Approximately 90% of piRNAs originate from specific genomic regions known as “piRNA clusters”, whose precursors are transcribed by RNA polymerase II and subsequently processed into mature piRNAs (23, 24). Based on their generation characteristics, piRNA clusters are classified as either single-stranded or double-stranded (25). Studies have shown that piRNA clusters can be widely expressed in different species, but the transcriptional mechanism of these clusters has been most intensively studied in the Drosophila germline (26, 27). Thus, studies of the Drosophila model have elucidated the transcriptional regulation of piRNA clusters ( Figure 1 ). Double-stranded piRNA clusters are predominantly found in germ cells, and their transcriptional regulation is dependent on the H3K9 trimethylation tag (28–30). The Drosophila HP1 homolog (Rhino) recognises this epigenetic tag and recruits Deadlock and Cutoff to form the Rhino-Deadlock-Cutoff (RDC) complex (31, 32). It activates RNA polymerase II (Pol II)-mediated transcription, while simultaneously suppressing transcript splicing to ensure stable elongation (33, 34). Subsequently, the RDC complex recruits the Nxf3-Nxt1 complex, which facilitates the transport of piRNA precursors to the nuage for further processing (35, 36).

Furthermore, single-stranded piRNA clusters are widely distributed in somatic cells and exhibit typical Pol II-dependent transcriptional characteristics. For instance, the Flamenco (Flam) locus contains a promoter enriched with H3K4me2 marks, which drive primary transcription (37, 38). The primary transcripts of single-stranded piRNA clusters are conventionally spliced, and their transcriptional regulation is dependent on the promoter signature and the activity of the key transcription factor, Cubitus interruptus (CI) (39). The precursor piRNAs then bind to nuclear export proteins (Nxf1 and Nxt1) and are transported to the Yb body for further processing under the guidance of the nuclear pore complex (40, 41). In mammals, piRNA clusters are similarly transcribed, but their expression in germ cells and somatic cells is tightly regulated.

The piRNA precursors are modified to mature piRNAs in the cytoplasm by ribonucleoprotein particles (42, 43). The study shows that piRNA production is highly conserved among species and proposes a unified model (44). The model consists of two linked mechanisms: a primary amplification pathway and a ‘ping-pong’ amplification pathway, and it has been fully validated in the Drosophila ( Figure 2 ). In primary amplification, due to the absence of PIWI-like protein Aubergine (Aub) and PIWI-like protein Argonaute 3 (Ago3), the precursor transcription product binds to the Yb body and is cleaved by the internal initiator piRNA to generate early intermediate piRNA (pre-pre-piRNA) (45, 46). Subsequently, it is transferred to the outer mitochondrial membrane by the RNA deconjugating enzyme Armitage (Armi) and cleaved by the endonuclease Zucchini (Zuc), the nucleic acid exonuclease (Trimmer), as well as 2’-O-methylated by the Methyltransferase Hen 1(Hen 1) to obtain mature piRNA (47, 48).

At the beginning of the ‘ping-pong’ cycle, the Ago3-initiator piRNA complex generates a new pre-pre-piRNA by cleaving the transcriptional precursor (49, 50). Subsequently, the molecule is transferred to Aub under the guidance of the RNA helicase Vasa to generate intermediate piRNA (pre-piRNA), and then pre-piRNA is modified by Trimmer and Hen 1 to generate mature piRNA (51, 52). Furthermore, the primary amplified pre-pre-piRNA also participates in the ‘ping-pong’ cycle. It binds to Armi and translocates to the outer mitochondrial membrane, where it is processed and modified to form a mature piRNA (53, 54). Next, the mature piRNA returns to the “ping-pong” cycle as an initiator piRNA that binds and cleaves to the complementary primary transcription precursor to form a new pre-pre-piRNA (16, 55). After binding to Ago3, it undergoes a series of modifications to generate a new piRNA, which has the same sequence as the starting piRNA, thus completing the ‘ping-pong’ cycle (15).

Although this mechanism was first elucidated in Drosophila, accumulating evidence indicates that the fundamental principles of piRNA biogenesis are conserved in mammals, including humans. In mammals, the PIWIL1–4 proteins function similarly to Drosophila’s PIWI, Aub, and Ago3. During spermatogenesis, the primary processing pathway remains dominant, while the ping-pong cycle also contributes to piRNA amplification, despite species-specific modifications (56). These similarities suggest that the Drosophila model provides a valuable framework for understanding piRNA biogenesis in humans and other mammals.

Increasing evidence suggests that the highly conserved piRNA biogenesis pathway may be dysregulated in cancer. For instance, abnormal expression of PIWI proteins (such as PIWIL1 and PIWIL4) in various cancers leads to altered piRNA production and function. Furthermore, auxiliary factors like HENMT1, Arx, and CCR4 play crucial roles in piRNA maturation. Their abnormal expression in tumors leads to defective piRNA biogenesis, impairing both primary processing and the efficiency of the ping-pong cycle. Consequently, this process contributes to the dysregulation of oncogenes and genomic instability. Abnormal piRNA biogenesis not only elucidates the link between piRNAs and cancer initiation and progression but also provides a basis for understanding their multifaceted roles in cancer (57). This topic will be thoroughly examined in the subsequent section.

Transcriptional gene silencing (TGS) mediated by the piRNA/PIWI complex occurs primarily at the chromatin level, involving DNA methylation and histone modifications that promote heterochromatin formation and transcriptional repression ( Figure 3A ). This process is initiated when the piRNA/PIWI complex interacts with Asterix (Arx) and Panorama (Panx), recognizing nascent transcripts and anchoring them to specific genomic loci, thereby triggering TGS (61). A key component of this silencing mechanism is histone H3 lysine 9 di- and tri-methylation (H3K9me2/3), catalyzed by the histone methyltransferase Eggless (Egg) and its cofactor Windei (Wde) (62). The deposition of these repressive marks facilitates the recruitment of heterochromatin protein 1 (HP1), which further reinforces heterochromatin architecture and stabilizes transcriptional repression (63). Additionally, the removal of the activating H3K4me2 mark by lysine-specific demethylase 1 (Lsd1) promotes DNA methylation, thereby preventing RNA polymerase II (RNA Pol II) from initiating transcription (64). Beyond histone modifications, the piRNA/PIWI complex also recruits DNA methyltransferases (DNMTs) to CpG sites, leading to DNA methylation and further consolidating transcriptional silencing (64, 65). Research indicates that PIWIL1 methylates the Gem Interacting Protein (GMIP) gene by the RASSF1C-PIWIL1-piRNA signaling axis, leading to down-regulation of the expression of this gene, thereby enhancing NSCLC progression (62).

In contrast, post-transcriptional gene silencing (PTGS) regulates gene expression at the RNA level, affecting mRNA stability and translation efficiency ( Figure 3B ). Research shows the piRNA/PIWI complex can regulate post-transcriptional gene networks through piRNA-RNA interactions (66). These RNAs include mRNAs (67), long-chain non-coding RNAs (68), and small interfering RNAs. piRNA binds to PIWI proteins to form the piRNA-induced silencing complex (piRISC), which further associates with other inhibitory factors to mediate mRNA degradation, ultimately executing PTGS (69). To illustrate, the piRNA/PIWI complex binds to the CCR 4-NOT complex to form a specific piRISC, which promotes RNA silencing (70). In mouse muscle, piRISC recruits CAF 1, resulting in extensive mRNA degradation and thus facilitating spermatogenesis (71). A study demonstrated that piR-55490 forms incomplete base pairing with the mTOR 3′-UTR, which results in the cleavage and degradation of mTOR mRNA and subsequent inhibition of NSCLC progression (17). Furthermore, piR-211106 was also found to inhibit pyruvate Carboxylase (PC) expression at the mRNA level, which inhibits NSCLC progression. Recent studies have demonstrated that piRNAs can participate in post-transcriptional gene regulation through m⁶A RNA methylation, which is one of the epigenetic regulatory mechanisms currently receiving much attention (72). Notably, this modification requires the specific binding of the corresponding methylase or demethylase post such as m⁶A methyltransferase complex (MTC), to exert a regulatory function on mRNA. For example, piR-27222 inhibited Casp8 expression by enhancing the level of m⁶A methylation modification of Casp8 mRNA through up-regulation of Wilms’ Tumor 1-Associated Protein (WTAP) expression. This process enhanced PANoptosis resistance, which in turn promoted the proliferation and invasion of NSCLC cells (73).

The third major regulatory mechanism of the piRNA/PIWI complex involves direct interaction with multiple proteins and regulation of downstream pathways ( Figure 3C ). To illustrate, the piR-54265/PIWIL2 complex interacts with signal transducer and activator of transcription 3 (STAT3) and phosphorylated-SRC (p-SRC), mediating SRC phosphorylation via the PAZ structural domain of PIWIL2, which in turn leads to STAT3 phosphorylation activation. This process not only promotes colorectal cancer (CRC) cell tumourigenesis but is also associated with enhanced chemotherapy resistance. Similarly, piR-L-138 can effectively inhibit cisplatin-induced apoptosis by binding to p60 isoform of Mouse double minute 2 homolog (p60-MDM2), a mechanism that significantly contributes to the development of chemoresistance in NSCLC (74). In addition, study reveals that piR-211106 can directly interact with Pyruvate Carboxylase (PC) protein and affect its function at the protein level, thus inhibiting the progression of NSCLC (75).

The majority of piRNAs exert their gene regulation function in the form of piRNA/PIWI complexes. However, it has been shown that piRNA can also bind to other co-factors to form complexes that can lead to the silencing of target genes or the activation of transcription ( Figure 3D ). In addition, piRNA can directly bind to target genes and inhibit them in a sequence-specific manner (76). For example, piR-L-163 is closely associated with NSCLC, binds directly to phosphorylated ERM protein (p-ERM), and affects p-ERM binding to filamentous actin (F-actin) and ERM-binding phosphoprotein 50 (EBP 50), which consequently affects cell migration and invasive capacity (77). Recent studies have shown that piRNA-PMLCPIR binds to nucleolin (NCL) and promotes the upregulation of integrin subunit beta 1 (ITGB1) expression, which in turn activates the PI3K/AKT signaling pathway and inhibits apoptosis, thereby decreasing the sensitivity of NSCLC to chemotherapy (78).

Cancer cells exhibit anti-aging and infinitely proliferative properties. Tumor proliferation is achieved through the regulation of the cell cycle, induction of angiogenesis, inhibition of apoptosis and modulation of cell metabolism. Evidence indicates that the abnormal expression of PIWI proteins and piRNAs can disrupt these regulatory mechanisms, thereby promoting the sustained proliferation of NSCLC.

Xie et al. (81) have observed that the activation of PIWIL1 enhances the sensitivity to teratoma-derived growth factor 1 (TDGF1), which combines and participates in downstream signaling pathways, thereby promoting the proliferation of NSCLC cells. It is noteworthy that the high expression of PIWIL1 is not attributable to gene mutation, but rather results from a reduction in the level of DNA methylation in its promoter region. Furthermore, RAS-associated structural domain family 1 member A splice variant (RASSF1C) has been demonstrated to facilitate lung cancer cell proliferation. It activates the MEK-ERK1/2 signaling pathway, promotes ERK1/2 phosphorylation, and upregulates PIWIL1 gene expression (82). Cell cycle proteins and their coordinated family of dependent kinases (CDKs) represent a key regulatory mechanism of cell cycle progression (91). Qu et al. (86) revealed that PIWIL2 plays a key role in NSCLC cell proliferation. This study showed that overexpression of PIWIL2 in H449 and A549 cell lines significantly increased the expression of the CDK2/Cyclin A complex. This contributed to the transition of the cells from the G1 phase to the S phase, accelerated DNA replication and cell cycle progression, and ultimately induced cell proliferation.

Li et al. (19) demonstrated that piR-651 influences the transition from the G1 to S phase by up-regulating the levels of cell cycle proteins D1 and CDK4. This resulted in a decrease in G0/G1-phase cells and an increase in G2/S-phase cells, thereby inhibiting the proliferation process of NSCLC. Furthermore, piR-651 has been demonstrated to regulate proliferation by affecting mitochondria-related apoptotic signaling pathways. Research demonstrated that piR-651 activates Caspase-3 after transfection to promote the proliferation of A549 and HCC827 cells. The process was achieved by mitochondrial inhibition of reactive oxygen species (ROS), up-regulation of Bcl-2 family proteins, and down-regulation of Bax protein (88). In another study, piR-55490 expression was significantly reduced in NSCLC tissues and cells. This small RNA exerts a silencing effect by binding to the 3′ untranslated region (3′UTR) of its target mTOR, which activates the AKT/mTOR signaling pathway, accelerates cell cycle progression, and enhances the efficiency of glucose absorption, collectively leading to the proliferation of NSCLC cells (89). Furthermore, the overexpression of RASSF1C in NSCLC cells has been observed to affect the expression of specific piRNAs, which in turn regulate downstream signaling pathways and involve in the tumor proliferation process. For instance, in the RASSF1C-overexpressed H1299 cell line, piR-34871 and piR-52200 were observed to be overexpressed, whereas the expression of piR-35127 and piR-46545 was downregulated. By knocking down piR-34871 and piR-52200 as well as overexpressing piR-35127 and piR-46545, tumor cells were found to have a significant decrease in their proliferative capacity (84). Further studies showed that RASSF1C could regulate piRNA expression by blocking AMPK phosphorylation and enhancing epidermal growth factor receptor (EGFR) signaling, thereby affecting the proliferation process of NSCLC cells.

The local invasion and distant metastasis of tumors represent crucial elements of tumor progression and are closely related to patient prognosis. Evidence suggests that the occurrence of metastasis not only increases the malignancy of the tumor but also has a significant negative impact on the five-year survival rate of patients (92). Studies have shown that piRNA and PIWI proteins are important in the metastasis of NSCLC. Therefore, a deeper understanding of the metastatic mechanisms of PIWI and piRNA is expected to provide the latest research basis for early diagnosis and precision treatment of tumors.

Xie et al. observed that in PIWIL1-overexpressing A549 cells, the transwell experiments demonstrated significant enhancement in cell migration and invasion, indicating that PIWIL1 facilitates the invasion and metastasis of NSCLC cells (81). Furthermore, PIWIL1 interacting with TDGF1 may further enhance tumor metastasis, and both drive the progression of tumor invasion and metastasis through synergistic effects. Recent studies have demonstrated that PIWIL1 regulates the methylation status of the oncogene GMIP through the RASSF1C/PIWIL1/piRNA signaling axis, which in turn affects tumor progression (55, 83). This study indicated that the overexpression of RASSF1C resulted in elevated methylation levels of the GMIP gene, which subsequently inhibited GMIP expression and promoted the metastasis of NSCLC cells to distant regions. Furthermore, RASSF1C has been demonstrated to upregulate the expression of prolyl 4-hydroxylase α-2 (P4HA2) and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) through the PIWIL1-piRNA pathway. This promotes extracellular matrix (ECM) remodeling in NSCLC and enhances tumor invasion and metastasis (84).

Research has demonstrated that piR-651 was highly expressed in NSCLC tissues and its expression level was positively correlated with distant tumor metastasis. The results showed that knockdown of piR-651 inhibited the invasion and metastasis of HCC827 and A549 cells, and down-regulation of piR-651 in 95-D cells also reduced their invasive ability (88, 93). Thus, piR-651 can act as an oncogenic factor and also plays an important regulatory role in tumor metastasis. Daugaard et al. identified significant downregulation of piR-57125 in metastatic lung adenocarcinoma, indicating its potential function as a metastasis suppressor in regulating tumor progression (90). Furthermore, Amaar et al. demonstrated that the expression of piR-35127 and piR-46545 was negatively regulated by RASSF1C/PIWIL1 pathway and associated with enhanced invasion and migration of NSCLC cells (22, 84). This indicates that piR-35127 and piR-46545 may function as tumor-suppressive factors that inhibit metastasis. Pyruvate carboxylase (PC), a vital enzyme that connects glycolysis to the tricarboxylic acid cycle, provides the energy for cell migration. Recent studies have demonstrated that piR-211106, acting as an upstream regulatory molecule of PC, reduces the expression level of its genes or proteins by targeting PC mRNA or directly interacting with PC proteins, thereby suppressing the malignant migration phenotype of lung adenocarcinoma cells (75). Another study showed that piR-27222 inhibited Casp8 expression by directly binding to Eukaryotic translation initiation factor 4B (eIF4B) and promoting WTAP expression, which then enhanced the m6A methylation level of Casp8 mRNA. This process inhibited cell death caused by PANoptosis, thereby promoting the proliferation and invasion of NSCLC (73). Notably, the piRNA-like small RNAs (piRNA-Ls), a class of short-stranded non-coding RNAs with structural and functional similarities to piRNAs. They has expanded the diversity of the piRNA family and offers a novel perspective for the understanding of the functional mechanisms of piRNAs (77, 94). There is evidence to suggest that piRNA-Ls are expressed in NSCLC cells and play a role in promoting tumor progression. For instance, Mei et al. demonstrated that piR-L-163 is closely associated with NSCLC and can directly bind to the phosphorylated ERM protein (p-ERM), thereby regulating the functional activity of ERM, which in turn affects the migratory and invasive capacity of cells (77).

Drug resistance is closely linked to poor clinical prognosis, which often leads to treatment failure and ultimately to patient death. Therefore, reducing the patient’s drug resistance response is important for improving the efficacy and prolonging the patient’s survival time. Recent studies have shown that piRNAs play a vital role in lung cancer progression and are critical in cancer drug resistance. Specifically, piRNA promotes drug resistance by inhibiting chemotherapy-induced apoptosis or inhibiting immune targeting by PD-1/PD-L1 inhibitors. In addition, PIWI proteins play an important regulatory role in NSCLC drug resistance. In recent years, intensive studies on the functions and mechanisms of piRNA/PIWI proteins have provided new insights into the understanding of drug resistance. Next, we will focus on the specific roles and mechanisms of piRNA/PIWI in NSCLC drug resistance.

It has been demonstrated that piR-211106 remarkably enhances the sensitivity of tumor cells to cisplatin (CDDP), thereby inhibiting tumor progression (75). This phenomenon may be related to the ability of piRNA to enhance chemotherapy-induced apoptosis. Furthermore, piR-211106 exerts a synergistic effect with cisplatin, thereby enhancing the efficacy of the drug. Its use as an adjuvant treatment can effectively inhibit tumor growth when chemotherapy is ineffective. In other chemotherapy resistance study of NSCLC, it was found that piR-L-138 is upregulated in cisplatin-treated NSCLC cells and xenograft mouse models (74). Subsequently, this study observed a significant increase in apoptosis by knocking down piR-L-138 expression. Upon further validation, the upregulated piR-L-138 in p53 mutant Lung squamous cell carcinoma(LSCC) was found to bind directly to the p60-MDM2 axis, inhibiting the cisplatin-induced apoptotic pathway and enhancing chemoresistance (74). This indicates that piR-L-138 may contribute to chemoresistance by inhibiting the apoptotic pathway. Furthermore, Xu et al. found that piRNA-PMLCPIR upregulated ITGB1 expression by binding to NCL and inhibiting the interaction between NCL and ITGB1. This process resulted in activating the PI3K/AKT signalling pathway and inhibiting apoptosis, thereby reducing chemosensitivity to cisplatin (78). In addition to influencing chemotherapy resistance, recent studies have shown that piRNA/PIWI proteins can contribute to the emergence of resistance to PD-1/PD-L1 inhibitors by modulating lipid metabolism. Specifically, the piRNA-137463/PIWIL2 complex reduces INSIG1 expression by regulating the LOC100128494/miR-24-3p/INSIG1 pathway, which then promotes cholesterol synthesis and the upregulation of PD-1/PD-L1 (87). Elevated levels of PD-1/PD-L1 enhance the immune evasion of tumor cells and diminish the therapeutic effect of immune checkpoint inhibitors, thus leading to the development of immune resistance.

Cancer stem cells (CSCs) are critical in oncogenesis, progression and recurrence, and the regulatory mechanisms of their stemness characteristics have become a hotspot in cancer research. Recent studies have confirmed the involvement of the piRNA/PIWI protein complex in the maintenance and regulation of tumour stemness, thereby providing a theoretical basis for targeted therapeutic strategies against CSCs.

PIWIL1, a gene associated with tumor stem cells, is significantly expressed in NSCLC and regulates tumor stem cell proliferation by activating the Wnt signaling pathway to inhibit β-catenin degradation. In addition, the overexpression of RASSF1C upregulates PIWIL1 (85). However, when RASSF1C is co-expressed with insulin-like growth factor-binding protein-5 (IGFBP-5). It regulates PIWIL1 through a post-transcriptional mechanism and potentiates the inhibitory effect on PIWIL1, thus attenuating tumour stemness (76). Currently, the study of piRNAs in tumor stemness in NSCLC is still in the preliminary exploration stage. Therefore, further exploration of the regulatory mechanism of piRNA on cancer stemness will become the highlight of future research.

Taken together, piRNA and PIWI proteins exhibit duality in the malignant phenotype of NSCLC. They not only act as oncogenic factors promoting tumour progression but also exert tumour-suppressing functions under specific conditions. Oncogenic piRNAs, such as piR-651, piR-L-138, and PIWIL2, enhance malignant traits including proliferation, metastasis, drug resistance, and stemness. In contrast, tumor-suppressive piRNAs, such as piR-35127, piR-211106, and piR-46545, restrain these processes and may counterbalance oncogenic signals.The dual role of piRNA/PIWI appears to be subject to multiple regulatory mechanisms, including cofactor interactions, signaling network modulations, and epigenetic modifications. Consequently, this mechanism reveals the intrinsic complexity of piRNAs in NSCLC and broadens its potential as a therapeutic target.