Emerging functions of piwi‐interacting RNAs in diseases

Abstract PIWI‐interacting RNAs (piRNAs) are recently discovered small non‐coding RNAs consisting of 24‐35 nucleotides, usually including a characteristic 5‐terminal uridine and an adenosine at position 10. PIWI proteins can specifically bind to the unique structure of the 3′ end of piRNAs. In the past, it was thought that piRNAs existed only in the reproductive system, but recently, it was reported that piRNAs are also expressed in several other human tissues with tissue specificity. Growing evidence shows that piRNAs and PIWI proteins are abnormally expressed in various diseases, including cancers, neurodegenerative diseases and ageing, and may be potential biomarkers and therapeutic targets. This review aims to discuss the current research status regarding piRNA biogenetic processes, functions, mechanisms and emerging roles in various diseases.


| INTRODUC TI ON
piRNAs in the heart have numerous potential regulatory effects that need to be further explored. 2 RNA interference (RNAi) was discovered in the late 1990s, 3 and it significantly changed our understanding of the regulation of gene expression. These non-coding RNAs (ncRNAs) are not translated into proteins but instead work through pairing with complementary bases of targeted RNA or binding to targeted proteins. 4 In 2006, Aravin et al 5 isolated a small RNA from the vas deferens of 3-month-old C57 mice and found that the highly expressed small RNA interacted with MILI (a PIWI subprotein); they named this small RNA piRNA. During the study of piRNA biosynthesis, it was found that a large number of piRNA clusters in the intergenic region were transcribed to form piRNA precursors by bidirectional or unidirectional transcription.
Endonucleases then digested these piRNA precursors to produce piRNAs. Additionally, piRNAs can also be produced in the mRNA 3′ untranslated region (3′ UTR) and some long non-coding regions in the genome. 6 Sixteen years have passed since the first discovery of piRNAs, but our understanding of the functions of piRNAs and PIWI proteins remains unclear. In this review, we discussed the current research status regarding piRNA biogenetic processes, functions and mechanisms, along with their roles in diseases.

| B I OG ENE S IS OF PIRNA S
Newly transcribed piRNAs are usually transported out of the nucleus, processed and matured in the cytoplasm, and then perform their biological function. The mechanism of piRNA production is divided into the primary and secondary piRNA pathways, which mainly revolves around the ping-pong amplification cycle process ( Figure 1). 7 In a further study of the piRNA ping-pong mechanism, it was found that the piRNA synthesis pathway exists not only in animal germ cells but also in somatic cells and that there are some differences in the piRNA ping-pong mechanism process between germ cells and somatic cells. 8,9 The piRNA somatic pathway can directly rely on PIWI proteins and piRNA clusters of flamenco fragments to produce piRNAs. First, the piRNA precursor binds to the cytoplasmic Yb body to form the 5′ U site of the intermediate piRNA under the action of Zucchini (Zuc) and cofactors (Vret, Mino, Gasz, etc). 10 The secondary structure of the piRNA is generated, and Zuc cleaves the PIWI-piRNA sequence to form a 3′ end. The exonuclease trimer and Papi cleave and further mature the PIWI-piRNA. 11 Then, Hen1-mediated methylation occurs, and finally, the mature PIWI-piRNA complex enters the nucleus to play a silencing role. Extracellular factors of mitochondria also play an important role in this pathway ( Figure 2). 8

| CHAR AC TERIS TI C S OF PIRNA S
Compared with siRNAs and miRNAs, piRNAs are greater in number, with approximately 5 × 10 4 species. The length is slightly longer than the first two, generally within the range of 24~35 nt.
They are methylated at the 3′ end, the first nucleotide at the 5′ end is typically uridine, and the 10th base is typically adenine. In mice, piRNAs are mainly distributed on the X chromosome and rarely on the Y chromosome, but most piRNAs in the human body are distributed on autosomal chromosomes and are rarely on sex chromosomes. 12 In Table 1, we summarize the many kinds of small non-coding RNAs according to their differences in size and function.

| piRNAs participate in transcriptional silencing
Previous studies on small RNA-mediated transcriptional gene silencing (TGS) in plants and yeasts have shown that the existence of nuclear PIWI proteins, and various coincidences of inhibitory histone labelling or DNA methylation in some piRNA pathway mutants, eventually lead to PIWI-dependent transcriptional silencing in animals. 13 However, as research continues, the direct pathways of transposon silencing in the piRNA pathway have also been found. 14,15 Among them, PIWI proteins mainly act at the transcriptional level ( Figure 3).
Studies suggest that mature piRNA-PIWI/Asterix (Arx) complexes scan for and detect nascent transposon transcription. 8,16 After detection, transcriptional inhibition is enforced by the formation of heterochromatin. In addition, it is speculated that piRNAs inhibit the insertion of foreign sequence fragments. 17

| piRNAs participate in post-transcriptional gene silencing (PTGS)
Many piRNAs regulate the post-transcriptional network to inhibit target function through piRNA-RNA interactions, and the mechanism is similar to that of miRNAs. 18,19 These RNA packets include transcribed F I G U R E 1 The mechanism of piRNA production and amplification. The piRNAs that are related to PIWI and Aub are usually antisense and complementary to transposon transcripts. PIWI/Aub starts from the 5′ end of the antisense piRNA and cuts transposon transcripts between 10 and 11 nt to generate Ago3-related piRNA. Ago3 identifies piRNA cluster transcripts and generates more PIWI/Aubrelated antisense strand piRNAs pseudogenes, 20 long non-coding RNAs (lncRNAs), 21 and mRNAs. 22 The piRNA interaction requires base pairing at the 5′ end of the piRNA, which exhibits strict base pairing in the 2-11 nt range and loose base pairing in the 12-21 nt range. 23 The piRNA-PIWI complex recruits carbon catabolite-repressed 4-negative TATA-less (CCR4-NOT) and Smaug (SMG) to form specific pi-RISCs, which can promote RNA inhibition through imperfect base pairing through miRNA-like mechanisms. [24][25][26] The piRNA-PIWI ribonucleoprotein complex can also lead to transposable element post-transcriptional silencing, thus maintaining the integrity of the genome. 25 This transposable element post-transcriptional F I G U R E 2 Hen1-mediated piRNA generation pathway. After transporting out of the nucleus, the piRNA precursor (with the red '?') is transferred and processed by Yb. Then, Zuc and its cofactors interact to produce piRNA intermediates. This processing step also requires Vret, Mino and Gasz. Armi seems to be involved in the decomposition of the secondary structure. After that, piRNA intermediates are loaded into PIWI. Zuc cleaves the piRNA and forms the 3′ end. Trimmer and its cofactor Papi participate in removing the piRNA intermediate and lead to the formation of the piRNA-PIWI complex. The piRNA-PIWI complex becomes mature after Hen1 methylation. Factors that participate in the biogenesis of newborn piRNAs are located on the outer membrane of mitochondria

| piRNA regulates protein and gene expression
piRNAs are regulators of proteins and genes. 19 The piRNA/PIWI complex binds directly to some proteins through piRNAs or the PIWI protein PAZ domain. 18

| piRNAs in cancer
Although recent studies have found that piRNA expression in somatic cells is relatively low, many piRNAs are involved in tumour occurrence and development (cancer cell proliferation, apoptosis, metastasis and invasion). These piRNAs are dysregulated in tumour tissue and play roles in tumour promotion or tumour inhibition. To stimulate more research to fully understand the molecular biological mechanisms of piRNAs in tumour diseases, we summarize some recent studies on piRNAs in multiple cancers for reference ( Table 2).

| piRNAs in ageing
piRNAs can maintain genome integrity through the PIWI-piRNA pathway, which plays an important role in ageing. TEs, also known as 'jumping genes', can move from one genome site to another, resulting in insertion mutations. 29 With organism ageing, TEs become increasingly active and multiply in the somatic cell genome. These TE characteristics highlight their decisive mutagenic role in the gradual decomposition of genetic information, a molecular marker related to ageing. 30 Therefore, TE-mediated genomic instability may greatly promote the ageing process. The PIWI-piRNA pathway can inhibit the activity of TEs and then delay ageing to a certain extent. 6

| piRNAs in the heart
An increasing number of studies have found that piRNAs exist in many somatic cells in addition to germ cells, including the heart, which may be related to many heart-related pathophysiological processes. 2 piRNAs are expressed during the process of cardiomyocyte differentiation. Their expression levels change during different developmental stages. 32 Additionally, piRNAs exist in cardiac progenitor cells, 33 suggesting that piRNA may play an important role in the process of heart regeneration and participate in the maintenance F I G U R E 3 piRNA participates in methylation regulation. The complex that includes piRNA, PIWI, Arx and Panx induces cotranscriptional inhibition. Then, the targeted transposon will be labelled with histone 3 lysine 9 trimethylation (H3K9me3), which is a modification produced by anovulatory (Egg) and its cofactor Windei (Wde). The subsequent recruitment of H3K9me3 by HP1 leads to the formation of heterochromatin. Lysine-specific demethylase 1 (Lsd1) may remove active histone 3 lysine 4 dimethylation (H3K4me2) labelling in the transposon promoter region, thus effectively inhibiting transposons at the transcriptional level. Maelstrom (Mael) blocks the transmission of H3K9me3 Expression disordered after drinking [42] Bladder cancer piRABC (DQ594040) Affected the expression of TNFSF4 protein and played an important role in the development of bladder cancer [43] Breast cancer piR-21285 Functioned in the occurrence and development of breast cancer through related epigenetic mechanisms [44] piR-4987; piR-20365; piR-20485; piR-20582 Up-regulated in breast cancer and may be used as a biomarker of breast cancer [45] piRNA-36712 A new tumour suppressor gene that can be used as a promising predictor of breast cancer prognosis [46] piR-36026 Plays a role in the regulation of tumour suppressor genes and mediates the progression of breast cancer in vivo and in vitro [47] piR-016658; piR-016975 Associated with tumour initiation and progression [48] Colorectal cancer piR-15551 Produced by LNC00964-3 and participates in the occurrence and development of colorectal cancer [49] piR-1245 Targeted tumour suppressor gene has a carcinogenic effect and can be used as a potential prognostic biomarker of colorectal cancer [50] piR-5937; piR-28876 Can be used as a potential biomarker for early detection of colon cancer [51] piR-54265 By promoting the formation of PIWIL2/STAT3/phosphorylated SRC (p-SRC) complex, activates the STAT3 signal pathway, promoting the proliferation, metastasis and chemotherapy resistance of colorectal cancer cells, thus playing a carcinogenic role; may become a therapeutic target for colorectal cancer [28] piR-020619; piR-020450 Has potential as a specific marker for early detection of colorectal cancer [10] piR-24000 High expression of PIR-24000 was significantly associated with aggressive colorectal cancer phenotypes [52] Oesophageal cancer (EC) piR-823 The level of piR-823 was significantly associated with lymph node metastasis [10] Fibrosarcoma piR-39980 Has a strong antitumor effect [53] Gastric cancer (GC) piR-651; piR-823 Can be used as a valuable biomarker for detecting circulating gastric cancer cells [54,55] piR-59056; piR-32105; piR-58099 Could be used as tumour markers in gastric cancer, furthermore, could effectively stratify GC patients into low-and high-risk recurrence groups [56] piR-1245 Associated with overall survival (OS) and progression-free survival (PFS) Glioblastoma piR-8041 Inhibits cell proliferation, induce cell cycle arrest and apoptosis, and inhibit cell survival pathway [58] Hepatocellular carcinoma piR-Hep1 Silencing piR-Hep1 inhibits the viability and invasiveness of cells and may lead to a decrease in the level of phosphorylation of active AKT [59] Lung cancer (LC) piR-34871; piR-52200 Correlated with RASSF1C expression; promoted cell proliferation and colony formation by reducing AMPK phosphorylation of the ATM-AMPK-p53-p21cip pathway [60] piR-55490 Inhibits the activation of Akt/mTOR pathway and inhibits the growth of lung cancer [22] piR-651 Inhibits cell proliferation, migration and invasion and induces apoptosis, thereby regulating the carcinogenic activity of NSCLC [54] piRNA/piRNA-L Interaction with proteins under pathophysiological conditions [61] Multiple myeloma (MM) piRNA-823 Promoted angiogenesis and played a carcinogenic role in MM [62] piR-004800 Participates in the carcinogenesis of the PI3K/AKT/mTOR pathway [63] (Continues) and differentiation of cardiomyocytes. It has also been found that piRNAs exist in the hypertrophied heart. 34 There is also a significant expression of piRNA in the serum exosomes of patients with heart failure. piRNAs might also be potential factors in markers of heart failure. 35

| PIRNA ONLINE DATABA S E S
Researchers may need to refer to many resources to better design their experiments and choose appropriate research models before carrying out piRNA projects. To facilitate future research, we collated free online databases to provide valuable piRNA information (Table 3). There is not yet a single fully featured database, so researchers should make use of each database according to their different functional features. Ovarian cancer piR-33733 Inhibits apoptosis by binding to targeted mRNA [64] piR-52207 Promotes cell proliferation, migration and tumorigenesis by binding to targeted mRNA [64] Osteosarcoma (OS) piR-39980 Related to the ability of cell migration and invasion [65] Oral squamous cell carcinoma (OSCC) piR-1037 Enhances the chemoresistance and motility of OSCC cells [66] Pancreatic cancer piR-017061 Expression is down-regulated in cancer cells [67] Prostate cancer piR-31470 Increased vulnerability to oxidative stress and DNA damage in human prostate epithelial RWPE1 cells.

| D ISCUSS I ON
[68] Thyroid cancer (TC) piR-13643; piR-21238 Expected to be a new biomarker for accurate detection of PTC. [69] Urinary tumours piR-32051; piR-39894; piR43607 Highly associated with clear cell renal cell carcinoma (ccRCC) metastasis, late clinical-stage and poor cancer-specific survival [67] piR-823 Detection of piR-823 in urine is helpful for the diagnosis of renal cell carcinoma [62] piR-57125; piR-30924; piR-38756 Abnormal expression in renal cell carcinoma can be used as a potential biomarker to judge the prognosis of renal cell carcinoma [70] TA B L E 2 (Continued) Provides comprehensive data on piRNA clusters in multiple species, tissues and developmental stages [77] piRTarBase http://cosbi6.ee.ncku.edu.tw/piRTa rBase/ Predicts binding sites of piRNAs to miRNAs [78] knowledge network to explain the biogenesis and function of piR-NAs and their relationships with related diseases, hoping to identify common targets among age-related diseases and shed new light on their clinical application.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.