Piwi-interacting RNAs Research Articles

Exposure to elevated temperature affects the expression of PIWI-interacting RNAs and associated transcripts in mouse testes.

Exposure to heat waves could result in adverse effects on human health, especially in male testicles. PIWI-interacting RNA (piRNA) is a novel type of small non-coding RNA, which can notably impact mRNA turnover and preserve germline maintenance in germline cells. However, piRNA's expression status when adapting to testicular heat stress remains largely unclear. To investigate the function and mechanisms of relevant piRNAs during testicular heat stress. In this study, a mouse testicular heat stress model was constructed, and the testes were removed for piRNA-sequencing. Bioinformatics analysis was used to discover the differential expressed piRNAs, piRNA clusters, and enriched pathways. A cell heat stress model was constructed to validate the top five upregulated piRNAs. Proliferation and apoptosis assays were utilized to validate the function of selected piRNA. Bioinformatics prediction, western blotting, and immunohistochemistry were used to illustrate the downstream mechanisms. Through the bioinformatics analysis, we identified the differential expression profile and enriched pathways of piRNAs and piRNA clusters during testicular hyperthermia. Besides, piR-020492 was proved to be upregulated in heat stress mouse testes and a germ cell model. A series of in vitro assays illustrated that an overexpression of piR-020492 could restrain the proliferation and promote the apoptosis of mouse germ cells. Kyoto Encyclopedia of Genes and Genomes analysis of piRNA-generating genes in the testicular heat stress model and piR-020492 targeting genes showed that the overlap pathways are adenosine monophosphate-activated protein kinase (AMPK) and insulin pathways. Validation experiments demonstrated that the key genes of AMPK and insulin pathway exhibit differential expression after an overexpression of piR-020492 or testicular heat stress. In conclusion, our findings revealed the expression profile of piRNAs in testicular heat stress and illustrated the function and mechanisms of piR-020492 in germ cells, which could provide novel insights into the mechanism of hyperthermia-induced testicular injury.

Emerging roles and potential application of PIWI-interacting RNA in urological tumors.

The piRNA (PIWI-interacting RNA) is P-Element induced wimpy testis (PIWI)-interacting RNA which is a small molecule, non-coding RNA with a length of 24-32nt. It was originally found in germ cells and is considered a regulator of germ cell function. It can interact with PIWI protein, a member of the Argonaute family, and play a role in the regulation of gene transcription and epigenetic silencing of transposable factors in the nucleus. More and more studies have shown that piRNAs are abnormally expressed in a variety of cancer tissues and patient fluids, and may become diagnostic tools, therapeutic targets, staging markers, and prognostic evaluation tools for cancer. This article reviews the recent research on piRNA and summarizes the structural characteristics, production mechanism, applications, and its role in urological tumors, to provide a reference value for piRNA to regulate urological tumors.

PDA-PRGCN: identification of Piwi-interacting RNA-disease associations through subgraph projection and residual scaling-based feature augmentation

BackgroundEmerging evidences show that Piwi-interacting RNAs (piRNAs) play a pivotal role in numerous complex human diseases. Identifying potential piRNA-disease associations (PDAs) is crucial for understanding disease pathogenesis at molecular level. Compared to the biological wet experiments, the computational methods provide a cost-effective strategy. However, few computational methods have been developed so far.ResultsHere, we proposed an end-to-end model, referred to as PDA-PRGCN (PDA prediction using subgraph Projection and Residual scaling-based feature augmentation through Graph Convolutional Network). Specifically, starting with the known piRNA-disease associations represented as a graph, we applied subgraph projection to construct piRNA-piRNA and disease-disease subgraphs for the first time, followed by a residual scaling-based feature augmentation algorithm for node initial representation. Then, we adopted graph convolutional network (GCN) to learn and identify potential PDAs as a link prediction task on the constructed heterogeneous graph. Comprehensive experiments, including the performance comparison of individual components in PDA-PRGCN, indicated the significant improvement of integrating subgraph projection, node feature augmentation and dual-loss mechanism into GCN for PDA prediction. Compared with state-of-the-art approaches, PDA-PRGCN gave more accurate and robust predictions. Finally, the case studies further corroborated that PDA-PRGCN can reliably detect PDAs.ConclusionPDA-PRGCN provides a powerful method for PDA prediction, which can also serve as a screening tool for studies of complex diseases.

The piRNA pathway is required for nucleopolyhedrovirus replication in Lepidoptera.

The Piwi-interacting RNA (piRNA) pathway has been shown to be involved in the antiviral defense against RNA viruses, especially in mosquitoes, but its universality has been questioned. Here, we used the Bombyx mori nucleopolyhedrovirus (BmNPV) -infected silkworm as a model to explore the effects of the key factors of piRNA pathway, BmAgo3 and Siwi, on replication of a large DNA virus (belonging to the family of Baculoviridae). We demonstrated that BmAgo3 and Siwi could promote the replication of BmNPV through both overexpression and knockdown experiments in BmN cell lines and silkworm larvae. In addition, we also studied the effect of PIWI-class genes on Autographa californica nucleopolyhedrovirus (AcMNPV) replication in the Spodoptera frugiperda cell line Sf9. By knocking down the expression of PIWI-class genes in Sf9, we found that Piwi-like-1 and Piwi-like-2-3 could inhibit AcMNPV replication, while Piwi-like-4-5 promoted virus replication. Our study provides compelling evidence that the piRNA pathway affects host infection by exogenous viruses in Lepidoptera. Also, our results reflect the diversity of the roles of PIWI-class genes in virus infection of the host across species. This study is the first to explore the interaction of PIWI-class proteins with DNA viruses, providing new insights into the functional roles of the piRNA pathway.

Intermitochondrial cement (IMC) harbors piRNA biogenesis machinery and exonuclease domain-containing proteins EXD1 and EXD2 in mouse spermatocytes.

Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3'-5' domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.

The role of PIWI-interacting RNA in naringin pro-angiogenesis by targeting HUVECs

Angiogenesis is a biological process in which resting endothelial cells start proliferating, migrating and forming new blood vessels. Angiogenesis is particularly important in the repair of bone tissue defects. Naringin (NG) is the main active monomeric component of traditional Chinese medicine, which has various biological activities, such as anti-osteoporosis, anti-inflammatory, blood activation and microcirculation improvement. At present, the mechanism of naringin in the process of angiogenesis is not clear. PIWI protein-interacting RNA (piRNA) is a small noncoding RNA (sncRNA) that has the functions of regulating protein synthesis, regulating the structure of chromatin and the genome, stabilizing mRNA and others. Several studies have demonstrated that piRNAs can mediate the angiogenesis process. Whether naringin can interfere with the process of angiogenesis by regulating piRNAs and related target genes deserves further exploration. Thus, the purpose of this study was to validate the potential angiogenic and bone regeneration properties and related mechanisms of naringin both in vivo and in vitro.

Identification and functional characterization of developmental-stage-dependent piRNAs in Tibetan sheep testes.

The core function of the testes is to produce sperms, which is the prerequisite for maintaining male fertility. PIWI-interacting RNAs (piRNAs) are a class of non-coding small RNAs that are mainly enriched in the reproductive organ and play a key role in germ cell development and spermatogenesis. However, the expression and function of piRNAs in the testes of Tibetan sheep, a domestic animal endemic to the Tibetan Plateau, remain unknown. In this study, we evaluated the sequence structure, expression profile, and potential function of piRNAs in testicular tissues from Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years of age, respectively) by small RNA sequencing. Of the identified piRNAs, the sequence lengths of 24-26 nt and 29 nt dominate. Most piRNA sequences begin with uracil and have a distinct ping-pong structure which mainly distributes in exons, repeat regions, introns, and other unannotated regions of the genome. The piRNAs in the repeat region are primarily derived from the retrotransposons: long terminal repeats, long interspersed nuclear elements, and short interspersed elements. These piRNAs constitute 2,568 piRNA clusters, which mainly distribute on chromosomes 1, 2, 3, 5, 11, 13, 14, and 24, and of these clusters, a total of 529 piRNA clusters were differentially expressed in at least two age groups. Most of the piRNAs were expressed in a low abundance in the testes of developing Tibetan sheep. A total of 41,552 and 2,529 differential piRNAs were identified in testes from 3 months vs. 1 year, and 1 year vs. 3 years, respectively, presenting significantly increased abundance for most piRNAs in 1 year and 3 years compared with 3 months. The functional evaluation of the target genes showed that the differential piRNAs are mainly involved in regulating gene expression, transcription, protein modification, and cell development during spermatogenesis and testicular development. In conclusion, this study focused on the sequence structure and expression characteristics of piRNAs in the testis of Tibetan sheep and provided new insights into the functional mechanism of piRNAs in testicular development and spermatogenesis of sheep.

The extracellular RNA and drug resistance in cancer: a narrative review

Background and Objective: Extracellular RNA (exRNA) is an evolving concept. In one hand, more endogenous RNA types are detected in various biofluids or found in local extracellular space. These endogenous exRNAs include microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), piwi-interacting RNAs (piRNAs), transfer RNAs (tRNAs) and protein-coding messenger RNAs (mRNAs). ExRNA profilings are associated with many physiopathological conditions, including drug resistance in cancer. Endogenous exRNAs are also significantly involved in the molecular mechanisms of cancer drug resistance. In the other hand, exRNA types are expanded to include exogenous RNA therapeutics which started to headline in recent years with the approval of several RNA-based drugs. This review discusses current knowledge of exRNAs function in cancer drug resistance, including their value as biomarkers, the significant roles of modulating cancer drug resistance and the usage of exogenous RNA-based therapeutics to relieve cancer drug resistance.

PIWI-interacting RNA-17458 is oncogenic and a potential therapeutic target in cervical cancer.

Cervical cancer (CC) is one of the leading cancers among the female reproductive system. The piwi-interacting RNA (piRNA) function and biogenesis has been studied in various cancers, including CC. But the precise mechanism of piRNA in CC is still unknown. In our study, we found that piRNA-17458 was overexpressed in CC tissues and cells. piRNA-17458 mimic and inhibitor promoted and suppressed proliferation, migration and invasion ability of CC cells, respectively. We also demonstrated that piRNA-17458 mimic could contribute to tumor growth in mice xenograft models. Besides, we also found that the piRNA-17458 mimic could enhance mRNA N6-methyladenosine(m6A) levels and increase WTAP stability in CC cells, while the effects of the mimic was reversed by the WTAP knockdown. The results of dual luciferase reporter assay showed that WTAP was a direct target of piRNA-17458. Knockdown of WTAP attenuated proliferation, migration and invasion of CC cells in piRNA-17458 mimic group. Our finding not only demonstrates for the first time that piRNA-17458 is overexpressed in CC tissues and cells, but also shows that piRNA-17458 promotes tumorigenesis of CC in a WTAP-mediated m6A methylation manner.

A method for screening the suppressor genes of siRNA and piRNA pathways using cultured silkworm cells.

The BmN-4 cell line originates from the ovaries of silkworm, Bombyx mori , and possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. BmN-4 cells are latently infected with Bombyx mori latent virus (BmLV), an RNA virus whose replication is strictly controlled by both siRNA and piRNA pathways. Knockdown or knockout of the core factors of these two small RNA pathways increases BmLV RNA amount, which in turn inhibits cell growth. Here, we used the known RNAi suppressor CrPV-1A to assess whether the BmN-4 cell line can be used for screening the suppressors of siRNA and piRNA pathways.

Detection of MicroRNAs by Northern Blot.

Small RNAs (sRNAs) are key regulators of transcriptomes and proteomes of organisms through their sequence-specific interaction with complementary RNA targets. sRNAs can be classified according to their origin and mode of action into different classes such as: microRNAs (miRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). The abundance and specific spatio-temporal expression of many sRNAs, especially miRNAs, is relevant for their biological function. Northern blotting is a widely used technique to study sRNAs because it is quantitative, relatively inexpensive, and readily available for most laboratories. This chapter describes the protocols for radioactive and non-radioactive sRNA Northern blot analysis, which includes RNA extraction, polyacrylamide gel electrophoresis, membrane transfer, hybridisation and detection of sRNA using oligonucleotide probes. The protocol is described to prepare most of the reagents needed in the lab, but also timesaving commercial reagent alternatives are included. Suggestions and nuances obtained from experience are included as Notes.

Pan-cancer landscape of immunology PIWI-interacting RNAs

PIWI-interacting RNAs (piRNAs), an emergent type of non-coding RNAs during oncogenesis, play critical roles in regulating tumor microenvironment. Systematic analysis of piRNAs’ roles in modulating immune pathways is important for tumor immunotherapy. In this study, in-depth analysis of piRNAs was performed to develop an integrated computational algorithm, the immunology piRNA (ImmPI) pipeline, for uncovering the global expression landscape of piRNAs and identifying their regulatory roles in immune pathways. The immunology piRNAs show a tendency towards overexpression patterns in immune cells, causing perturbations in tumors, being significantly associated with infiltration of immune cells, and having prognostic value. The ImmPI score can contribute to prioritizing tumor-related piRNAs and distinguish two subtypes of SKCM (immune-cold and hot phenotypes), as characterized by different prognoses, immunogenicity and antitumor immunity. Finally, we developed an interactive web resource (ImmPI portal: http://www.hbpding.com/ImmPi) for the biomedical research community, with several useful modules to browse, visualize, and download the results of immunology piRNAs analysis. Overall, our work provides a comprehensive landscape of piRNAs across multiple cancer types and sheds light on their regulatory and functional roles in tumor immunity. These findings pave the way for future research and development of piRNA-based immunotherapies for cancer treatment.

TarpiD, a database of putative and validated targets of piRNAs.

Piwi-interacting RNAs (piRNAs) are a novel class of 18-36 nts long small non-coding single-stranded RNAs that play crucial roles in a wide array of critical biological activities besides maintaining genome integrity by transposon silencing. piRNAs influence biological processes and pathways by regulating gene expression at transcriptional and post-transcriptional level. Studies have reported that piRNAs silence various endogenous genes post-transcriptionally by binding to respective mRNAs through interaction with the PIWI proteins. Several thousands of piRNAs have been discovered in animals, but their functions remain largely undiscovered owing to a lack of proper guiding principles of piRNA targeting or diversity in targeting patterns amongst piRNAs from the same or different species. Identification of piRNA targets is essential for deciphering their functions. There are a few tools and databases on piRNAs, but there are no systematic and exclusive repositories to obtain information on target genes regulated by piRNAs and other related information. Hence, we developed a user-friendly database named TarpiD (Targets of piRNA Database) that offers comprehensive information on piRNA and its targets, including their expression, methodologies (high-throughput or low-throughput) for target identification/validation, cells/tissue types, diseases, target gene regulation types, target binding regions, and key functions driven by piRNAs through target gene interactions. The contents of TarpiD are curated from the published literature and enable users to search and download the targets of a particular piRNA or the piRNAs that target a specific gene for use in their research. This database harbours 28 682 entries of piRNA-target interactions supported by 15 methodologies reported in hundreds of cell types/tissues from 9 species. TarpiD will be a valuable resource for a better understanding of the functions and gene-regulatory mechanisms mediated by piRNAs. TarpiD is freely accessible for academic use at https://tarpid.nitrkl.ac.in/tarpid_db/.

IL11 signaling mediates piR-2158 suppression of cell stemness and angiogenesis in breast cancer.

Emerging evidence has indicated the aberrant expression of PIWI-interacting RNAs (piRNAs) in human cancer cells to regulate tumor development and progression by governing cancer cell stemness. Herein, we identified downregulation of piR-2158 in human breast cancer tumors, especially in ALDH+ breast cancer stem cells (BCSCs) from patients and cell lines, which was further validated in two types of genetically engineered mouse models of breast cancer (MMTV-Wnt and MMTV-PyMT). Enforced overexpression of piR-2158 in basal-like or luminal subtypes of breast cancer cells suppressed cell proliferation, migration, epithelial-mesenchymal transition (EMT) and stemness in vitro. Administration of a dual mammary tumor-targeting piRNA delivery system in mice reduced tumor growth in vivo. RNA-seq, ChIP-seq and luciferase reporter assays demonstrated piR-2158 as a transcriptional repressor of IL11 by competing with AP-1 transcription factor subunit FOSL1 to bind the promoter of IL11. STAT3 signaling mediated piR-2158-IL11 regulation of cancer cell stemness and tumor growth. Moreover, by co-culturing of MDA-MB-231 and HUVECs in vitro and CD31 staining of tumor endothelial cells in vivo, we demonstrated inhibition of angiogenesis by piR-2158-IL11 in breast cancer. In conclusion, the current study not only reveals a novel mechanism through which piR-2158 inhibits mammary gland tumorigenesis via regulating cancer stem cells and tumor angiogenesis, but also provides a novel therapeutic strategy in treatment of breast cancer.

PS-B02-9: HEART RELATED CIRCULAR RNA (HRCR) INFLUENCES POLYGENIC CARDIAC HYPERTROPHY BY ALTERING CARDIAC MICRORNAS

Left ventricular hypertrophy (LVH) is the main risk factor for cardiovascular disease after age. Circular RNAs (circRNA), a novel and underexplored RNA type, are created by the back-splicing of pre-mRNA and have been proposed to regulate gene expression post-transcriptionally via targeting micro RNAs (miRNA). Heart related circRNA (Hrcr) has been proposed as an important regulator of LVH and has been shown to be downregulated in mice with cardiac hypertrophy induced by chronic infusion of isoproterenol. Our aim was to investigate the expression of Hrcr in the hypertrophic heart during development and at maturity. We used the hypertrophic heart rat (HHR), a unique normotensive polygenic model of LVH and heart failure, and its genetic control strain, the normal heart rat (NHR). We first measured the expression of Hrcr by quantitative real-time PCR in left ventricles of HHR and NHR at five ages (2 days old, 4-, 13-, 33- and 50-weeks old). Secondly, we silenced HRCR in human primary cardiomyocytes and performed small RNA sequencing to reveal its miRNA downstream targets. Lastly, we performed in silico investigations to predict miRNA-mRNA targets and explore enriched gene ontology terms in our dataset.We found that Hrcr is upregulated in the HHR at 13-, 33- and 50-weeks old (FC = 2.77-3.57; P < 0.05). The silencing of HRCR revealed novel downstream targets, including four miRNAs (miR-1-3p, miR-330, miR-27a-5p and miR-299-5p) and two noncoding RNAs (ribosomal RNA RNA5-8SP2 and piwi-interacting RNA piR-17153). The miRNA-mRNA interactions predicted a total of 359 mRNAs highly likely to be targeted by the differentially expressed miRNAs. Lastly, 206 gene ontology terms were enriched in our dataset, including terms related to heart development and cardiovascular disease.

Exosomal hsa-piR1089 promotes proliferation and migration in neuroblastoma via targeting KEAP1.

Neuroblastoma (NB) is a sympathetic nervous system tumor and one of the most common pediatric, extra-cranial, solid tumors, especially in early childhood. Its expression is heterogeneous and shows a unique clinical and prognostic feature. Due to its insidious onset, most diagnoses are accompanied by metastasis, making patient prognoses extremely poor. Novel biomarkers are urgently needed for easy diagnosis, metastasis detection, and investigation of potential mechanisms regulating NB tumor progression. Recent research highlights that circulating tumor markers could be used to diagnose and monitor prognosis in various tumors. Among them, exosomal genetic material has attracted much attention because of its tumor-secreted specificity and unique mechanism of action. In this study, we used next-generation sequencing to study PIWI-interacting RNAs (piRNAs) in exosomes derived from NB patient plasma. We found higher human piRNA 1089 (hsa-piR-1089) levels in exosomes from NB patients than from normal controls. Our receiver operating characteristic (ROC) curve analyses showed that hsa-piR-1089 had high diagnostic sensitivity and specificity. We also found that high hsa-piR-1089 expression in NB tumor tissues was associated with a high-risk Children's Oncology Group classification and metastasis. Our in vitro experiments showed that exosomal hsa-piR-1089 promoted NB cell proliferation and migration by inhibiting Kelch-like ECH-associated protein 1 (KEAP1) expression. Moreover, low KEAP1 expression was associated with NB progression in clinical samples. In conclusion, our data indicate that blood-borne exosomal hsa-piR-1089 is a diagnostic marker for NB and assessing metastasis. Our study provides a quick, simple, and noninvasive diagnostic method for NB and contributes to developing new treatment strategies.

Abstract 1211: Identification and functional analysis of Piwi-interacting RNAs in cervical cancer

Abstract 1211: Identification and functional analysis of Piwi-interacting RNAs in cervical cancer

The Role of Nucleases Cleaving TLR3, TLR7/8 and TLR9 Ligands, Dicer RNase and miRNA/piRNA Proteins in Functional Adaptation to the Immune Escape and Xenophagy of Prostate Cancer Tissue.

The prototypic sensors for the induction of innate and adaptive immune responses are the Toll-like receptors (TLRs). Unusually high expression of TLRs in prostate carcinoma (PC), associated with less differentiated, more aggressive and more propagating forms of PC, changed the previous paradigm about the role of TLRs strictly in immune defense system. Our data reveal an entirely novel role of nucleic acids-sensing Toll-like receptors (NA-TLRs) in functional adaptation of malignant cells for supply and digestion of surrounding metabolic substrates from dead cells as specific mechanism of cancer cells survival, by corresponding ligands accelerated degradation and purine/pyrimidine salvage pathway. The spectrophotometric measurement protocols used for the determination of the activity of RNases and DNase II have been optimized in our laboratory as well as the enzyme-linked immunosorbent method for the determination of NF-κB p65 in prostate tissue samples. The protocols used to determine Dicer RNase, AGO2, TARBP2 and PIWIL4 were based on enzyme-linked immunosorbent assay. The amount of pre-existing acid-soluble oligonucleotides was measured and expressed as coefficient of absorbance. The activities of acid DNase II and RNase T2, and the activities of nucleases cleaving TLR3, TLR7/8 and TLR9 ligands (Poly I:C, poly U and unmethylated CpG), increased several times in PC, compared to the corresponding tumor adjacent and control tissue, exerting very high sensitivity and specificity of above 90%. Consequently higher levels of hypoxanthine and NF-κB p65 were reported in PC, whereas the opposite results were observed for miRNA biogenesis enzyme (Dicer RNase), miRNA processing protein (TARB2), miRNA-induced silencing complex protein (Argonaute-AGO) and PIWI-interacting RNAs silence transposon. Considering the crucial role of purine and pyrimidine nucleotides as energy carriers, subunits of nucleic acids and nucleotide cofactors, future explorations will be aimed to design novel anti-cancer immune strategies based on a specific acid endolysosomal nuclease inhibition.

A combined miRNA–piRNA signature in the serum and urine of rabbits infected with Toxoplasma gondii oocysts

BackgroundIncreasing evidence has shown that non-coding RNA (ncRNA) molecules play fundamental roles in cells, and many are stable in body fluids as circulating RNAs. Study on these ncRNAs will provide insights into toxoplasmosis pathophysiology and/or help reveal diagnostic biomarkers.MethodsWe performed a high-throughput RNA-Seq study to comprehensively profile the microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) in rabbit serum and urine after infection with Toxoplasma gondii oocysts during the whole infection process.ResultsTotal RNA extracted from serum and urine samples of acutely infected [8 days post-infection (DPI)], chronically infected (70 DPI) and uninfected rabbits were subjected to genome-wide small RNA sequencing. We identified 2089 miRNAs and 2224 novel piRNAs from the rabbit sera associated with T. gondii infection. Meanwhile, a total of 518 miRNAs and 4182 novel piRNAs were identified in the rabbit urine associated with T. gondii infection. Of these identified small ncRNAs, 1178 and 1317 serum miRNAs and 311 and 294 urine miRNAs were identified as differentially expressed (DE) miRNAs in the acute and chronic stages of infections, respectively. A total of 1748 and 1814 serum piRNAs and 597 and 708 urine piRNAs were found in the acute and chronic infection stages, respectively. Of these dysregulated ncRNAs, a total of 88 common DE miRNAs and 120 DE novel piRNAs were found in both serum and urine samples of infected rabbits.ConclusionsThese findings provide valuable data for revealing the physiology of herbivore toxoplasmosis caused by oocyst infection. Circulating ncRNAs identified in this study are potential novel diagnostic biomarkers for the detection/diagnosis of toxoplasmosis in herbivorous animals.Graphical

First Identification and Investigation of piRNAs in the Larval Gut of the Asian Honeybee, Apis cerana.

Piwi-interacting RNAs (piRNAs), a class of small non-coding RNAs (ncRNAs), play pivotal roles in maintaining the genomic stability and modulating biological processes such as growth and development via the regulation of gene expression. However, the piRNAs in the Asian honeybee (Apis cerana) are still largely unknown at present. In this current work, on the basis of previously gained high-quality small RNA-seq datasets, piRNAs in the larval gut of Apis cerana cerana, the nominated species of A. cerana, were identified for the first time, followed by an in-depth investigation of the regulatory roles of differentially expressed piRNAs (DEpiRNAs) in the developmental process of the A. c. cerana. Here, a total of 621 piRNAs were identified in A. c. cerana larval guts, among which 499 piRNAs were shared by 4-(Ac4 group), 5-(Ac5 group), and 6-day-old (Ac6 group) larval guts, while the numbers of unique ones equaled 79, 37, and 11, respectively. The piRNAs in each group ranged from 24 nucleotides (nt) to 33 nt in length, and the first base of the piRNAs had a cytosine (C) bias. Additionally, five up-regulated and five down-regulated piRNAs were identified in the Ac4 vs. Ac5 comparison group, nine of which could target 9011 mRNAs; these targets were involved in 41 GO terms and 137 pathways. Comparatively, 22 up-regulated piRNAs were detected in the Ac5 vs. Ac6 comparison group, 21 of which could target 28,969 mRNAs; these targets were engaged in 46 functional terms and 164 pathways. The results suggested an overall alteration of the expression pattern of piRNAs during the developmental process of A. c. cerana larvae. The regulatory network analysis showed that piR-ace-748815 and piR-ace-512574 in the Ac4 vs. Ac5 comparison group as well as piR-ace-716466 and piR-ace-828146 in the Ac5 vs. Ac6 comparison group were linked to the highest number of targets. Further investigation indicated that targets of DEpiRNAs in the abovementioned two comparison groups could be annotated to several growth and development-associated pathways, such as the Jak/STAT, TGF-β, and Wnt signaling pathways, indicating the involvement of DEpiRNAs in modulating larval gut development via these crucial pathways. Moreover, the expression trends of six randomly selected DEpiRNAs were verified using a combination of stem-loop RT-PCR and RT-qPCR. These results not only provide a novel insight into the development of the A. c. cerana larval gut, but also lay a foundation for uncovering the epigenetic mechanism underlying larval gut development.