PIWI-interacting RNAs, called piRNAs, are single-stranded small RNAs with a size of 24–31 nucleotides (Iwasaki et al., 2015). They are bound by a special class of proteins, namely, PIWI - an abbreviation of P-element Induced Wimpy testis in Drosophila. (Lin and Spradling, 1997; Sohn and Oh, 2023). The piwi gene was first identified to be involved in germline stem cell division in Drosophila gonads, in both ovaries and testis (Lin and Spradling, 1997). Subsequently, piRNAs were discovered in mouse testis in 2006 and were defined as small non-coding RNAs that specifically interact with PIWI proteins (Aravin et al., 2006; Girard et al., 2006). For distinction, the name Piwi refers to one of the specific proteins in Drosophila, while PIWI refers to the protein subfamily (Ross et al., 2014). Additionally, PIWIs belong to the Argonaute proteins family, which encompasses another branch containing Ago proteins that bind to a different class of small RNAs, specifically, microRNAs (miRNAs) and small interfering RNAs (siRNAs) (Wang X. et al., 2023) (Figure 1A). Like the fellow members of Argonaute protein family, PIWIs have a bilobal structure, with lobes at both the N- and C-terminus binding to the 5′ and 3′ ends of piRNAs, like a pocket, protecting the piRNA (Matsumoto et al., 2016; Dayeh et al., 2018). The PIWI proteins are well conserved among different classes of animal species (Kim et al., 2018; Huang and Wong, 2021) (Table 1). The PIWI proteins consist of four main domains, namely, PIWI-Argonaute-Zwille (PAZ), N-terminal (N), PIWI, and middle (MID). The PAZ domain recognizes and binds to the 2′-O-methylated 3′end of piRNA, while the MID domain binds to the 5′uridine end, and the PIWI domain acts as an RNase H endonuclease to cleave the target transcript captured by piRNA through complementary base pairing (Litwin et al., 2017) (Figure 1B).

piRNAs are generated from specific gene loci called piRNA clusters (Parhad and Theurkauf, 2019). In the cell nucleus, piRNA clusters are transcribed by RNA Polymerase II (RNA Pol II), into piRNA precursors, then these transcripts are exported to the cytoplasm through nuclear pores and are processed by enzymes such as Zucchini to form smaller piRNA fragments called piRNA intermediates (Huang and Wong, 2021; Ramat and Simonelig, 2021; Allikka Parambil et al., 2024). Most of these fragments are bound by the homologous protein PIWIL2, enter the ping-pong cycle (a process described in detail later on, in this review), or are methylated at the 3′end by the methyltransferase Hen1, forming a mature piRNA complex with the PIWIL1 homolog (Gainetdinov et al., 2018; Huang and Wong, 2021, p. 20121) (Figure 1C). Here, it is worth mentioning that, unlike the other two well-known small RNAs (miRNA and siRNA), the precursor of piRNA is a single-stranded RNA (ssRNA) rather than double-stranded RNA (dsRNA), and thus the production of mature piRNAs is independent of the enzyme Dicer, an RNase III endoribonuclease (Iwasaki et al., 2015).

In humans, transposable elements (TEs), also called “jumping genes”, and their derivatives account for 44%–48% of the genome (Autio et al., 2021; Chénais, 2022). TEs are inserted into protein-coding genes in numerous ways, causing genetic mutations and destabilizing the gene structure (Parhad and Theurkauf, 2019). Briefly, the mature piRNA complex which is exported to the nucleus from the cytoplasm, binds to complementary sites in nascent target TE transcripts and silences their expression, thus protecting the cell (Parhad and Theurkauf, 2019; Ramat and Simonelig, 2021). Additionally, piRNA complexes can silence TE transcripts in the cytoplasm and can activate, protect, and localize a wider range of other messenger RNAs (mRNAs) (Ramat and Simonelig, 2021). Most piRNAs and PIWIs gather in the “Nuage” region of the perinuclear cytoplasm, where they undergo a ping-pong cycle (Pek et al., 2012; Ge et al., 2019). The ping-pong cycle is an essential mechanism widely observed in flies, fish, and mammals, playing a critical role in piRNA amplification and the silencing of transposon transcripts (Czech and Hannon, 2016; Huang and Wong, 2021). Initially, researchers found that, in Drosophila germ cells, the 10 nucleotides at the 5′end of piRNAs that bind to Aub and Ago3, respectively, showed strong complementarity, indicating that these two classes of piRNAs are likely to interact with each other, which was later defined as the “ping-pong signature” (Brennecke et al., 2007; Li et al., 2009; Rojas-Ríos and Simonelig, 2018). For instance, during the ping-pong cycle in Drosophila germ cells, the piRNA-Aub complex binds and cleaves the target TE transcript (Rojas-Ríos and Simonelig, 2018). The cleaved fragment of the transcript with the exposed 3′ end is loaded into the Ago3 protein with the help of the Tudor-domain protein Krimper (Czech and Hannon, 2016). The Ago3 protein, guided by the RNA fragment, forms a complex with the piRNA precursor. Subsequently, the precursor is cleaved, and the fragment with the exposed 3′ end is loaded into the Aub protein, forming a new piRNA-Aub complex, aided by the Trimmer proteins (Czech and Hannon, 2016) (Figure 1D). The ping-pong cycle is conserved in other animal species, such as mouse, where the counterparts of Aub and Ago3 are MILI and MIWI2, respectively (Ernst et al., 2017; Huang and Wong, 2021), and the auxiliary proteins involved in this cycle are yet to be confirmed.

Although initial studies in Drosophila led the researchers to believe that the piRNA pathway was limited to the germ line (Malone et al., 2009), recent studies have shown that piRNAs also function in mammalian and non-mammalian somatic tissues and stem cells. Further studies have discovered that depending on the context, for instance, in cancer cells, in addition to the piRNA-PIWI functional complex, piRNA and PIWIs can work independently of each other to control the expression of specific genes (Zhang et al., 2023).

In the current review, we explored the roles of piRNAs and PIWI proteins in stem cells other than germline, including Adult Stem Cells (ASCs), Embryonic Stem Cells (ESCs), Induced Pluripotent Stem Cells (iPSCs), etc. Additionally, we discussed the emerging roles of piRNA and PIWI proteins, both dependent and independent of each other, in regulating the expression of TE transcripts and mRNAs along with their translation in somatic cells and the development of numerous human diseases.

In planarian neoblasts, SMEDWI-1 and SMEDWI-3 were reported to be crucial for the localization of germinal histone H4 (gH4) mRNA to chromatoid bodies (CB), a type of ribonucleoprotein (RNP) granules located in the perinuclear cytoplasm, and these PIWIs are indispensable for the formation of CBs(Rouhana et al., 2014). Also, recent studies have found that the piRNA- SMEDWI-1 complex binds to and regulates non-polyadenylated mRNAs, including stem-loop transcripts of all five histones during the S phase of the cell cycle (Allikka Parambil et al., 2024). Interestingly, during the differentiation of neoblasts, both CB and neoblast histone transcripts are degraded. More than 50 piRNAs which match the coding sequences of the transcripts of five histones respectfully have been identified. These piRNAs are likely to be the derivatives of a cleaved histone mRNA, and may therefore serve as potential candidates for guiding PIWI cleavage (Rouhana et al., 2014). Yet, it remains to be elucidated which specific PIWI proteins are involved in the degradation of histone transcripts. Additional work has demonstrated that SMEDWI-3 is involved in the degradation of a large number of mRNAs, including the transcripts of Traf-6 and Npk1-like genes, in neoblasts. Traf-6 is closely linked to the differentiation and proliferation of planarian stem cells, while the Npk1-like gene has not been studied in animals yet. At the same time, for other mRNAs, such as the transcripts of Y2R and TEX2, the piRNA-SMEDWI-3 complex binds to them but does not yield any change in their abundance, suggesting a translational control of these transcripts (Figure 2C; Table 2) or the RNA stabilization by the bound PIWIs, to maintain their function in the regulation of cell cycle (Kim et al., 2019). Likewise, in human embryonic stem cell (hESC) lines (H1, H9), during the differentiation of these pluripotent cells, the expression of HILI mRNA was significantly downregulated, while that of HIWI2 was upregulated (Liu et al., 2017; La Greca et al., 2020). In addition, small RNA-seq data showed that multiple piRNAs, including piR-4403262 and piR-4424378, may be involved in the regulation of numerous mRNAs in cardiomyocytes, including transcripts of MALAT1, TTN, and PLN (La Greca et al., 2020). Additionally, studies have shown that piRNAs promote the differentiation of cardiac progenitor cells and the regeneration of cardiac cells (Rajan et al., 2014). There are hundreds of upregulated or downregulated piRNAs involved in this process, including piR-30444, piR-46538 as over-expressed, and piR-30136, piR-30368 down-expressed (Vella et al., 2016). These upregulated piRNAs silence LINE-1 and promote the differentiation of cardiac progenitor cells and the regeneration of cardiac cells by activating the AKT signalling pathway (Vella et al., 2016). Furthermore, it is evident that in the hippocampus of newborn mice, the adult neural progenitor cells (aNPCs) are rich in PIWIL2/MILI expression, and more than 570,000 putative piRNAs were identified from small RNA libraries, including one homologous to the human piR-61648. The reads of these putative piRNAs were significantly reduced, as aNPCs differentiated into neurons (Gasperini et al., 2020; Gasperini et al., 2023). Surprisingly, through in silico analysis, researchers discovered that repeats in 5S rRNA and tRNAs are important targets of piRNAs in aNPCs, in addition to the known targets such as mRNAs encoding ribosomal proteins, and transcripts including SINEB1 (Gasperini et al., 2020) (Figure 2D). By way of regulation, these piRNAs are anticipated to inhibit ribosome assembly, thereby reducing the rate of protein synthesis in aNPCs, and maintaining cell stemness (Gasperini et al., 2020). Moreover, the lack of MILI and piRNAs results in damage to neurogenesis, and forms reactive glia, which are commonly produced after damage to the nervous system, with its appearance often accompanied by inflammation and aging (Gasperini et al., 2023) (Table 2). Similarly, in the early Drosophila embryo, Aub was found to directly interact with the protein Wispy, a non-canonical poly(A) polymerase that polyadenylates the long poly(A) tails of maternal mRNAs related to reproduction in the early embryo, thereby protecting these mRNAs(Dufourt et al., 2017). The piRNA-Aub-dependent stabilization of mRNAs has already been shown to be necessary for protecting nanos in the early Drosophila embryo, which is considered as a germline stemness marker (Ramat and Simonelig, 2021; Rinkevich et al., 2022). Furthermore, maternal piRNA- Aub directly recruits the poly(A)-binding protein (PABP) that protects mRNA through binding to the poly(A) tail (Ramat and Simonelig, 2021). Here, Aub interacts with eIF3d, a subunit of eukaryotic initiation factor 3 complex (eIF3), initiating the translation of the target mRNAs including Nanos (Figure 3A). Nanos is closely related to the development of the germline, thereby bypassing the classic but rate-limiting protein synthesis mechanism, which is activated by eukaryotic initiation factor 4E (eIF4E) binding to the mRNA 5′cap (Ramat et al., 2020; Ramat and Simonelig, 2021).

Previous studies on Drosophila intestinal stem cells (ISCs) show that JAK/STAT signalling can activate ISC proliferation (Ayyaz and Jasper, 2013). Under acute stress, the JAK/STAT pathway not only induces ISC proliferation but also induces the increased expression of Drosophila Piwi protein, thereby silencing retrotransposons and ensuring gene stability (Sousa-Victor et al., 2017; Rojas-Ríos and Simonelig, 2018). Knocking down piwi led to the accumulation of multiple TE transcripts, such as Ty3 retrotransposon, mdg1 and hetA (Figure 3B; Table 2), while in contrast, the loss of Aub and Ago3, which belong to the same PIWI protein family, did not affect TE silencing (Sousa-Victor et al., 2017). It is still uncertain whether the functions of these PIWI proteins depend on piRNAs(Sousa-Victor et al., 2017; Tang et al., 2023). It is worth mentioning that recent small RNA-seq data from the Drosophila intestine also failed to provide evidence of piRNAs’ existence (Siudeja et al., 2021). Thus, provides for the hypothesis that PIWI proteins may act alone in Drosophila ISCs, independent of piRNA sequences. Moreover, the STAT signalling-dependent Piwi protein activation was detected in enteroblasts, the ISC daughter cells with pluripotency, further strengthening the case for piRNA-independent PIWI function in ISCs(Sousa-Victor et al., 2017). Likewise, under disease conditions, namely in glioblastoma (GBM), PIWIL1 plays a significant role by promoting the self-renewal of glioma stem cells (GSCs). Overexpression of PIWIL1 has been consistently observed in these GSCs, contributing to their maintenance and tumorigenic potential (Huang et al., 2021). However, knockdown experiments targeting PIWIL1 have revealed a contrasting effect. The knockdown of PIWIL1 triggers an increase in tumor suppressors BTG2 and FBXW7, leading to the destabilization of the oncogene c-Myc, which in turn diminishes the proliferation and self-renewal capabilities of GSCs (Figure 4A). This effect is mediated by the enhanced stability and expression of BTG2 and FBXW7 mRNAs, which are crucial for inhibiting cell cycle progression and facilitating the degradation of oncogenic proteins like c-Myc. Consequently, reducing key stem cell factors such as Olig2 and Nestin hampers the maintenance of GSCs. These findings suggest that PIWIL1 contributes to glioblastoma growth through its regulatory effects on mRNAs. While these observations provide insights into PIWIL1’s function in mRNA regulation, further studies are needed to clarify whether these effects occur independently of piRNAs or involve indirect mechanisms (Huang et al., 2021). Also, in studying the expression of piRNA pathway genes in cancer, Genzor et al. (2019) investigated whether these genes could restore functional piRNA silencing in non-germline cells. They found that while genes like PIWIL1, PIWIL2, PLD6 (Zucchini), and DDX4 (VASA) were expressed in some cancer cell lines, their expression alone was not enough to form functional piRNA-PIWI silencing complexes (piRISCs). For example, in COLO205 cells, which have high levels of PIWIL1 but lack DDX4 and other key components, small RNAs bound to PIWIL1 did not show the features of true piRNAs. Instead, they resembled other RNA fragments, such as microRNAs, and did not exhibit piRNA activity (Genzor et al., 2019). Additionally, knocking down or deleting PIWIL1 did not impact transposon transcript levels, indicating that the piRNA pathway was not functional in this setting. These findings highlight the importance of careful analysis to confirm true piRNA activity and avoid misinterpreting aberrant gene expression in cancer.

Bone marrow stromal cells (BMSCs) include multipotent cells such as osteochondral and adipocyte progenitors (Gao et al., 2022). Through studies on BMSCs in rats and other mammals, researchers found that piRNAs are crucial for osteoblast differentiation. For instance, the piRNA, piR-63049 plays a significant role in maintaining the stemness of BMSCs(Chen et al., 2021). Meanwhile, other piRNAs, such as piR-016735, were found in exosomes derived from BMSCs, and the piRNA overexpression inhibits the differentiation of BMSCs into osteoblasts (Wang et al., 2020; Chen et al., 2021). Correspondingly, studies on human BMSCs have revealed that the expression of another piRNA, piR-36741, promotes BMSC differentiation into osteoblasts (Liu et al., 2021; Mishra et al., 2023) (Figure 4B; Table 2). Another study on human dental apical papilla stem cells (SCAP) and bone marrow mesenchymal stem cells (BMMSCs) showed that piRNAs such as piR-007832 and piR-015026 are highly expressed in BMMSCs, and the target genes of these piRNAs are enriched in pathways such as p53 signalling and the cascade associated with Epstein-Barr virus infection (Wang et al., 2020), likely involved in osteogenic differentiation (Jiang et al., 2019). Whereas, the piRNAs reported in SCAP are associated with MAPK signalling, citrate cycle, and Ras signalling pathways (Wang et al., 2020), among which, the MAPK signalling was closely related to the odonto-/osteogenic differentiation of SCAP (Wang et al., 2018). Interestingly, BMMSCs are believed to communicate with hematopoietic stem cells (HSCs) via extracellular vesicles (EVs) (Batsali et al., 2020) (Table 2). By sequencing small RNAs in human EVs, De Luca, L. et al. found at least two piRNAs, namely piR-020814 and piR-002732 affected the expression of numerous proteins including MPO, SLC2A5, SLAMF8, and CYP1B1(De Luca et al., 2016). These proteins have various functions such as antibacterial activity, facilitation of fructose transport, and are involved in adaptive and innate immune responses, and participate in the oxidative metabolism of xenobiotics. Likewise, induced pluripotent stem (iPS) cells from mice express at least one piRNA, piR-mmu-64162, which regulates the expression of proteins, such as Ccnd1 and Hipk1, that promote cellular reprogramming and resist cell senescence (Zhu et al., 2019). However, previous triple knockout of PIWI proteins (MILI, MIWI, MIWI2) in mouse iPS cells showed that the lack of PIWI proteins did not affect the expansion and gene expression of iPSCs(Cheng et al., 2014), raising the possibility that piRNAs function independently of PIWIs in iPSCs, which requires further studies to strengthen the argument. Similarly, murine Neural Stem Cells (NSCs) were reported to attack and neutralize viruses by releasing exosomes/microvesicles (Ex/Mv) that are enriched in approximately 150 piRNAs, including piR-mmu-141534 and piR-mmu-429488, with potential antiviral capabilities, targeting the genomes of HIV, lentiviruses and SARS-CoV-2 (Yu et al., 2020) (Figure 4C; Table 2). Furthermore, the authors of the study detected PIWIL2/MILI in the NSC Ex/Mv, however, the specific roles of this protein and how or whether it regulates specific piRNA function in attributing antiviral functions of those NSC Ex/Mv remains to be determined (Yu et al., 2020).

PIWI proteins form a complex with piRNAs, namely piRNA-induced silencing complex (piRISC), to mediate both transcriptional and post-transcriptional gene silencing (Loubalova et al., 2023). Not all PIWI proteins operate similarly or at the same level in the regulation of gene expression. While MILI/PIWIL2 and MIWI/PIWIL1 are involved at the post-transcriptional level, MIWI2/PIWIL4 primarily engages in transcriptional gene silencing (Loubalova et al., 2023). Here, we will specifically discuss the role of the piRNA-PIWI complexes in the regulation of mRNAs and retrotransposon silencing at the post-transcriptional level.

Cancer remains one of the most prevalent and challenging health concerns globally, despite extensive research efforts over the decades (Sung et al., 2021). While significant progress has been made in understanding cancer biology, there are still many gaps in our knowledge that warrant further exploration. Amidst the heterogeneity of cancer, emerging evidence has highlighted the involvement of piRNAs and their associated PIWI proteins in the development and progression of various cancer types, including those affecting the stomach, lungs, pancreas, and liver (Liu et al., 2019). The multifaceted roles of piRNAs and PIWI proteins in cancer underscore the need for a comprehensive understanding of their mechanisms and potential as biomarkers and targets (Ameli Mojarad et al., 2022). Recent findings indicate that PIWI protein involvement in cancer can either depend on piRNA or function independently. Similarly, piRNAs can either partner with PIWI proteins or act autonomously within cancer pathways (Zhang et al., 2023). In 2020, over one million cases of stomach-related cancer were recorded, resulting in over 700 thousand reported fatalities (Sung et al., 2021). In the same year, Haifan et al. explored the independent function of PIWIL1/HIWI in gastric cancer (Lin et al., 2020), and demonstrated that PIWIL1 recruits the NMD (nonsense-mediated mRNA decay) machinery, a cellular pathway that degrades aberrant or selective mRNAs to prevent harmful protein production, involving components like SMG1, UPF2, and UPF1. This recruitment leads to the downregulation of tumor suppressor mRNAs, such as VCL, LAMC3, FLNA, MYO18B, SRCIN1, and TPM2, which are associated with cell adhesion and migration, facilitating gastric cancer progression. (Figure 7A; Table 3). However, PIWIL1 role in the upregulation of oncogene mRNAs such as CCND3, ORC6, PCNA, CDC25A, and MCM10 remains unexplored (Lin et al., 2020; Ameli Mojarad et al., 2022). Additionally, piR-1245 is in high concentration in gastric juice from gastric cancer patients, compared to healthy individuals, suggesting its potential as a biomarker (Zhou et al., 2020; Ameli Mojarad et al., 2022). Likewise, the upregulation of PIWIL1 was reported in Hepatocellular Carcinoma (HCC) (Xie et al., 2015; Xu et al., 2020). In 2023, Wu et al. revealed that PIWIL1’s interaction with piR-017724 was implicated in HCC development (Figure 7B; Table 3) (Wu et al., 2023). Interestingly, a paradoxical function of PIWIL1/HIWI has surfaced, where the upregulation of piR-017061, which binds with PIWIL1, inhibited the mRNA translation of Ephrin A5 (EFNA5), involved in regulating cell growth and proliferation in pancreatic cancer. Elevated levels of EFNA5 markedly enhance the growth of pancreatic cancer cells, whereas reducing its expression leads to diminished cell growth. These findings suggest that EFNA5 functions as an oncogene in the context of pancreatic cancer. Conversely, the downregulation of piR-017061 was shown to upregulate EFNA5, an oncogene in pancreatic cancer progression (Xie et al., 2021) (Figure 7C; Table 3). Thus, demonstrating that HIWI exhibits a multifaceted role, exerting a detrimental influence on cancer progression depending on the cell type, while concurrently fostering tissue health, a duality that underscores its complexity and importance in the regulation of cell development. On the other hand, PIWIL2/HILI has emerged as a pivotal player in lung cancer, the second most prevalent cancer worldwide, according to the 2020 statistics (Sung et al., 2021; Jian et al., 2022). PIWIL2 drives the progression of non-small cell lung cancer through the induction of CDK2 and Cyclin A expression, thereby inhibiting apoptosis, and G2/M cycle arrest (Qu et al., 2015) (Figure 7D; Table 3). Benzo(a)pyrene (BaP), classified as a carcinogen is linked to various forms of lung injuries, particularly lung cancer, through exposure from sources such as cigarette smoke and pollution from fine particles in the air (measured as PM_2.5). Recent epidemiological studies revealed that PM_2.5 is involved in the suppression of three piRNAs (piR-004153, piR-020326 and piR-020388), which in turn promoted lung injuries associated with BaP exposure (Chen et al., 2019; Lei et al., 2024). Recent studies have emphasized the importance of rigorous annotation when studying piRNAs in somatic cells, as misannotations can lead to misleading conclusions. Tosar et al. (2021) pointed out that SNORD57, a small nucleolar RNA, is often misidentified as piR-54265, previously proposed as a biomarker for colorectal cancer. Their findings stress that SNORD57, rather than piR-54265, is a more reliable biomarker, highlighting the need for careful evaluation of piRNA data in somatic research (Tosar et al., 2021). This underscores the complexity of distinguishing bona fide piRNAs from other small RNAs in somatic tissues and the importance of robust in silico analysis for accurate identification. Such methodological precision is critical for understanding the diverse roles of piRNAs in diseases, as their functions extend beyond canonical germline processes to influence mRNA regulation and transposon silencing in somatic contexts.

Cardiovascular disease (CVD) causes around 18.6 million deaths annually worldwide and encompasses myocardial issues such as hypertrophy, infarction, and atherosclerosis, aggravated by risk factors including hyperlipidemia, hypertension, and obesity, particularly prevalent in type 2 diabetes (Liu et al., 2019). In recent years, there has been growing interest in exploring the role of piRNAs concerning these cardiovascular conditions. Research studies thus far mainly focused on identifying piRNAs as biomarkers for these diseases. For instance, recently, a novel aortic valve calcification-associated piRNA has been identified, called AVCAPIR which increases valvular calcification and promotes the progression of calcific aortic valve disease (CAVD) (Han et al., 2024). Additionally, a highly upregulated piRNA, namely piRNA-000691, known as CFRPi, is reported to be abundant in the heart and is expressed specifically in cardiac fibroblasts, promoting cardiac fibrosis under chronic pressure overload, thereby contributing to heart failure (Chen et al., 2024). Moreover, the piRNAs, namely, piR-hsa-9010, piR-hsa-28646, and piR-hsa-23619 are significantly upregulated in acute myocardial infarction (AMI) patients, with piR-hsa-28646 and piR-hsa-23619 showing higher expression in Primary Human Umbilical Vein Endothelial Cells (HUVEC) (Figure 8A), with involvement in key signalling pathways such as Wnt and TNF signalling. Wnt pathway is activated in endothelial cells of the infarct area and plays a role in angiogenesis following myocardial infarction. The TNF pathway is crucial in mediating the inflammatory response and affecting vascular function after myocardial infarction (Huang et al., 2023). Despite these intriguing findings, there is a lack of studies on the expression and function of PIWI proteins in mammalian model systems including mice, which is crucial for understanding the implications of the piRNA pathway in cardiac development. However, A recent study has highlighted potential interactions between PIWI proteins, including PIWIL4 and PIWIL2, with a newly identified piRNA known as Heart Necroptosis-Associated piRNA (HNEAP). In 2023, Wang et al. demonstrated that HNEAP plays a crucial role in regulating cardiomyocyte necroptosis by influencing the 5-methylcytosine (m⁵C) modification of the mRNA of Activating Transcription Factor 7 (Atf7). Specifically, HNEAP inhibits the DNA (Cytosine-5)-Methyltransferase 1 (DNMT1)-mediated methylation of Atf7 mRNA. This inhibition leads to increased ATF7 expression, which in turn suppresses CHMP2A, a key anti-necroptotic factor, highlighting HNEAP’s significance in cardiac pathophysiology (Wang K. et al., 2023) (Figure 8B; Table 3). Notably, HNEAP exhibits specific enrichment in heart tissue and cardiomyocytes, suggesting potential interactions with PIWI proteins, namely PIWIL4 and PIWIL2 (Wang K. et al., 2023). These findings illuminate novel mechanisms of piRNA-PIWI-mediated post-transcriptional gene regulation and propose HNEAP as a promising therapeutic target for addressing ischemic heart injury (Wang K. et al., 2023).

piRNAs and PIWI proteins are implicated in different neurodegenerative diseases, including Parkinson’s, Alzheimer’s, and Amyotrophic lateral sclerosis (ALS) (Sato et al., 2023). In ALS, there is a loss of motor neurons in the brain and spinal cord, leading to muscle weakness, paralysis, and eventually death (Hardiman et al., 2017). In 2022, the study conducted by Abdelhamid et al. found that, in sporadic ALS samples, three piRNAs (hsa-piR-000578, hsa-piR-020871, and hsa-piR-022184) were upregulated, while two (hsa-piR-009294 and hsa-piR-016735) were downregulated (Abdelhamid et al., 2022). They also observed dysregulation of PIWI proteins: PIWIL4 was downregulated, while PIWIL1 was upregulated, and PIWIL2 and PIWIL3 were not detectable in the samples (Figure 8A; Table 3) (Abdelhamid et al., 2022). These findings suggest the potential of piRNAs as biomarkers for treatment options, where, additional research is required to substantiate this claim. Similarly, Alzheimer’s is the most common of all the other neurodegenerative diseases followed by Parkinson’s, and is characterized by memory loss and cognitive decline (Masters et al., 2015; Bloem et al., 2021). In Alzheimer’s disease (AD), dysregulated piRNAs have been identified as potential biomarkers and therapeutic targets. Jain et al. (2019) found altered levels of piR-019324, piR-019949, and piR-020364 in cerebrospinal fluid (CSF) exosomes of AD patients. Roy et al. (2017) observed the upregulation of piRNAs in AD brains, correlating with AD risk single nucleotide polymorphisms (SNPs). Additionally, increased expression of HIWI/PIWIL1 was noted in AD hippocampal neurons, possibly linked to tau pathology-induced chromatin changes. These findings highlight the role of piRNAs and PIWI proteins in AD pathogenesis (Roy et al., 2017; Jain et al., 2019; Sato et al., 2023). In contrast, the role of piRNAs and PIWI proteins in Parkinson’s disease (PD) remains largely unexplored, with limited findings reported in the literature. According to the review by Sato et al. (2023), recent studies, such as those by Zhang and Wong (2022), have begun to shed light on this area. They explored the role of piRNAs in PD where they identified over 200 piRNAs in the prefrontal cortex and over 500 piRNAs in the amygdala, a region often affected by Lewy bodies (Zhang and Wong, 2022). Among these, more than 15 piRNAs in the prefrontal cortex, and over 50 in the amygdala showed differential expression in PD patients. Notably, those 50 piRNAs differentially expressed in the amygdala were predicted to target over 20 protein-coding genes, including those critical for mitochondrial function, such as MT-CO1 and MT-CO3, involved in the onset of PD (Zhang and Wong, 2022; Sato et al., 2023).

The roles of piRNAs and PIWI proteins have been noted in a spectrum of respiratory tract diseases, including pneumonia, asthma, tuberculosis, pulmonary arterial hypertension, and interstitial lung disease (ILD) (Yao et al., 2023). In pneumonia, PIWIL2 and PIWIL4 are upregulated in response to endoplasmic reticulum (ER) stress, and MIWI2 modulates immune responses in multiciliated airway epithelial cells in mice (Figure 8A) (Hogan et al., 2014; Wasserman et al., 2017; Gebert et al., 2018). PIWIL2 in ILD, interacts with heat shock protein 90 (Hsp90) to modulate TGF-β signalling and fibrosis (Zou et al., 2020). In tuberculosis (TB), the increased activity of PIWIL2 and PIWIL4, along with the significant presence of piRNAs such as piRNA-1007467, piR-hsa-1344, and piR-hsa-1944, highlights the complex involvement of the piRNA-PIWI axis in TB development. These piRNAs are implicated in influencing disease progression and modulating the host immune response (Zhang et al., 2019). We believe future studies on the mechanisms underlying the changes in PIWI expression and the targets, namely protein-coding genes might shed light on the pivotal information necessary for developing accurate and effective piRNA-PIWI-based treatment strategies for ILD and TB.