The Biogenesis and Functions of piRNAs in Human Diseases.

As reported, more than three-quarters of the human genome is capable of being transcribed into RNAs, but only 2% ultimately encodes proteins. 1 , 2 , 3 Those RNAs that do not encode proteins are called noncoding RNAs (ncRNAs). With the establishment and development of high-throughput genome sequencing technology, it has been widely recognized that ncRNAs are of great importance in cellular activities and individual health. Basically, ncRNAs can be divided, based on the length, into long ncRNAs (>200 nt) and small ncRNAs (≤200 nt). 4 Furthermore, small ncRNAs can be classified into several subclasses such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small cytoplasmic RNAs (scRNAs), transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs). 5 , 6 piRNAs were first isolated and purified from rat testes in four independent laboratories in 2006. 7 , 8 , 9 , 10 piRNAs are 26–30 nt in length, with 2′- O -methylation at their 3′ end. 11 Actually, piRNAs promote gene silencing processes via interacting with Piwi proteins. 7 , 12 , 13 There are four isoforms of Piwi proteins in human beings, i.e., PIWIL1 (Hiwi), PIWIL2 (Hili), PIWIL3, and PIWIL4 (Hiwi2); three in rodents, i.e., PIWIL1 (Miwi), PIWIL2 (Mili), and PIWIL4 (Miwi2); and three in Drosophila , i.e., PIWI, Aub, and Ago3. 14 , 15 The initially identified functions of piRNAs included transposon silencing and gene integrity maintained in germline cells. To date, piRNAs have been detected both in the germline cells and somatic cells in many animals, such as nematodes, insects, fish, and mammals. 16 It is apparent that piRNAs not only serve as transposon silencers but they also play crucial roles in regulating gene expression. 17 , 18 , 19 , 20 Aberrant piRNA expressions have been detected in diverse diseases, especially in neoplasms and reproductive system diseases. piRNAs are promising novel biomarkers for early diagnosis and therapeutic targets for precise medicine. This review explains the biogenesis, functions, changes, and probable roles of piRNAs in diseases of different systems. A large proportion of piRNA precursors are produced from genetic regions named piRNA clusters, which can be divided into uni-strand or dual-strand clusters. Uni-strand clusters give rise to precursors mapping only to one strand, whereas dual-strand clusters produce precursors mapping to both genomic strands. Additionally, some piRNA precursors can be generated from the 3′ UTR of protein-coding genes, or from individual transposons. 21 , 22 The transcription of uni-strand clusters is similar to the canonical mRNA transcription. Uni-strand clusters harbor the transcription-associated histone 3 lysine 4 demethylation (H3K4me2) mark at promoters. Additionally, piRNA precursors are 5′ methyl-guanosine capped and 3′ terminated. 23 , 24 On the contrary, dual-strand clusters lack clear signatures of RNA polymerase II (RNA Pol II) promoters, such as H3K4me3 and peaks of RNA Pol II, and non-polyadenylated piRNA precursors are produced ( Figure 1 ). 23 , 25 , 26 Figure 1 Difference between Uni-Strand and Dual-Strand Cluster Transcription Uni-strand clusters (left) have distinct promoter regions, featured by H3K4me2, and generate 5′-methyl-guanosine-capped and 3′-terminated precursors from one direction. Dual-strand clusters (right) have no distinct promoter regions and generate non-polyadenylated piRNA precursors from two directions. Difference between Uni-Strand and Dual-Strand Cluster Transcription Uni-strand clusters (left) have distinct promoter regions, featured by H3K4me2, and generate 5′-methyl-guanosine-capped and 3′-terminated precursors from one direction. Dual-strand clusters (right) have no distinct promoter regions and generate non-polyadenylated piRNA precursors from two directions. Dual-strand clusters are transcribed into piRNA precursors with the involvement of several proteins, including RNA Pol II, Rhino-Cutoff-Deadlock complex (RDC complex), Moonshiner, TATA-box binding protein-related factor (TRF2), and three prime repair exonuclease (TREX). First, RNA Pol II is recruited onto piRNA clusters. Then, the transcription is facilitated by the RDC complex, which includes three subunits: Rhino (Rhi), Deadlock (Del), and Cutoff (Cuff). Rhi, the paralog of heterochromatin protein HP1, binds to the histone 3 lysine 9 trimethylation (H3K9m3) mark. Although the H3K9me3 mark is usually known to inhibit transcription in heterochromatin, it is essential in the process of piRNA precursor biogenesis. Meanwhile, Rhi inhibits inaccurate splicing between dual-strand cluster transcripts and represses the use of canonical cleavage and polyadenylation sequence motifs. 27 Cuff prevents premature termination and protects precursors from degradation. 23 , 25 Moonshiner, the paralog of the germline-specific transcription initiation factor IIA subunit 1, also facilitates the initiation of transcription and represses the H3K9me3 mark by interacting with the RDC complex and TRF2. 28 TREX prevents R-loop formation, the hybrids between nascent RNA and DNA. 29 , 30 , 31 The nuclear DEAD box RNA helicase 56-kDa U2AF-associated protein (UAP56) suppresses the splicing of dual-strand cluster precursors ( Figure 2 A). 27 , 32 Figure 2 The Procedure of piRNA Production (A) Biogenesis of piRNAs precursors from dual-strand clusters. piRNA precursors are produced with the involvement of RNA polymerase II, the Rhino (Rhi)-Cutoff (Cuff)-Deadlock (Del) complex, Moonshiner (Moon), TATA-box binding protein (TBP)-related factor 2 (TRF2), three prime repair exonuclease (TREX), and 56-kDa U2AF-associated protein (UAP56). Rhi binds to the H3K9me3 mark and inhibits transcript splicing, as well as represses the use of canonical cleavage and polyadenylation sequence motifs. Cuff prevents premature termination and protects RNA from degradation. Moonshiner promotes initiation of transcription and represses the H3K9me3 mark by interacting with the RDC complex and TRF2. TREX prevents the formation of R-loops. UAP56 suppresses the splicing of dual-strand cluster. (B) Biogenesis of mature piRNAs. The red dotted box represents the primary biogenesis of piRNAs. First, secondary structures of piRNA precursors are resolved by the RNA helicase Armitage (Armi). After despiralization, precursors are processed into pre-piRNAs with a 5′ monophosphate by the endonuclease Zucchini (Zuc). Then, pre-piRNAs are loaded onto the PIWI and trimmed by the 3′ to 5′ exonuclease Nibbler (Nbr). Concomitantly, the newly formed 3′ terminal ends are methylated at the 2′ oxygen by the small RNA 2′- O -methyltransferase Hen1. This procedure is called primary biogenesis. The dark blue dotted box represents the secondary biogenesis of piRNAs (the ping-pong cycle). Aub binds to antisense-strand piRNAs and then cleaves sense piRNA precursors, giving rise to sense piRNAs loaded into Ago3. In contrast, Ago3 binds to piRNAs from sense clusters. The Ago3/piRNA complex cleaves antisense piRNA precursors and produces antisense piRNAs that are loaded into Aub. The round of cleavage and trimming repeats and produces a number of piRNAs in a short time. The Procedure of piRNA Production (A) Biogenesis of piRNAs precursors from dual-strand clusters. piRNA precursors are produced with the involvement of RNA polymerase II, the Rhino (Rhi)-Cutoff (Cuff)-Deadlock (Del) complex, Moonshiner (Moon), TATA-box binding protein (TBP)-related factor 2 (TRF2), three prime repair exonuclease (TREX), and 56-kDa U2AF-associated protein (UAP56). Rhi binds to the H3K9me3 mark and inhibits transcript splicing, as well as represses the use of canonical cleavage and polyadenylation sequence motifs. Cuff prevents premature termination and protects RNA from degradation. Moonshiner promotes initiation of transcription and represses the H3K9me3 mark by interacting with the RDC complex and TRF2. TREX prevents the formation of R-loops. UAP56 suppresses the splicing of dual-strand cluster. (B) Biogenesis of mature piRNAs. The red dotted box represents the primary biogenesis of piRNAs. First, secondary structures of piRNA precursors are resolved by the RNA helicase Armitage (Armi). After despiralization, precursors are processed into pre-piRNAs with a 5′ monophosphate by the endonuclease Zucchini (Zuc). Then, pre-piRNAs are loaded onto the PIWI and trimmed by the 3′ to 5′ exonuclease Nibbler (Nbr). Concomitantly, the newly formed 3′ terminal ends are methylated at the 2′ oxygen by the small RNA 2′- O -methyltransferase Hen1. This procedure is called primary biogenesis. The dark blue dotted box represents the secondary biogenesis of piRNAs (the ping-pong cycle). Aub binds to antisense-strand piRNAs and then cleaves sense piRNA precursors, giving rise to sense piRNAs loaded into Ago3. In contrast, Ago3 binds to piRNAs from sense clusters. The Ago3/piRNA complex cleaves antisense piRNA precursors and produces antisense piRNAs that are loaded into Aub. The round of cleavage and trimming repeats and produces a number of piRNAs in a short time. Uni-strand piRNA cluster transcriptions have been observed in many metazoans, such as sponges, hydras, silk worms, fruit flies, zebrafish, mice, and humans, which appear to be evolutionarily conserved. 7 , 8 , 9 , 24 , 33 , 34 , 35 , 36 , 37 , 38 Although dual-strand piRNA cluster transcriptions have been reported in Drosophila , whether they occur in other organisms needs more study. 24 Once the transcription is finished, piRNA precursors are transported out of the nucleus ( Figure 2 B). First, the secondary structures are resolved by the RNA helicase Armitage (Armi). After despiralization, piRNA precursors are processed into pre-piRNAs with a 5′ monophosphate through the cleavage of the mitochondria-associated endonuclease Zucchini (Zuc). Then, pre-piRNAs are loaded onto PIWI proteins and are trimmed at the 3′ ends by a 3′ to 5′ exonuclease, Nibbler (Nbr). Concomitantly, the newly formed 3′ terminal ends are methylated at the 2′ oxygen by the small RNA 2′- O -methyltransferase Hen1. 33 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 It has been suggested that 2′- O -methylation may enhance piRNA stability. 41 , 45 , 46 , 47 This procedure is called primary biogenesis of piRNAs, and piRNAs produced in this way are named primary piRNAs. Primed by primary piRNAs, the generation of piRNAs is amplified with the involvement of Ago3 and Aub proteins. Aub binds to antisense-strand piRNAs and then cleaves sense piRNA precursors, giving rise to sense piRNAs bound by Ago3. In contrast, Ago3 binds to sense-strand piRNAs and cleaves antisense piRNA precursors, producing antisense piRNAs that load onto Aub. 48 , 49 The round of cleavage repeats and produces a number of piRNAs in a short time. The way in which large amounts of piRNAs are produced is called secondary biogenesis of piRNAs, as well as the ping-pong cycle. These piRNAs are then bound by PIWI proteins and transported back to the nucleus to silence target genes. The exact procedure of piRNA biogenesis remains elusive, and most of our understanding comes from Drosophila germline cells. A similar pathway has been observed in mice just with the involvement of different Piwi proteins. Meanwhile, there are clues indicating that this procedure in humans may be similar to Drosophila . 50 , 51 It was initially reported that piRNAs defended against transposon mobilization in fly germline cells, which was later verified in organisms ranging from hydras to humans. 15 , 33 , 40 , 52 , 53 , 54 , 55 , 56 , 57 Transposons, known as jumping genes, are similar to endogenous viruses. They threaten the stability of genes as they “copy and paste” their own DNA in the host genome to reproduce themselves, which causes a series of consequences. Exon insertions hinder the coding sequence, and the insertion into introns may change splicing patterns, which may lead to the production of novel and potentially deleterious fusion proteins. 58 If transcripts of transposons are inserted into promoter or enhancer regions, gene transcriptions can also be transformed. Meanwhile, insertions into 5′ UTRs or 3′ UTRs can affect post-transcriptional gene regulation. Transposon insertions also lead to DNA nicks and double-strand breaks; moreover, errors in the repair of these lesions can lead to recombination between transposon repeats, triggering chromosomal duplication, deletion, translocation, and inversion. 59 At the transcriptional level, piRNAs and Piwi proteins directly modify chromatin structure and histone proteins in the nucleus via the regulation of DNA methyltransferase (DNMT). DNMT methylates CpG islands in promoter regions, which suppresses the initiation of transcription. Piwi proteins guide DNMT to bind with transposable elements or target genes. As a consequence, transposable elements or target genes are silenced. However, little is known about the exact mechanisms by which piRNAs and Piwi proteins regulate DNMT protein expression. piRNAs and Piwi proteins also interact with histone methylation machinery and regulate the methylation of histone lysine residues (H3K and H4K). They recruit histone methyltransferases onto target transcripts to repress transcription. 60 , 61 Currently, the relationships between histone modification and piRNAs remain unresolved. At the post-transcriptional level, piRNAs and Piwi proteins induce mRNA degradation via mRNA decay machinery. The major way to decay mRNA and impede translation is to shorten the poly(A) tail of mRNAs, which is called deadenylation. 51 As piRNAs are involved in gene regulation, there has been a budding interest in defining their roles in human diseases. A number of studies have revealed that the dysregulation of piRNAs can both promote or suppress the occurrence and development of various diseases, especially cancers. 62 Lung cancer ranks first with highest incidence and mortality among all cancers in both sexes worldwide. 63 Unlike miRNAs and circRNAs, which have been largely studied in lung cancer pathogenesis, there have been limited studies demonstrating the relationship between piRNAs and lung cancer. 64 , 65 However, current studies can extend our insights into the occurrence and development of lung cancer and can provide new therapeutic strategies. A recent study compared piRNA expression profiles between eight non-small-cell lung cancer (NSCLC) tissue samples and three human bronchial epithelial cell samples. 66 piR-L-163 in NSCLC cells, the top downregulated one, inhibited cell migration and invasion by maintaining the function of phosphorylated ezrin-radixin-moesin (p-ERM), which connects transmembrane proteins, such as ERM-binding phosphoprotein 50 (EBP50) and filamentous actin (F-actin). 66 In another study, piR-55490 was found to suppress lung cancer cell proliferation by binding to the 3′ UTR of mammalian target of rapamycin (mTOR) mRNA. 67 It was identified that upregulated piR-651 promoted cell proliferation, migration, and invasion, as well as suppressed cell death in NSCLC A549 and HCC827 cell lines, but its mechanism needs further study. 68 Moreover, piR-57125 was significantly downregulated in metastatic lung adenocarcinoma cells compared with primary cancer cells. 69 Other piRNAs, such as piR-34871, piR-35127, piR-46545, piR-52200, and piR-57125, were also determined to be downregulated in lung cancer cells, and they comprehensively promoted the proliferation of lung cancer cell lines. 70 These studies suggest that aberrant piRNA expressions are involved in the tumorigenesis, invasion, and metastasis of lung cancer, which may provide potential functional targets and novel biomarkers for precise medicine. Gastric cancer remains an important cancer, making it the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide, 63 while it is the second most commonly diagnosed cancer in males and third in females, and it is the second leading cause of cancer death in both men and women in China. 71 It was identified that piR-823 significantly downregulated in gastric cancer tissues. Cell proliferation was inhibited with piR-823 mimics, and a xenograft nude mouse model verified that piR-823 suppressed tumor growth. 72 Moreover, patients with gastric cancer had low expression of piR-651 and piR-823 in peripheral blood. piR-32105, piR-58099, and piR-59056 were reported to increase in gastric cancer tissues. 73 It is well known that the early stage of gastric cancer can be cured by surgery or endoscopic treatment, but the prognosis of advanced gastric cancer is unsatisfied. Immunotherapy, similar to anti-programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) therapy, is a promising therapy in cancer, while patients with advanced gastric cancer are not very sensitive to immunotherapy alone or combined with chemotherapy in first-line therapy. More studies should be performed to identify whether piRNAs are related to immune escape, and whether those piRNAs, similar to piR-823, which can be detected in peripheral blood, could be promising biomarkers for predicting immunotherapy outcomes and for diagnosis in gastric cancer. Liver cancer is predicted to be the sixth most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide. Rates of both incidence and mortality are 2- to 3-fold higher among men in most world regions. 63 Additionally, liver cancer is the third most commonly diagnosed cancer in men and the third leading cause of cancer-related deaths among both men and women in China. 71 The formation of liver cancer can be divided into several steps: cirrhotic nodules (CNs), low-grade dysplastic nodules (LGDNs), high-grade dysplastic nodules (HGDNs), early hepatocellular carcinoma (eHCC), and progressed HCC (pHCC). 74 Each stage was characterized by several piRNAs. For example, piR_LLi_24894 was only expressed in CNs; piR-LLi-30552 and hsa-piR-020498 were mainly expressed in HGDNs, eHCCs, and pHCCs; and hsa-piR-013306 was accumulated only in HCC. Downregulated piRNAs in early HCC-targeted genes such as tumor necrosis factor (TNF) receptor, phosphatidylinositol 3-kinase (PI3K)/AKT (serine/threonine kinase), WNT/β-catenin, growth arrest and DNA damage-inducible protein 45 (GADD45), adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), high mobility group box 1 (HMGB1), and phosphatase and tensin homolog (PTEN), which control cell cycle regulation, telomerase activity, protein ubiquitination, DNA methylation, and apoptosis. 74 Furthermore, piR-823 promoted the production of α-smooth muscle actin (α-SMA), and collagen type I alpha 1 (COL1a), as well as components of extracellular matrix, which finally resulted in cirrhosis. 75 piR-Hep1 may have a role in suppressing cell death by activating the signal transducer and activator of transcription 3 (Stat3)/Bcl-x L signaling pathway. 76 Actually, most HCCs develop from cirrhosis for which hepatitis B virus infection and alcohol abuse are the most common causes. It is important to explore potential relationships between piRNAs and these two kinds of cirrhosis, which are valuable for the prevention of cirrhosis transforming into HCC. Colorectal cancer (CRC) is among the five most commonly diagnosed cancers, and the incidence rate increases year by year. 71 piR-1245 was predominantly overexpressed in CRC tissues and regulated the expression of vital genes that control cellular processes such as cell death and survival, DNA replication and repair, and cell-cell communication. 77 These genes included activating transcription factor 3 (ATF3), BTG anti-proliferation factor 3 (BTG1), dual specificity phosphatase 1 (DUSP1), Fas cell surface death receptor (FAS), nuclear factor κB (NF-κB) inhibitor α (NFκBIα), uridine phosphorylase 1 (UPP1), sestrin 2 (SESN2), and tumor protein p53 inducible nuclear protein 1 (TP53INP1). 77 Consistently, the downregulation of piR-1245 induced cell apoptosis, while the overexpression of piR-1245 promoted cell proliferation. 77 It was found that upregulated piR-54265 promoted CRC cell proliferation and invasiveness through the STAT3 pathway, which resulted in the increase of metastasis-causing molecules, such as matrix metallopeptidase (MMP)2 and MMP9. Moreover, piR-54265 hindered chemotherapy, and patients with higher serum piR-54265 levels presented a significantly poorer response to chemotherapy. 78 piR-823 was reported to activate the expression of heat shock transcription factor 1 (HSF1) by promoting its phosphorylation at Ser326, and promote colorectal carcinogenesis. 79 piR-5937, piR-001311, piR-004153, piR-017723, piR-017724, piR-020365, piR-28876, piR-32105, piR-58099, and piR-59056 were also downregulated in CRC patients. 80 , 81 , 82 Patients with different primary sites of CRCs had different clinical manifestations, therapeutic responses, prognosis, and other characteristics. As many piRNAs are involved in the development of CRC, whether piRNAs present divergent expressions in left colon cancer, right colon cancer, or rectal cancer and whether they are associated with prognosis are worthy of further study. There are several types of kidney cancers, and most cases are clear cell renal cell cancers (ccRCCs). 83 piR-34536 and piR-51810 were downregulated in ccRCC tumor tissues, and their low expression was related to shorter disease-free survival (DFS) and overall survival (OS). piR-823 was significantly decreased in tumor tissues, and a low level was associated with a longer DFS. 84 , 85 Additionally, piR-32051, piR-39894, and piR-43607 were detected to be significantly upregulated in embryonic renal cancer and all ccRCC cell lines, accompanied by a shorter cancer-specific survival and a higher chance of tumor metastasis. 86 Immune checkpoint inhibitors are effective in the treatment of advanced kidney cancer, yet there appear to be no studies about piRNAs and kidney immunotherapy. 87 The relationship between piRNAs and immunotherapy can be further explored. It was reported that the mortality of bladder cancer ranked ninth in China in 2016, and the overall incidence of bladder cancer is increasing in men. 88 Up to date, there have been very few studies illustrating roles of piRNAs in bladder cancer, to which more attention could be paid. A total of 197 piRNAs were differentially expressed in bladder cancer cells compared with normal cells, among which piR-60152 was the most downregulated. 89 It was inferred that piR-60152 suppressed bladder cancer EJ cell apoptosis by targeting TNFSF4, which is a binding partner for the TNF superfamily member, OX40. However, it was not observed that piR-60152 promoted cell cycle progression, cell migration, and invasion. 89 Breast cancer is the most common cancer diagnosed among women. 90 Breast cancers can be classified into molecular subtypes according to different expressions of cellular markers, such as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2), Ki-67, and basal cytokeratin (CK). The six subtypes are luminal A, luminal B, luminal-HER2, HER2-enriched, basal-like, and TNP (triple-negative phenotype)-nonbasal. 91 It was found that piR-36712 may suppress breast cancer cell proliferation by inhibiting selenoprotein W1 (SEPW1) expression through a retroprocessed pseudogene of SEPW1, SEPW1P. SEPW1 inhibited the expression of p53 and p21 by inducing mRNA degradation, and the knockdown of SEPW1 resulted in G 1 cell cycle arrest. A lower piR-36712 level was related to a shorter progression-free survival (PFS) and a higher risk of axillary lymph node metastasis. 92 Moreover, piR-36712 appeared to enhance the effect of chemotherapeutic agents. 93 piR-021285 promoted invasion through methylating the 5′ UTR first exon of the pro-invasive Rho GTPase activating protein 11A (ARHGAP11A) gene. 94 In another study, upregulated piR36026 promoted cancer cell proliferation by inhibiting serpin family A member 1 (SERPINA1) and lecithin retinol acyltransferase (LRAT) gene expression, which are known for their tumor-suppressing function. 95 It was found that piR-932 was significantly overexpressed in breast cancer cells, which led to low latexin (a type of tumor-suppressing protein) expression. 96 High expression of piR-20365, piR-20485, and piR-4987 was correlated with metastasis. 97 In addition, piR-23662, piR-26526, piR-26527, piR-26528, piR-30293, piR-34377, piR-34736, piR-35407, piR-36318, and piR-36249 were significantly downregulated, while piR-651, piR-1282, piR-21131, piR-23672, piR-31106, piR-32745, and piR-36743 were upregulated in breast cancer cells. 98 , 99 The specific mechanisms behind those changes are still unknown. Although many piRNAs were dysregulated in breast cancers, these studies were not histologically classified. Many questions remain unknown, such as, whether there is any difference between piRNA expressions in different luminal subtypes; whether aberrant piRNA expressions are associated with the phenotypic impact of genomic alterations; and whether piRNAs influence responses to treatment in different subtypes of breast cancer. Ovarian cancer accounts for 2.5% of all female malignancies, but 5% of female cancer deaths. 100 In endometrioid ovarian cancer (ENOCa) samples, the upregulation of piR-52207 repressed four target genes, nudix (nucleoside diphosphate linked moiety X)-type motif 4 (NUDT4), methionine synthetase (MTR), eukaryotic translation initiation factor 2 subunit 3 (EIF2S3), and M-phase phosphoprotein 8 (MPHOSPH8), which are involved in 3-phosphoinositide biosynthesis, D-myo-inositol-5-phosphate biosynthesis, folate transformation, vascular endothelial growth factor (VEGF), and EIF2 signaling pathways, respectively. 101 In serous ovarian cancer (SOCa) samples, piR-52207 repressed the expression of actin-related protein 10 (ACTR10) and pleckstrin homology domain-containing family A member 5 (PLEKHA5), which are associated with the PI3K/Akt signaling pathway and dynactin’s pointed-end complex biogenesis, respectively. 101 Furthermore, piR-33733 was identified to target the lipoic acid synthase (LISA) gene, which is involved in the synthesis of lipoic acid, a well-known antioxidant that induces apoptosis. 101 It is suggested that mutations of breast cancer susceptibility genes (BRCA1 and BRCA2) account for almost 40% of ovarian cancer cases in women with a family history of the disease. 102 It is worth of learning whether piRNAs are related to BRCA mutation. If piRNAs do associate with BRCA mutations, they can serve as assistant factors for genomic evaluation and indicators for anti-BRCA drug resistance. Neurogliomas are the most common primary malignant brain tumors in adults. Neuroglioma can be divided into four grades (I–IV) based on malignant behavior. 103 piR-598 was identified as a tumor suppressor because its target genes were related to cell death, such as BAX, which regulates p53-mediated cell death. 104 It was found that piR- DQ593109 regulated blood-tumor barrier (BTB) permeability through the MEG3/miR-330-5p/RUNX3 axis. 105 Additionally, piR-39980 promoted tumor growth and facilitated drug resistance, yet piR-8041 suppressed glioma growth. 28 , 106 Neuroblastoma is a complex and therapeutically challenging malignant cancer in children. Seven piRNAs (piR-32287, piR32512, piR-36095, piR-38581, piR-52205, piR-52206, and piR-57816) were enriched in neuroblastoma cells, and their target genes included BRCA1, eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), histone deacetylase 2 (HDAC2), and peroxisome proliferator activated receptor alpha (PPARA), which are demonstrated to promote tumorigenesis. 107 Currently, gliomas are treated with surgery and lack effective drugs, as drugs can hardly penetrate the blood-brain barrier. If more piRNAs are detected in cerebral spinal fluid or embed with nanoparticle biointerfacing by platelet membrane cloaking, such as piR- DQ593109 , which could increase the permeability of the blood-brain barrier, the drug treatment to neurological cancer will be more efficient. Multiple myeloma (MM) is a commonly diagnosed hematological neoplasm and is a progressive and mostly incurable disease. 108 MM cells are characterized by permeating into end organs and growing into tumors with the help of endothelial cells (ECs), an important type of cells in tumor microenvironments. Increased piR-823 promoted EC proliferation by repressing the caspase-3/Bax signaling pathway, and it enhanced angiogenesis by inducing the secretion of VEGF and interleukin (IL)-6. 109 Additionally, piR-823 induced intercellular adhesion molecule 1 (ICAM-1) and C-X-C motif chemokine receptor 4 (CXCR4) secretion to promote EC invasion. 109 In another study, piRNA-823 induced DNA methylation via DNMTs (DNMT3A and DNMT3B) and suppressed a methylation-silenced tumor suppressor, p16INK4A. 110 Meanwhile, piRNA-4987 and piRNA-Hep1 were overexpressed in MM cells. 111 As piRNAs could result in the development of MM through DNA methylation, and histone-lysine N -methyltransferase EZH2 inhibitors have been applied for MM treatment, whether piRNAs interact with EZH2 in MM pathogenesis is worth further study. Head and neck squamous cell carcinoma (HNSCC) can occur in the oral cavity, oropharynx, hypopharynx, larynx, and nasopharynx. It is well known that virus infection and tobacco and alcohol consumption increase the risk of development of HNSCC. 112 In HPV16/18-associated HNSCC tissues, the loss of the piRNA FR140858 could potentially promote the expression of minichromosomal maintenance complex component 7 (Mcm7), which controls the initiation and elongation steps of eukaryotic DNA replication. 113 In smoking-related HNSCC tissues, four upregulated piRNAs (NONHSAT015828, NONHSAT081250, NONHSAT113708, and NONHSAT123636) are connected with advanced tumor stage and metastasis, and the downregulated NONHSAT067200 was related to longer patient survival. 114 Similarly, in alcohol-related HNSCC tissues, piR-34946, piR-38034, piR-43219, and piR-58510 were upregulated, and piR-70732 was downregulated. 115 More efforts should be made so that deeper knowledge between piRNAs and HNSCC can be identified and more therapeutic opportunities will be available. Osteosarcoma is the most common primary solid tumor of bone in childhood and adolescence and mainly occurs in the second decade of life. 116 Current therapeutic methods include surgical resection of all cancer tissues and systemic chemotherapy. The 5-year event-free survival rate of patients with localized osteosarcoma is approximately 70%, while that of patients with metastatic or recurrent osteosarcoma is less than 20%. 117 Few studies showed that piRNAs are associated with osteosarcoma. piR-39980 was reported to promote the apoptosis of fibrosarcoma cells and suppress cell proliferation by inhibiting ribonucleoside-diphosphate reductase subunit M2 expression, an enzyme indispensable in DNA synthesis and repair. 118 Because most sarcomas are not sensitive to chemotherapy, it is far-reaching to explore how piRNAs weaken the efficacy of systemic chemotherapy to sarcomas. Overall, intensive studies have indicated that aberrant piRNA expressions may lead to tumorigenesis. It has been proposed that cancer cells present certain hallmarks, such as sustaining proliferative signaling, evading growth suppressors, inducing angiogenesis, activating invasion and metastasis, and others. 119 Those aberrant piRNAs may influence the acquisition of hallmark capacities ( Figure 3 ). For example, piR-L-163 inhibited cell migration and invasion by maintaining the function of p-ERM and F-actin; 66 piR-823 enhanced angiogenesis by inducing the secretion of VEGF and IL-6. 109 piRNAs also are strongly correlated with tumor cell malignant phenotype, clinical stage, as well as clinical response, and they can be a predictive factor for prognosis. Most piRNAs are unique to individual cancer types, while several piRNAs are common to many cancer types, such as piR-823. piR-823 suppressed the development of gastric cancer and kidney cancer, yet it promoted the development of liver cancer, MM, and CRCs. Specific piRNAs in particular cancers can serve as particular biomarkers, and those common ones can be complementary to other tests. However, specific mechanisms by which piRNAs lead to tumorigenesis remain unknown, such as whether piRNAs influence those targets transcriptionally or post-transcriptionally. Moreover, the relationship between cancer sensitivity to chemoradiotherapy and piRNAs is also worth further discussion. In recent years, immunotherapy has been effective in NSCLC, malignant melanoma, and kidney cancer, whereas it has had almost no effect in gastric, colorectal, and breast cancer. Is there any difference in piRNA expression profiles between immunotherapy-sensitive and -insensitive cancers? More studies are needed to decipher underlying molecular functions and deepen our understanding of therapeutic methods of piRNAs in cancer. Figure 3 piRNAs Related with Cancer Hallmarks piRNAs in inner circle promote normal cells acquiring these cancer hallmark capabilities, and others in outer circle prevent normal cells from obtaining these cancer hallmarks. piRNAs Related with Cancer Hallmarks piRNAs in inner circle promote normal cells acquiring these cancer hallmark capabilities, and others in outer circle prevent normal cells from obtaining these cancer hallmarks. An evaluation published by World Health Organization in 2004 reported that one in every four couples in developing countries had been found to be affected by infertility. The report published at the end of 2012 showed that the overall burden of infertility remained similar. 120 Infertility patients are under great psychological pressure and can finally result in mental illness. Additionally, infertility will lead to low birth rate and aggravate the aging of society. Premature ovarian insufficiency, polycystic ovary syndrome, endometriosis, uterine fibroids, and endometrial polyps are the main causes of female infertility. Testicular and post-testicular deficiencies may lead to male infertility. 120 As a highly regulated developmental process, mammalian spermatogenesis consists of three steps in chronological order: mitosis, meiosis, and differentiation. This process results in a haploid gamete for sexual reproduction. Aberrant piRNA expression in testicular cells may cause spermatogenic failure. Five piRNAs ( DQ589977 , DQ591415 , DQ598918 , DQ601291 , and DQ601609 ) were found to be downregulated in spermatogenic failure samples, and they targeted PIWI2 and tudor domain-containing protein 1 (TDRD1) gene promoters. Their expression was negatively correlated with the methylation degree of the PIWI2 and TDRD1 gene promoters. 121 Other proposed target genes induced IL-16, kallikrein 1 (KLK1), G protein-coupled receptor 156 (GPR156), histone cluster 1 H2aa (HIST1H2AA), RAB24 (a member of the RAS oncogene family), sphingomyelin phosphodiesterase 3 (SMPD3), and dolichyl-phosphate mannosyltransferase polypeptide 1 (DPM1). 121 It was observed that the rs508485 variation in the 3′ UTR of the HIWI2 gene led to idiopathic nonobstructive azoospermia. The mutation also affected mRNA stability or altered the binding affinity of regulatory miRNAs. 122 , 123 hsa-piR-26399 was also expressed differently between sera of subfertile and control men. 124 piRNAs were originally found in mouse testis cells and were confirmed to be key players in germ cell maintenance in mouse and Drosophila models. Because piRNAs act as versatile regulators of gene expression and epigenetic events during spermatogenesis, they may provide a potent tool for the diagnosis of infertility. It will be useful to establish cell and stage-specific expression profiles of piRNAs during mammalian spermatogenesis using microarray or real-time RT-PCR. Furthermore, piRNAs could be promising targets of novel drugs for treatment. Oxidative stress is the most relevant biochemical cause of retinitis pigmentosa, and retinal pigment epithelium cells are very sensitive to oxidative stress. 125 piRNAs, such as piR-41220 and piR-46956, were globally downregulated in retinal pigment epithelium cells treated with oxidized low-density lipoprotein (oxLDL). Specially, they defended against oxidative stress by targeting genes linked with visual function. 126 piRNA expressions were profiled in cardiac progenitor cells (CPCs), such as cardiofibroblasts (CFs), cardiosphere-derived cells (CDCs), and cardiospheres. 127 Upregulated piRNAs (such as DQ591926 , DQ593270 , and DQ593595 ) targeted transposons throughout the human genome, including LINE retrotransposons, of which LINE-1 was most targeted. 127 Additionally, it was found that piRNAs stimulated cell viability through the AKT signaling pathway in the cardiac system. 128 In another study, many piRNAs were downregulated in heart failure patients, among which hsa-piR-020009 and hsa-piR-006426 were the top two. 129 piR-2106027 was increased significantly in myocardial infarction (MI) patients, except in troponin-I-negative MI patients. 130 It was found that approximately 100 piRNAs were dysregulated in Alzheimer’s disease (AD) patients, and most AD-associated piRNAs were correlated with genome-wide single-nucleotide polymorphisms (SNPs). 131 Roy et al. 132 identified that three piRNAs (piR-38240, piR-34393, and piR-40666) that participated in maintaining hemostasis in the nervous system. Their overexpression may lead to cell apoptosis and neurodegeneration and affect the trafficking pathway regulating Aβ levels. Their target genes were cytochrome C somatic (CYCS), karyopherin subunit α6 (KPNA6), and Ras-related protein Rab-11A (RAB11A), which induce apoptosis and maintain cellular homeostasis via oxidative stress-induced modulation of the Nrf2 (nuclear factor E2-related factor 2)-dependent antioxidant response and recycled endosomes and vesicular trafficking. piRNAs or piRNA-like molecules derived from the LINE and SINE genes were significantly downregulated and failed to silence LINE and SINE transposons in patients with Parkinson’s disease (PD). 133 So far, there have been few studies on other human diseases. The analysis of piRNA roles in these non-neoplastic diseases is far less thorough than that of tumors. Because it is difficult to obtain tissue samples, most studies are performed on model animal tissues. Actually, several studies suggested that piRNAs can be detected in body fluid. If relationships between piRNAs in body fluid and these diseases can be summarized, there will be more options for clinical diagnosis and treatment. Although many piRNAs are dysregulated in cancers and other diseases, most mechanisms behind these changes are elusive. Those piRNAs with potential mechanisms are listed in Table 1 and are shown in Figure 4 . Table 1 piRNAs in Different Diseases and Potential Mechanisms Behind Them Disease piRNA Changes Mechanism Role Reference Lung cancer piR-L-163 downregulated bind to 3′ UTR of mTOR mRNA suppressor 66 piR-55490 maintain the function of phospho-ezrin-radaxin-moesin (p-ERM) 67 Liver cancer piR-823 upregulated increase the production of α-SMA and COL1a1 promotor 75 piR-Hep1 activate the Stat3/Bcl-x L signaling pathway 76 Colorectal cancer piR-1245 upregulated regulate the expression of ATF3, BTG1, DUSP1, FAS, NFKBIA, UPP1, SESN2, TP53INP1, and MDX1 promotor 77 piR-54265 regulate the STAT3 signaling pathway 78 piR-823 activate the expression of HSF1 79 Bladder cancer piR-60152 downregulated regulate the expression of TNFSF4 suppressor 89 Breast cancer piR-021285 upregulated methylate 5′ UTR/first exon of the proinvasive ARHGAP11A gene promotor 94 piR-36026 suppress the expression of SERPINA1 and LRAT 95 piR-932 suppress the expression of Latexin 96 piR-36712 downregulated suppress the expression of SEPW1 suppressor 92 Ovarian cancer piR-52207 upregulated regulate the expression of NUDT4, MTR, EIF2S3, MPHOSPH8, ACTR10, and PLEKHA5 promotor 101 piR-33733 regulate the expression of LIAS promotor 101 Neurological cancer piR-32287 upregulated regulate the expression of four transfactors: BRCA1, EIF2AK2, HDAC2, and PPARA promotor 107 piR-32512 piR-36095 piR-38581 piR-52205 piR-52206 piR-57816 DQ593109 regulate the blood-tumor barrier (BTB) permeability promotor 106 piR-598 downregulated regulate the expression of genes related with cell death, such as BAX suppressor 105 MM piR-823 upregulated suppress the caspase-3/Bax signaling pathway promotor 109 , 110 induce the secretion of VEGF, IL-6, ICAM-1, and CXCR4 secretion induce DNA methylation suppress the methylation of p16INK4A HNSCC FR140858 downregulated regulate the expression of the minichromosomal maintenance complex component 7 (MCM7) suppressor 113 Osteosarcoma piR-39980 downregulated suppress the expression ribonucleoside-diphosphate reductase subunit M2 suppressor 118 Male infertility DQ589977 downregulated enhance the methylation level of several genes’ promoters, including PIWI2 and TDRD1, IL16, KLK1, GPR156, HIST1H2AA, SMPD3, and DPM1 suppressor 121 DQ591415 DQ598918 DQ601291 DQ601609 Retinitis pigmentosa piR-41220 downregulated regulate the expression of genes vital in the pathway involved in visual function suppressor 126 piR-46956 Alzheimer’s disease piR-34393 upregulated suppress the expression of CYCS, KPNA6, and RAB11A promotor 132 piR-38240 piR-40666 Parkinson’s disease piRNAs derived from LINE and SINE genes downregulated silence LINE and SINE transposons suppressor 133 Figure 4 Aberrant piRNAs with Potential Mechanisms in Different Diseases Various piRNAs are dysregulated in human diseases. piRNAs in red are upregulated and those in black are downregulated in different diseases. piRNAs in Different Diseases and Potential Mechanisms Behind Them Aberrant piRNAs with Potential Mechanisms in Different Diseases Various piRNAs are dysregulated in human diseases. piRNAs in red are upregulated and those in black are downregulated in different diseases. Owing to the emergence of next-generation sequencing technologies, the expression of piRNAs can be readily observed. The production of piRNAs is complicated, and many vital proteins are involved. piRNAs regulate gene expression in a transcriptional or post-transcriptional manner and closely influence human health. A number of studies have reported dysregulated expressions of piRNAs in samples of different diseases and proposed their potential mechanisms. Most piRNAs are confined to particular disease models, while some are likely incidental, such as piR-651 and piR-823, which can be observed in different cancers. They could be either suppressors or promotors in different cancers because of the heterogeneity of cancers. Actually, the exact mechanism by which piRNAs regulate human health are still unresolved. Many questions remain unknown, such as whether the aberrant expression of piRNAs is the real cause of these diseases or just a byproduct of other molecular activities. What are the ideal thresholds for distinguishing healthy individuals from patients? Such ideas still face challenges, which will need to be addressed before piRNA-related therapies can transition from bench to bed. Further studies and multicenter clinical trials should be performed in the future to fully understand the basic biological mechanisms of piRNAs and their disruption.

X.W., Y.P., and F.Y. drafted the manuscript. J.Z., M.X., F.Y., T.U., P.M., and W.L. discussed and revised the manuscript. Y.S., P.M., and W.L. designed the research and drafted the manuscript. All authors read and approved the final manuscript.

The authors declare no competing interests.