expression profile of piRNAs in 3 of the prostate cancer tissues and matched normal adjacent tissues by piRNA sequencing. The aim of this analyse was to find the different expressed piRNAs in prostate cancer.
Project description:During embryonic germ cell development in mice, transposon-enriched, piwi-interacting RNAs (piRNAs) guide MILI and MIWI2 to direct silencing of potentially active mobile element families. In contrast, we know much less about the function of the highly abundant and extremely diverse class of piRNAs, which partner with MIWI and MILI during meiosis. Both MIWI and its catalytic activity are required for successful spermatogenesis, strongly indicating that piRNA-guided cleavage is critical for germ cell development. To gain an understanding of meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing an entire human meiotic piRNA cluster. This triggered a spermatogenesis defect, presumably by inappropriately targeting the piRNA machinery to mouse RNAs essential for germ cell development. Through an analysis of such de novo targets, we derived a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein coding genes as targets of native piRNAs. Cleavage of genic targets begins at the pachytene stage when meiotic piRNAs first appear. As such, target mRNA levels attenuate starting from the pachytene stage and are further repressed throughout meiosis. Target mRNA-piRNA pairs also show evidence of an ongoing cleavage-dependent amplification cycle, which is not normally a strong feature of meiotic piRNAs. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells. 48 samples
Project description:During embryonic germ cell development in mice, transposon-enriched, piwi-interacting RNAs (piRNAs) guide MILI and MIWI2 to direct silencing of potentially active mobile element families. In contrast, we know much less about the function of the highly abundant and extremely diverse class of piRNAs, which partner with MIWI and MILI during meiosis. Both MIWI and its catalytic activity are required for successful spermatogenesis, strongly indicating that piRNA-guided cleavage is critical for germ cell development. To gain an understanding of meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing an entire human meiotic piRNA cluster. This triggered a spermatogenesis defect, presumably by inappropriately targeting the piRNA machinery to mouse RNAs essential for germ cell development. Through an analysis of such de novo targets, we derived a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein coding genes as targets of native piRNAs. Cleavage of genic targets begins at the pachytene stage when meiotic piRNAs first appear. As such, target mRNA levels attenuate starting from the pachytene stage and are further repressed throughout meiosis. Target mRNA-piRNA pairs also show evidence of an ongoing cleavage-dependent amplification cycle, which is not normally a strong feature of meiotic piRNAs. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.
Project description:The piRNA pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. piRNAs are maternally deposited and required for proper transposon silencing in adult offspring. However, a long-standing question in the field is the precise function of maternally deposited piRNAs and its associated factors during embryogenesis. Here, we probe the spatio-temporal expression patterns of several piRNA pathway components during early stages of development. Amongst those, factors required for transcriptional gene silencing (TGS) showed ubiquitous abundance in somatic and pole cells throughout the first half of embryogenesis. We further analysed the transcriptomes of various embryo stages and correlated these with the presence of selected chromatin marks. We found that a number of transposon families show bursts of transcription during early embryonic stages. Transposons heavily targeted by maternally deposited piRNAs accumulated repressive chromatin marks following their spike in expression. Furthermore, depletion of maternally deposited Piwi protein in early embryos resulted in increased expression of transposons targeted by inherited piRNAs and was accompanied by a strong loss of repressive chromatin marks at coding sequences. Overall, our data suggests a pivotal role for the piRNA pathway in transposon defence during Drosophila embryogenesis in somatic cells.
Project description:In animals, a discrete class of small RNAs, the piwi-interacting RNAs (piRNAs), guard germ cell genomes against the activity of mobile genetic elements. piRNAs are generated, via an unknown mechanism, from apparently single-stranded precursors that arise from discrete genomic loci, termed piRNA clusters. The content of piRNA clusters, determines the capacity of the system to respond to a given element, in essence comprising an organism's evolving molecular definition of transposons. Presently, little is known about the signals that distinguish a locus as a source of piRNAs and about how abundant piRNAs are selected. To address these questions, we inserted new sequence information into piRNA clusters in mice and flies. In all cases, this information was incorporated into the piRNA repertoire and in one instance was shown to confer the ability to recognize and silence a corresponding element. Notably, patterns of piRNA abundance suggested that both intrinsic sequence and context with the cluster inform piRNA generation. Though piRNAs themselves are not conserved between species, the genomic location of clusters is often retained. We were able to create artificial piRNA clusters in non-native contexts in both mice and flies, indicating that the signals that define these as generative loci must lie within the clusters themselves rather than being implicit in their genomic position.
Project description:In animals, a discrete class of small RNAs, the piwi-interacting RNAs (piRNAs), guard germ cell genomes against the activity of mobile genetic elements. piRNAs are generated, via an unknown mechanism, from apparently single-stranded precursors that arise from discrete genomic loci, termed piRNA clusters. The content of piRNA clusters, determines the capacity of the system to respond to a given element, in essence comprising an organism's evolving molecular definition of transposons. Presently, little is known about the signals that distinguish a locus as a source of piRNAs and about how abundant piRNAs are selected. To address these questions, we inserted new sequence information into piRNA clusters in mice and flies. In all cases, this information was incorporated into the piRNA repertoire and in one instance was shown to confer the ability to recognize and silence a corresponding element. Notably, patterns of piRNA abundance suggested that both intrinsic sequence and context with the cluster inform piRNA generation. Though piRNAs themselves are not conserved between species, the genomic location of clusters is often retained. We were able to create artificial piRNA clusters in non-native contexts in both mice and flies, indicating that the signals that define these as generative loci must lie within the clusters themselves rather than being implicit in their genomic position. Total RNA and RNA associated with Piwi proteins were isolated and size-fractionated by PAGE into 19-33nt. These were processed and sequenced on the Illumina GA2 platform.
Project description:Germ cells of most animals critically depend on piRNAs and Piwi proteins. Surprisingly, piRNAs in mouse oocytes are relatively rare and dispensable. We present compelling evidence for strong Piwi-piRNA expression in oocytes of other mammals. Human fetal oocytes express PIWIL2 and transposon-enriched piRNAs. Oocytes in adult human ovary express PIWIL1 and PIWIL2, while those in bovine ovary just express PIWIL1. In human, macaque and bovine ovaries we find piRNAs that resemble testis-borne pachytene piRNAs. Isolated bovine follicular oocytes were shown to contain abundant, relatively short piRNAs that preferentially target transposable elements. Using label-free quantitative proteome analysis we show that these maturing oocytes strongly and specifically express the thus-far uncharacterized PIWIL3 protein, alongside other known piRNA-pathway components. In bovine early embryos these piRNAs are still abundant, revealing a potential impact of piRNAs on mammalian embryogenesis. Our results reveal unexpected, highly dynamic piRNA pathways in mammalian oocytes and early embryos.
Project description:To determine the altered microRNA expression signature in human prostate cancer compared to benign prostate tissue. To determine the altered mRNA expression signatures upon overexrpession miR-31 in prostate cancer cells. Two condition experiments:1) Total RNA from 21 pairs of prostate cancers and matched benign prostate tissues were collected and processed for microRNA detection. 2) In LNCaP prostate cancer cells, miR-31 was overexpressed and compared to control miR-NC.
Project description:To elucidate potential roles of piRNA in lung cancer, we analyzed global piRNA expression profiles in 8 NSCLC and 3 HBE cell lines. RNA-seq results showed that >99% of the approximately 4.5 million reads were between 26 and 32 bases. Most of the piRNA loci are represented by two or more identical reads. We observed a total 555 expressed mature piRNAs, distributed among chromosomes and mitochondria with bias in chromosomes 1 and 6. 99% of the piRNAs are mapped to intergenic regions (64%) or introns (35%) and 1% to exons. Of the 555 mature piRNAs, 260 (47%) have been recorded in NCBI whereas 295 (53%) are novel. These results provide a comprehensive view of piRNA expressed in human bronchial epithelial cells and NSCLC cells. To get the purified mature piRNAs, small RNA (<200nt) was separated from total RNAs firstly, and then piRNA were purified in one nucleotide resolution gel. A unique 6 nt barcode for every cell line to distinguish each reads from each specific cell line, and the right size of small RNAs were used library for Illumina sequencing.
Project description:To elucidate potential roles of piRNA in lung cancer, we analyzed global piRNA expression profiles in 8 NSCLC and 3 HBE cell lines. RNA-seq results showed that >99% of the approximately 4.5 million reads were between 26 and 32 bases. Most of the piRNA loci are represented by two or more identical reads. We observed a total 555 expressed mature piRNAs, distributed among chromosomes and mitochondria with bias in chromosomes 1 and 6. 99% of the piRNAs are mapped to intergenic regions (64%) or introns (35%) and 1% to exons. Of the 555 mature piRNAs, 260 (47%) have been recorded in NCBI whereas 295 (53%) are novel. These results provide a comprehensive view of piRNA expressed in human bronchial epithelial cells and NSCLC cells.