piRNA Expression Research Articles

Dynamics of cattle sperm sncRNAs during maturation, from testis to ejaculated sperm

BackgroundDuring epididymal transit, spermatozoa go through several functional maturation steps, resulting from interactions with epididymal secretomes specific to each region. In particular, the sperm membrane is under constant remodeling, with sequential attachment and shedding of various molecules provided by the epididymal lumen fluid and epididymosomes, which also deliver sncRNA cargo to sperm. As a result, the payload of sperm sncRNAs changes during the transit from the epididymis caput to the cauda. This work was designed to study the dynamics of cattle sperm sncRNAs from spermatogenesis to final maturation.ResultsComprehensive catalogues of sperm sncRNAs were obtained from testicular parenchyma, epididymal caput, corpus and cauda, as well as ejaculated semen from three Holstein bulls. The primary cattle sncRNA sperm content is markedly remodeled as sperm mature along the epididymis. Expression of piRNAs, which are abundant in testis parenchyma, decreases dramatically at epididymis. Conversely, sperm progressively acquires miRNAs, rsRNAs, and tsRNAs along epididymis, with regional specificities. For instance, miRNAs and tsRNAs are enriched in epididymis cauda and ejaculated sperm, while rsRNA expression peaks at epididymis corpus. In addition, epididymis corpus contains mainly 20 nt long piRNAs, instead of 30 nt in all other locations. Beyond the bulk differences in abundance of sncRNAs classes, K-means clustering was performed to study their spatiotemporal expression profile, highlighting differences in specific sncRNAs and providing insights into their putative biological role at each maturation stage. For instance, Gene Ontology analyses using miRNA targets highlighted enriched processes such as cell cycle regulation, response to stress and ubiquitination processes in testicular parenchyma, protein metabolism in epididymal sperm, and embryonic morphogenesis in ejaculated sperm.ConclusionsOur findings confirm that the sperm sncRNAome does not simply reflect a legacy of spermatogenesis. Instead, sperm sncRNA expression shows a remarkable level of plasticity resulting probably from the combination of multiple factors such as loss of the cytoplasmic droplet, interaction with epididymosomes, and more surprisingly, the putative in situ production and/or modification of sncRNAs by sperm. Given the suggested role of sncRNA in epigenetic trans-generational inheritance, our detailed spatiotemporal analysis may pave the way for a study of sperm sncRNAs role in embryo development.

PiRNA-mediated gene regulation and adaptation to sex-specific transposon expression in D. melanogaster male germline.

Small noncoding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of the piRNA pathway in transposon silencing and therefore genome defense. However, the ability of the piRNA program to respond to different transposon landscapes and the role of piRNAs in regulating host gene expression remain poorly understood. Here, we comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, we identified new piRNA clusters and established complex satellites as dual-strand piRNA clusters. While sharing most piRNA clusters, the two sexes employ them differentially to combat the sex-specific transposon landscape. We found two piRNA clusters that produce piRNAs antisense to four host genes in testis, including CG12717/pirate, a SUMO protease gene. piRNAs encoded on the Y chromosome silence pirate, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pirate is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-coding gene.

Analysis Pipeline of PIWI‐Interacting RNA (piRNA) in an Inflammatory Parasitic Infectious Disease

Small noncoding RNAs (sncRNA) have been implicated as major contributors to the pathogenesis of various diseases. PIWI-Interacting RNA (piRNA) are a type of sncRNA. with several regulatory roles across the genome. They are primarily expressed from piRNA clusters within the genome, where they go on to regulate and maintain genomic stability. They are capable of silencing transposable elements and inducing epigenetic changes. Some studies also suggest that piRNAs can regulate gene transcription. This gene regulatory mechanism is thought to be similar to that of miRNAs. These sncRNA play important roles in regulating gene expression by forming complexes with Argonaute proteins to recognize specific target sequences. The specific goal of this project was to develop a method of computationally analyzing piRNA in the context of their diverse functions and relate them to biological responses occurring in disease. To accomplish this, we created a pipeline to analyze dysregulation of piRNA expression and predict how these changes contribute to Trypanosoma cruzi-induced cardiac pathology. We used primary human cardiac myocytes (PHCM) challenged by T. cruzi infection and computationally assessed the impact of piRNA during inflammatory response. Small RNA from PHCM challenged by T. cruzi infection was extracted and sequenced. The piRNA annotation program Piano was used to predict known piRNAs via a support vector machine algorithm with transposon interaction informatics. The NOISeq method was used to determine differentially expressed piRNAs. Prediction of binding interactions between coding transcripts and piRNAs was done by adapting the miRanda and RNA22 algorithms using strict pairing thresholds. Predicted target transcripts were then mapped to enriched KEGG pathways using WEBGESTALT. Mapped genes were then queried for biological interaction network construction with the GeneMANIA algorithm. In combination, these analyses were used to produce biological interaction networks involving piRNAs and predicted target genes related to inflammation, specifically pro-inflammatory AP-1 transcription factor genes. We demonstrate a method of ascertaining the dynamics of changes in piRNA expression using this disease model, from which more directed biological inquiries can subsequently be made.

Dysregulation of piwi‐interacting RNA expression in primary human cardiac myocytes during early phase of Trypanosoma cruzi infection

Trypanosoma cruzi, the causative agent of Chagas Disease, is an obligate intracellular parasite that has caused severe mortality and morbidity historically in Central and South America. Due to international travel, globalization, and migration, this neglected tropical disease is now present in most industrialized countries. About 30% of those who develop chronic T. cruzi infection will exhibit various pathologies, including cardiomyopathies, neurological and/or gastrointestinal diseases. We and others have implicated gene dysregulation as necessary in facilitating T. cruzi infection. However, the role of small noncoding RNAs during infection is still being investigated. Hence, the molecular mechanisms that contribute to chronic chagas cardiomyopathies remains to be elucidated. In this study, we aim to delineate dysregulation of a class of small non-coding RNAs, piwi-interacting RNAs (piRNAs), during the early phase of T. cruzi infection in primary human cardiomyocytes (PHCM). We hypothesize that dysregulated piRNAs target genes that may play important roles in infection. After parasite challenge, we extracted PHCM RNA for subsequent RNA sequencing. We then utilized bioinformatic tools in order to analyze differential expression of piRNAs, to predict mRNA targeting, and to map piRNAs to biological pathways. We found an average of 21,595,866 of clean reads (88.4%) mapped to the human reference genome. During the course of infection, T. cruzi induced differential expression of 217 unique piRNAs. In silico analysis predicted that both dysregulated known and novel piRNAs could target several genes of interest, including NFATC2, FOS, and TGF-b1, and related genes known to interact with these targets. These novel findings are the first to implicate that Trypanosoma cruzi induces differential expression of piRNAs in PHCM, which advances our knowledge of piRNAs in infectious disease pathogenesis.

HRV16 Infection Induces Changes in the Expression of Multiple piRNAs.

Human rhinovirus (HRV) is one of the most important cold-causing pathogens in humans. Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNAs whose best-understood function is to repress mobile element (ME) activity in animal germline. However, the profile of human/host piRNA during HRV infection is largely unknown. Here we performed high-throughput sequencing of piRNAs from H1-HeLa cells infected with HRV16 at 12h, 24h, and 36h. The results showed that22,151,664, 24,362,486 and 22,726,546 piRNAs displayed differential expression after HRV16 infection for three time points. A significant differential expression of 21 piRNAs was found in all time points and further verified by RT-qPCR, including 7 knownpiRNAs and 14 newly found piRNAs. In addition, piRNA prediction was performed on Piano using the SVM algorithm and transposon information. It found that novel_pir78110, novel_pir78107, novel_pir78097, novel_pir78094 and novel_pir76584 are associated with the DNA/hobo of Drosophila, Ac of maize and Tam3 of snapdragon (hAT)-Charlie transposon. The novel_pir97924, novel_pir105705 and novel_pir105700 recognize long interspersed nuclear elements 1 (LINE-1). The novel_pir33182 and novel_pir46604 are related to the long terminal repeat (LTR)/(Endogenous Retrovirus1) ERV1 repetitive element. The novel_pir73855 is related to the LTR/ERVK repetitive element. Both novel_pir70108 and novel_pir70106 are associated with the LTR/ERVL-MaLR repetitive element. The novel_pir15900 is associated with the DNA/hAT-Tip100 repetitive element. Overall, our results indicated that rhinovirus infection could reduce theexpression of some piRNAs to facilitate upregulation of LINE-1 transcription or retrotransposons' expression, which is helpful to further explore the mechanism of rhinovirus infection.

Abnormal PIWI-interacting RNA profile and its association with the deformed extracellular matrix of oocytes from recurrent oocyte maturation arrest patients

To depict the PIWI-interacting RNA (piRNA) profile in oocytes from patients with recurrent oocyte maturation arrest (ROMA) and explore the piRNA candidates associated with the disease. An observational study. Academic research unit. Sixteen ROMA patients who provided 140 immature oocytes that arrested at metaphase I, and 146 control patients who provided 420 oocytes for invitro culture that were collected at the stages of germinal vesicle (GV), metaphase I (MI), and MII. None. Expression profiles of piRNA and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) validating data of piR-hsa-17139 and its target genes. After the piRNA profile was established using piRNA sequencing and hierarchical clustering, the target genes of the piRNA were predicted by bioinformatics databases and matched with mRNA sequencing data. The piRNA expression profiles showed a greater quantity of differentially expressed piRNAs in the older-stage oocytes compared with the early-stage oocytes. The piRNA and mRNA sequencing data indicated that the most affected genes were mainly concentrated in the extracellular matrix (ECM) pathway. Based on the comparison of the piRNA and mRNA sequencing data, four differentially expressed piRNAs were associated with modulation of those ECM pathway genes. The qRT-PCR validation confirmed that piR-hsa-17139 was the only up-regulated piRNA, and its target ECM genes were suppressed in ROMA oocytes. The expression level of piR-hsa-17139 declined slightly while the expression of its target ECM genes plunged dramatically during the development of normal oocytes. As the important genome monitors in gametogenesis, abnormally expressed piRNAs may affect the expression of ECM modulating genes, which subsequently contributes to ROMA.

SNPC-1.3 is a sex-specific transcription factor that drives male piRNA expression in C. elegans.

Piwi-interacting RNAs (piRNAs) play essential roles in silencing repetitive elements to promote fertility in metazoans. Studies in worms, flies, and mammals reveal that piRNAs are expressed in a sex-specific manner. However, the mechanisms underlying this sex-specific regulation are unknown. Here we identify SNPC-1.3, a male germline-enriched variant of a conserved subunit of the small nuclear RNA-activating protein complex, as a male-specific piRNA transcription factor in Caenorhabditis elegans. SNPC-1.3 colocalizes with the core piRNA transcription factor, SNPC-4, in nuclear foci of the male germline. Binding of SNPC-1.3 at male piRNA loci drives spermatogenic piRNA transcription and requires SNPC-4. Loss of snpc-1.3 leads to depletion of male piRNAs and defects in male-dependent fertility. Furthermore, TRA-1, a master regulator of sex determination, binds to the snpc-1.3 promoter and represses its expression during oogenesis. Loss of TRA-1 targeting causes ectopic expression of snpc-1.3 and male piRNAs during oogenesis. Thus, sexually dimorphic regulation of snpc-1.3 expression coordinates male and female piRNA expression during germline development.

Extracellular vesicles piwi-interacting RNAs from skin mucus for identification of infected Cynoglossus semilaevis with Vibrio harveyi

Extracellular vesicles play a regulatory role in intracellular and intercellular transmission through a variety of biological information molecules, including mRNA, small RNAs and proteins. piRNAs are one kind of regulatory small RNAs in the vesicles at the post transcriptional level. Hereby, we isolated the extracellular vesicles from skin mucus and screened the piRNA profiles of these vesicles, aiming at developing biomarkers related to bacterial infections in Cynoglossus semilaevis. The different profilings of piRNAs in mucous extracellular vesicles of C. semilaevis were compared through small RNA sequencing, between fish infected with Vibrio harveyi and healthy ones. The number of clean reads on the alignment of exosome sick (ES) group was 105, 345 and that of exosome control (EC) group was 455, 144. GO and KEGG pathway enrichment analysis showed that most of the target genes were involved in cellular process, response to stimulus, biological regulation, immune system process and signal transduction, signal molecular and interaction, transport and catabolism. The 45 final candidate piRNAs related to immunity or infectious diseases included 20 piRNAs with high expression in the ES group and 25 piRNAs with a low expression in the ES group. After verification by qRT-PCR, there was significant difference of five piRNAs expression level between infected fish and healthy fish, in line with the sequencing. The expression level of piR-mmu-16401212, piR-mmu-26829319 and piR-gga-244092 in infected fish were significantly lower than that of control group, while piR-gga-71717 and piR-gga-99034 were higher, which implying that these piRNAs in mucous extracellular vesicles can be used to identify diseased fish from normal ones. This work supplied a novel class of biomarker for infection diagnosis in fish, and it will be benefit for screening disease resistant breeding of C. semilaevis.

Global 3’UTR shortening and down-regulation of repeated element related piRNA play crucial roles in boys with cryptorchidism

BackgroundCryptorchidism is the most common congenital defect in children's genitourinary system. Decades of research have identified both environmental and genetic factors contribute to the etiology. MethodsSmall-RNA/mRNA-seq were performed on testicular tissues from cryptorchidism patients. Downstream analysis included mRNA expression, piRNA expression and miRNA expression. ResultsWe find a global downregulation of repeated element related piRNA expression as well as a global 3’UTR shortening of mRNAs in patients with cryptorchidism. We also find that genes with shortened 3’UTR which are highly enriched in vascular endothelial growth and protein ubiquitination, tend to be up-regulated in cryptorchidism. These results indicate that boys with cryptorchidism may not have normal piRNA functions to protect developmental tissues from transposon invasion. Dysregulated shortened 3’UTR genes may affect normal testicular tissue development. ConclusionIn summary, our findings also provided the first landscape of gene regulation in cryptorchidism, especially in terms of post-transcriptional regulations.

PiRNA-Independent Transposon Silencing by The  <i>Drosophila</i> THO Complex

piRNAs guide Piwi/Panoramix-dependent H3K9me3 chromatin modification and transposon silencing in the Drosophila germline. THO is a heteropenameric RNA export complex composed of Hpr1, Tho2, and Thoc5-7. Null thoc7 mutations displace Thoc5 and 6 from a Tho2-Hpr1 sub-complex, reduce expression of a subset ofgermline piRNAs, and increase steady state expression of a subset of transposon families, suggesting that THO silences transposons by promoting piRNA biogenesis. Here we show that thoc7 mutations increase transposon transcription, but do not reduce anti-sense piRNAs targeting half of the transcriptionally activated elements, disrupt piRNA-guided H3K9me3 chromatin modification, or block Panoramix-dependent silencing of a reporter transgene. In addition, unspliced transposon transcripts co-precipitate with THO through a Piwi- and Panoramix-independent mechanism, and piwi mutations dominantly enhance germline defects associated with thoc7 null alleles. We therefore propose that THO silences transposons through a piRNA-independent mechanism, which functions cooperatively with piRNAs to support germline development.

Circulating SNORD57 rather than piR-54265 is a promising biomarker for colorectal cancer: common pitfalls in the study of somatic piRNAs in cancer.

There is increasing interest among cancer researchers in the study of Piwi-interacting RNAs (piRNAs), a group of small RNAs important for maintaining genome stability in the germline. Aberrant expression of piRNAs in cancer could imply an involvement of these regulatory RNAs in neoplastic transformation. On top of that, it could enable early cancer diagnosis based on RNA analysis in liquid biopsies, as piRNAs are not expected to widely circulate in the bloodstream of healthy individuals. Indeed, it has recently been shown that serum piR-54265 allows for excellent discrimination between colorectal cancer patients and healthy controls. However, we have also shown that most somatic piRNAs reported to date in mammals are actually fragments of other noncoding RNAs. Herein, we show that reports positioning piR-54265 as a noninvasive biomarker for colorectal cancer were actually measuring variations in the levels of a full-length (72 nt) small nucleolar RNA in serum. This should place a cautionary note for future research in somatic and cancer-specific piRNAs. We deeply encourage this line of research but discuss proper ways to identify somatic piRNAs without the interference of erroneous entries contained in piRNA databases. We also introduce the concept of miscellaneous-piRNAs (m-piRNAs) to distinguish between canonical piRNAs and other small RNAs circumstantially associated with PIWI proteins in somatic cells.

Systematic evaluation of the effects of genetic variants on PIWI-interacting RNA expression across 33 cancer types.

PIWI-interacting RNAs (piRNAs) are an emerging class of non-coding RNAs involved in tumorigenesis. Expression quantitative trait locus (eQTL) analysis has been demonstrated to help reveal the genetic mechanism of single nucleotide polymorphisms (SNPs) in cancer etiology. However, there are no databases that have been constructed to provide an eQTL analysis between SNPs and piRNA expression. In this study, we collected genotyping and piRNA expression data for 10 997 samples across 33 cancer types from The Cancer Genome Atlas (TCGA). Using linear regression cis-eQTL analysis with adjustment of appropriate covariates, we identified millions of SNP-piRNA pairs in tumor (76 924 831) and normal (24 431 061) tissues. Further, we performed differential expression and survival analyses, and linked the eQTLs to genome-wide association study (GWAS) data to comprehensively decipher the functional roles of identified cis-piRNA eQTLs. Finally, we developed a user-friendly database, piRNA-eQTL (http://njmu-edu.cn:3838/piRNA-eQTL/), to help users query, browse and download corresponding eQTL results. In summary, piRNA-eQTL could serve as an important resource to assist the research community in understanding the roles of genetic variants and piRNAs in the development of cancers.

Trypanosoma cruzi Modulates PIWI-Interacting RNA Expression in Primary Human Cardiac Myocytes during the Early Phase of Infection.

Trypanosoma cruzi dysregulates the gene expression profile of primary human cardiomyocytes (PHCM) during the early phase of infection through a mechanism which remains to be elucidated. The role that small non-coding RNAs (sncRNA) including PIWI-interacting RNA (piRNA) play in regulating gene expression during the early phase of infection is unknown. To understand how T. cruzi dysregulate gene expression in the heart, we challenged PHCM with T. cruzi trypomastigotes and analyzed sncRNA, especially piRNA, by RNA-sequencing. The parasite induced significant differential expression of host piRNAs, which can target and regulate the genes which are important during the early infection phase. An average of 21,595,866 (88.40%) of clean reads mapped to the human reference genome. The parasite induced 217 unique piRNAs that were significantly differentially expressed (q ≥ 0.8). Of these differentially expressed piRNAs, 6 were known and 211 were novel piRNAs. In silico analysis showed that some of the dysregulated known and novel piRNAs could target and potentially regulate the expression of genes including NFATC2, FOS and TGF-β1, reported to play important roles during T. cruzi infection. Further evaluation of the specific functions of the piRNAs in the regulation of gene expression during the early phase of infection will enhance our understanding of the molecular mechanism of T. cruzi pathogenesis. Our novel findings constitute the first report that T. cruzi can induce differential expression of piRNAs in PHCM, advancing our knowledge about the involvement of piRNAs in an infectious disease model, which can be exploited for biomarker and therapeutic development.

Hepatoselüler karsinom tedavisi için etkili terapötikler PIWI Interacting RNA ifadelerini nasıl değiştirir?

Aim: PIWI interacting RNAs (piRNAs) are novel members of small non-coding RNAs that cannot produce proteins but are effective on transcription and post-transcriptional mechanisms of cells. Nowadays, the application of both natural compounds and vitamins is essential for treatment of cancer cells instead of chemical compounds. In this study, we aimed to detect possible expression changes of piRNAs in order to compare 4- Hydroxycoumarin to the active form of vitamin D (1.25-Dihydroxyvitamin D) in hepatocellular carcinoma. Methods: According to our previous study, HePG2 cells were treated with 4- Hydroxycoumarin, 1.25-Dihydroxyvitamin D and drug form of vitamin D at the optimal time and concentration. After treatment, the total RNA was isolated and expressions of piR-Hep-1 and piR-651 were determined by using Real Time Polymerase Chain Reactions. Results: According to our obtained data, statistically significant upregulation of piR-651 expression was observed in 4-Hydroxycoumarin-treated HePG2 cells compared to control (p<0.001). However, the expression of piR-Hep-1 statistically was not affected from 4- Hydroxycoumarin treatment (p>0.05). In contrast, 1.25-dihydroxyvitamin treatment downregulated the expression of piR-Hep-1 statistically significant in HePG2 cells (p<0.001). piR-Hep-1 was not statistically significant effected from drug form of vitamin D treatment (p>0.05). Conlusion: Our results indicated that some of the piRNAs might have special expression patterns in hepatocellular carcinoma and these expression patterns can be regulated by treated natural compounds. We suggest that substances that are observed to be effective in hepatocellular carcinoma individually may result in different piRNA expression changes contrary to the expectations.

Expression of Pirna in Tissues of Ovarian Endometriosis Cysts

Study Objective To identify the expression of PIWI associated RNA (piRNA) in tissues of endometrium and endometrioid ovarian cysts and to evaluate differences in the expression of piRNA between eutopic and ectopic endometrium. Design Prospective observational cohort study; Level II-1. Setting Federal State Budget Institution «The National Medical Research Center for Obstetrics, Gynecology and Perinatology», Moscow. Patients or Participants The study included 21 patients of reproductive age (30.27 ± 3.78 years) with endometrioid ovarian cysts. Paired samples of endometrial tissues were collected during surgery in the proliferative phase of menstrual cycle. Tissue samples for further analysis were selected after histological examination. The analysis of piRNA expression was carried out by the new generation sequencing on the Illumina platform, followed by processing of the data by bioinformatics analysis. Interventions Laparoscopic surgery for endometriod ovarian cysts and endometrial biopsy followed by histopathologic and piRNA expression analysis of obtained tissue. Measurements and Main Results According to the sequencing data, 197 piRNA were identified in the tissues of the eutopic endometrium and endometrioid ovarian cysts. The analysis of differential expression revealed predominantly reduced expression of piRNA in the ectopic endometrium relative to the eutopic endometrium. In total 30 differentially expressed piRNA were identified, including 4 with increased and 26 with reduced expression. Using bioinformatic methods, possible gene targets of differentially expressed piRNA were found. Functional analysis of potential targets of differentially expressed piRNA revealed signaling pathways and cellular processes that may contribute to the development of endometriosis. Conclusion The data obtained suggest that alterations in piRNA expression of ectopic endometrium and subsequent disturbances in the expression of their target genes may be significant link in our search for the pathogenesis of endometriosis.

Global Analyses of Expressed Piwi-Interacting RNAs in Gastric Cancer.

Gastric cancer (GC) represents a notable amount of morbidity and mortality worldwide. Understanding the molecular basis of CG will offer insight into its pathogenesis in an attempt to identify new molecular biomarkers to early diagnose this disease. Therefore, studies involving small non-coding RNAs have been widely explored. Among these, PIWI-interacting RNAs (piRNAs) are an emergent class that can play important roles in carcinogenesis. In this study, small-RNA sequencing was used to identify the global piRNAs expression profile (piRNome) of gastric cancer patients. We found 698 piRNAs in gastric tissues, 14 of which were differentially expressed (DE) between gastric cancer (GC), adjacent to gastric cancer (ADJ), and non-cancer tissues (NC). Moreover, three of these DE piRNAs (piR-48966*, piR-49145, piR-31335*) were differently expressed in both GC and ADJ samples in comparison to NC samples, indicating that the tumor-adjacent tissue was molecularly altered and should not be considered as a normal control. These three piRNAs are potential risk biomarkers for GC, especially piR-48966* and piR-31335*. Furthermore, an in-silico search for mRNAs targeted by the differentially expressed piRNAs revealed that these piRNAs may regulate genes that participate in cancer-related pathways, suggesting that these small non-coding RNAs may be directly and indirectly involved in gastric carcinogenesis.

PIWI-interacting RNAs piR-13643 and piR-21238 are promising diagnostic biomarkers of papillary thyroid carcinoma.

Emerging studies demonstrate that PIWI-interacting RNAs (piRNAs) participate in the development of cancers. 75 pairs of papillary thyroid carcinoma (PTC) samples and 31 benign thyroid nodule samples were included in this three-phase biomarker identifying study. First, piRNA expression profiles of five pairs of PTC samples were acquired piRNA sequencing. The expression of all upregulated piRNAs were further validated by RT-qPCR. Paired t and nonparametric test were used to evaluate the association between all upregulated piRNAs and clinic stage. The expression levels of key piRNAs were corrected by demographic data to construct a multivariate model to distinguish malignant nodules from benign. Additionally, the intersection between target genes of key piRNAs and differentially expressed genes in The Cancer Genome Atlas (TCGA) PTC samples were used to perform enrichment analysis. Only piR-13643 and piR-21238 were significantly upregulated in PTC and associated with clinic stage. Moreover, both piR-13643 (Area Under Curve (AUC): 0.821) and piR-21238 (AUC: 0.823) showed better performance in distinguishing malignant nodules from benign than currently used biomarkers HBME1 (AUC: 0.590). Based on our findings, piR-13643 and piR-21238 were observed to be significantly upregulated in human PTC. PIWI-interacting RNAs could serve as promising novel biomarkers for accurate detection of PTC.

Diversification of piRNAs expressed in PGCs and somatic cells during embryonic gonadal development

ABSTRACT piRNAs are small non-coding RNAs known to play a main role in defence against transposable elements in germ cells. However, other potential functions, such as biogenesis and differences in somatic and germline expression of these regulatory elements, are not yet fully unravelled. Here, we analysed a variety of piRNA sequences detected in mouse male and female primordial germ cells (PGCs) and gonadal somatic cells at crucial stages during embryonic differentiation of germ cells (11.5–13.5 days post-coitum). NGS of sncRNA and bioinformatic characterization of piRNAs from PGCs and somatic cells, in addition to piRNAs associated with TEs, indicated functional diversification in both cell types. Differences in the proportion of the diverse types of piRNAs are detected between somatic and germline during development. However, the global diversified patterns of piRNA expression are mainly shared between germ and somatic cells, we identified piRNAs related with molecules involved in ribosome components and translation pathway, including piRNAs derived from rRNA (34%), tRNA (10%) and snoRNA (8%). piRNAs from both tRNA and snoRNA are mainly derived from 3ʹ and 5ʹ end regions. These connections between piRNAs and rRNAs, tRNAs or snoRNAs suggest important functions of specialized piRNAs in translation regulation during this window of gonadal development.

Serum PIWI-Interacting RNAs piR-020619 and piR-020450 Are Promising Novel Biomarkers for Early Detection of Colorectal Cancer

Early diagnosis can significantly reduce colorectal cancer deaths. We sought to identify serum PIWI-interacting RNAs (piRNAs) that could serve as sensitive and specific noninvasive biomarkers for early colorectal cancer detection. We screened the piRNA expression profile in sera from 7 patients with colorectal cancer and 7 normal controls using small RNA sequencing. Differentially expressed piRNAs were measured in a training cohort of 140 patients with colorectal cancer and 140 normal controls using reverse transcription quantitative PCR. The identified piRNAs were evaluated in two independent validation cohorts of 180 patients with colorectal cancer and 180 normal controls. Finally, the diagnostic value of the identified piRNAs for colorectal adenoma (CRA) was assessed, and their expression was measured in 50 patients with lung cancer, 50 with breast cancer, and 50 with gastric cancer. The piRNAs piR-020619 and piR-020450 were consistently elevated in sera of patients with colorectal cancer as compared with controls. A predicative panel based on the two piRNAs was established that displayed high diagnostic accuracy for colorectal cancer detection. The two-piRNA panel could detect small-size and early-stage colorectal cancer with an area under the ROC curve of 0.863 and 0.839, respectively. Combined use of the two piRNAs could effectively distinguish CRA from controls. Aberrant elevation of the two piRNAs was not observed in sera of patients with lung, breast, and gastric cancer. Serum piR-020619 and piR-020450 show a strong potential as colorectal cancer-specific early detection biomarkers. The field of circulating piRNAs could allow for novel tumor biomarker development.

Identification and application of piwi-interacting RNAs from seminal plasma exosomes in Cynoglossus semilaevis

BackgroundPiwi-interacting RNAs (piRNAs) have been linked to epigenetic and post-transcriptional gene silencing of retrotransposons in germ line cells, particularly in spermatogenesis. Exosomes are important mediators of vesicle transport, and the piRNAs in exosomes might play an important role in cell communication and signal pathway regulation. Moreover, exosomic piRNAs are promising biomarkers for disease diagnosis and physiological status indication. We used Cynoglossus semilaevis because of its commercial value and its sexual dimorphism, particularly the sex reversed “pseudomales” who have a female karyotype, produce sperm, and copulate with normal females to produce viable offspring.ResultsTo determine whether piRNAs from fish germ line cells have similar features, seminal plasma exosomes from half-smooth tongue sole, C. semilaevis, were identified, and their small RNAs were sequenced and analysed. We identified six signature piRNAs as biomarkers in exosomes of seminal plasma from males and pseudomale C. semilaevis. Bioinformatic analysis showed that all six signatures were sex-related, and four were DNA methylation-related and transposition-related piRNAs. Their expression profiles were verified using real-time quantitative PCR. The expression of the signature piRNAs was markedly higher in males than in pseudomales. The signature piRNAs could be exploited as male-specific biomarkers in this fish.ConclusionsThese signatures provide an effective tool to explore the regulatory mechanism of sex development in C. semilaevis and may provide guidance for future research on the function of piRNAs in the generative mechanism of sex reversed “pseudomales” in C. semilaevis.