P-element-induced wimpy testis-interacting RNA: A biomarker for gastric cancer diagnosis, progression, and prognosis

Abstract

P-element-induced wimpy testis (PIWI)-interacting RNAs are a class of small non-coding RNAs, typically 24–31 nucleotides in length. They function by binding to PIWI proteins and play important roles in gene regulation. Initially identified in germline cells, PIWI-interacting RNAs (PiRNAs) mediate transposon silencing and maintain genomic integrity. Accumulating evidence indicates that, beyond their roles in mammalian germ cells, PiRNAs are also aberrantly expressed in a variety of human cancers, where they participate in tumorigenesis and progression through epigenetic and post-transcriptional regulatory mechanisms. In gastric cancer, several PiRNAs and their associated PIWI proteins exhibit distinct expression patterns and influence malignant behaviors, such as cancer cell proliferation, invasion, metastasis, and chemotherapy resistance. Moreover, due to their high stability and detectability in bodily fluids and gastric mucosal tissues, PiRNAs show great potential as novel biomarkers for diagnosis, prognosis assessment, and treatment monitoring of gastric cancer. This review systematically summarizes the biogenesis, functional mechanisms, and molecular networks of PiRNAs in gastric cancer, discusses their clinical prospects for early detection, prognostic evaluation, and targeted therapy, and outlines current research limitations and future directions.

Key Words: P-element-induced wimpy testis-interacting RNA; P-element-induced wimpy testis complex; Gastric cancer; Function; Biomarker

Core Tip: Gastric cancer is a highly lethal digestive tract malignancy with unclear pathogenesis. Non-coding RNAs are closely related to its occurrence and development, among which P-element-induced wimpy testis (PIWI)-interacting RNAs have great research potential. PIWI-interacting RNAs (PiRNAs) regulate gene expression by binding to PIWI proteins and are abnormally expressed in gastric cancer tissues. This review focuses on PiRNAs and PIWI proteins, clarifies their molecular mechanisms in gastric cancer, and discusses their value as novel biomarkers for early diagnosis and targeted therapy.

INTRODUCTION

Gastric cancer is a common digestive tract malignancy, ranking third in mortality rate among all cancers, with approximately 800000 deaths reported worldwide annually[1]. The occurrence of gastric cancer is associated with factors such as genetics, Helicobacter pylori infection, poor dietary habits, and environmental pollution[2]. Nevertheless, the specific mechanism underlying its development remains unclear. Due to the lack of obvious early symptoms and limited clinical screening methods, most patients are diagnosed at an advanced stage, with a 5-year survival rate below 10%[3,4]. Therefore, exploring novel biomarkers and therapeutic targets for gastric cancer holds significant clinical importance.

Non-coding RNAs (ncRNAs) are RNA transcripts that do not encode proteins, including microRNAs (miRNAs), small nucleic acids, and P-element-induced wimpy testis-interacting (PIWI-interacting) RNAs. They play crucial roles in modifying epigenetics - heritable changes in gene expression without altering the DNA sequence and regulating gene expression at both the cellular and chromosomal levels to control cellular differentiation. The miRNA is implicated with the initiation, progression, and drug resistance of gastric cancer. However, due to its inherent instability and susceptibility to degradation, quality control poses a critical challenge. Even minor adjustments in detection procedures may compromise miRNA stability and functionality, thereby limiting its application in gastric cancer diagnosis[5,6]. In contrast, small nucleic RNA is a class of ncRNAs ranging 50-200 nucleotides in length, primarily involved in the processing of precursor mRNA in eukaryotic organisms. Its low concentration and instability in circulating body fluids make it difficult to obtain, limiting its clinical application. PIWI-interacting RNAs (PiRNAs), ranging 24-31 nucleotides in length, have emerged as a novel class of ncRNAs and serve as important cellular biological mediators[7]. Thus far, more than 30000 PiRNAs have been identified in the human genome, far exceeding the total number of miRNAs (approximately 2000)[8]. These molecules play multiple roles in gene regulation.

PiRNAs exert their gene regulatory functions by binding to PIWI proteins of the argonaute (AGO) family, forming a silencing ribonucleoprotein complex capable of recognizing and silencing complementary sequences. This complex plays a crucial role in maintaining the stability and integrity of the germline genome[9,10]. High-throughput sequencing technology reveals that PiRNA-related pathways are widely expressed in germ cells and actively present in somatic cells, particularly in human cancers[11,12]. Among these, gastric cancer tissues contain 698 types of PiRNAs, with 14 exhibiting differential expression between gastric cancer, adjacent gastric tissues, and non-cancerous tissues. Computer-simulated searches of PiRNA-targeted mRNA indicate that these PiRNAs may directly and indirectly contribute to gastric carcinogenesis. This review compiles relevant research and knowledge on PiRNAs and PIWI proteins, explores their molecular mechanisms in gastric cancer, and discusses their potential as biomarkers. The aim of this review is to provide insights for early diagnosis, pathogenesis research, prognosis assessment, and personalized treatment strategies in gastric cancer.

OVERVIEW AND SYNTHESIS MECHANISM OF PIRNAS

PiRNAs are small ncRNAs that were first identified in Drosophila melanogaster by Aravin et al[13]. They are characterized by their interaction with the PIWI protein family, a subfamily of proteins known as piwi proteins (PIWI), which are specific to germ cells. PiRNAs and PIWI proteins are highly conserved among amphibians and play crucial biological roles. Mature PiRNAs form a complex, i.e., PiRNA-induced silencing complex (piRISC), with PIWI proteins. This complex cleaves transposon mRNA in the cytoplasm and silences transposon gene loci in the nucleus through epigenetic modifications, such as DNA methylation and histone modifications. Thus, these molecules jointly play a crucial role in maintaining genomic stability at both cytoplasmic and nuclear locations (Figure 1).

Figure 1 P-element-induced wimpy testis-interacting RNA synthesis mechanism. A: Within the cell nucleus, double-stranded P-element-induced wimpy testis (PIWI)-interacting RNA clusters undergo transcription processes to generate PIWI-interacting RNA (PiRNA) precursors, which can further participate in post-transcriptional silencing regulation; B: In mitochondria-associated processes, Zucchini endoribonuclease protein facilitates the processing of PiRNA precursors into intermediate PiRNA molecules. These intermediates form PiRNA/PIWI complexes with PIWI protein, enabling protein interactions in the nucleus or cytoplasm and post-transcriptional silencing; C: Transposon-related sequences contribute to the generation of PiRNA precursors, which subsequently enter subsequent pathways such as the ping-pong mechanism; D: In the ping-pong mechanism, Aubergine (AUB) protein binds to PiRNA to form AUB-PiRNA complexes; E: These AUB-PiRNA complexes interact with Argonaute 3 protein and other factors, achieving target transposon sequence silencing and PiRNA sequence amplification through the ping-pong cycle, thereby maintaining PiRNA biosynthesis and functional regulation; F: AUB-piRNA complexes participate in the ping-pong cycle with AGO3 to cleave transposon transcripts, amplifying piRNA pools and reinforcing transposon silencing. AGO3: Argonaute 3; Aub: Aubergine; piRISC: P-element-induced wimpy testis-interacting RNA-induced silencing complex; PiRNA: P-element-induced wimpy testis-interacting RNA; PIWI: P-element-induced wimpy testis; Zuc: Zucchini endoribonuclease.

Primary maturation pathway: From genomic transcription to functional complex assembly

The primary maturation pathway of PiRNAs can directly generate PiRNAs from PiRNA clusters containing PIWI proteins and flamenco fragments[14,15]. Primary PiRNA precursors are predominantly long single-stranded RNA, transcribed by RNA polymerase II, and matured through 5' capping and 3' polyadenylation[16]. Subsequently, these mature precursors are processed by the transcription factor Cubitus interruptus and exported to the Yb body, where they are cleaved by the Zucchini endoribonuclease (Zuc), also termed phospholipase D family member 6 (PLD6), to generate primary PiRNA precursor fragments containing 5' monophosphate[17]. The cleavage site of Zuc exhibits sequence specificity, typically cleaving downstream of the uracil residue to generate primary PiRNA fragments approximately 26-31 nucleotides in length[18]. Subsequently, mature PiRNA is ultimately formed through 3' end trimming mediated by exonucleases, such as PARN-like domain-containing 1 (PNLDC1) and methylation catalyzed by Hua Enhancer 1 (HEN1). Notably, primary maturation pathways differ across species. For example, in nematodes, PiRNA precursor processing involves the Integrator complex[19]; in mammals, mitochondrial phospholipase D (MITOPLD) may regulate processing through mitochondrial membrane localization[20]. Such interspecies differences in piRNAs biogenesis pathways lead to distinct regulatory effects on genome stability and cellular development[21]. However, relevant comparative studies remain insufficient and warrant further in-depth investigation.

Ping-pong mechanism: A feedback loop for sub-target PiRNA amplification and transposon silencing

The ping-pong cycle, also known as the secondary pathway, is the core amplification mechanism for PiRNA-mediated transposon silencing. It relies on the coordinated action of Aubergine-piRISCs and AGO3 in the cytoplasm to initiate the ping-pong cycle, which generates secondary PiRNAs[20]. This mechanism relies on the cyclic cleavage of two PIWI proteins. Primary PiRNAs (typically derived from antisense-transcribed transposon RNA) are loaded into Aub, where they recognize and cleave the complementary antisense strand of the transposon RNA, producing secondary PiRNA precursors with 10-nucleotide complementarity. Subsequently, the precursor is captured by AGO3 and processed into mature secondary PiRNAs. These AGO3-PiRNA complexes subsequently target and cleave antisense transposon RNA, generating new Aub-binding PiRNAs. This creates a ‘ping-pong cycle’ that exponentially amplifies PiRNAs while efficiently suppressing transposon activity[9,22]. Furthermore, Zuc participates in the formation of the 3' end of secondary PiRNAs and generates diverse PiRNAs through phase cutting[23], thereby enhancing arge recognition capabilities[7,24-26]. Recent studies indicate that the ping-pong mechanism may also form PiRNA processing bodies (nuage) through phase separation. Nuage enhances PiRNA-mediated transposon silencing to maintain genomic stability and regulates epigenetic memory and transgenerational inheritance[27].

PIWI/PIRNA BIOLOGICAL FUNCTIONS

Extensive research demonstrated the role of PiRNA in animal germ cells[6,28], with studies showing that PiRNA maintains genomic integrity by suppressing transposable elements[29]. In human genetic disorders, the phenomenon of transposable elements silencing has also been observed[30]. PiRNAs can guide transposon suppression by recognizing numerous complementary sequences. They achieve transcriptional silencing through epigenetic mechanisms[31,32], and post-transcriptional inhibition via PiRNA/PIWI complex formation[33,34], thereby regulating cancer progression across multiple omics levels. Research indicates that PiRNAs and their PIWI proteins promote demethylation of retrotransposons by forming complexes (piRISC), thereby inhibiting transposon retrotranscription and maintaining genomic stability; owing to this activity, they are termed “the cellular immune system”[35,36]. Loss of piRISC function or reduced expression enables unrestricted transposon activity, inducing greater genomic damage and abnormal expression of disease-associated genes[37] (Figure 2).

Figure 2 P-element-induced wimpy testis-interacting RNA biological functions. A: Transcriptional gene silencing (TGS): P-element-induced wimpy testis (PIWI)-initiated signals recruit regulatory factors to nucleosomal chromatin, thereby driving chromatin condensation into heterochromatin and repressing gene transcription; B: Post-transcriptional gene silencing (PTGS): PIWI proteins associate with mRNA molecules (spanning 5'UTR, exons, intron, and 3'UTR regions), and coordinate with modifications to inhibit gene expression at the post-transcriptional stage (via translational repression or mRNA degradation); C: PIWI-protein interactions: PIWI proteins engage in protein–protein interactions with protein 1 and protein 2 through their structural motifs; this process supports their regulatory functions in both TGS and PTGS. PiRNA: P-element-induced wimpy testis-interacting RNA; PIWI: P-element-induced wimpy testis; UTR: Untranslated region; DNMT: DNA methyltransferases.

PiRNA/PIWI complex mediates target gene silencing at the transcriptome level

The PIRNA/PIWI complex regulates gene transcription through two mechanisms. The first mechanism is transcriptional-level gene silencing (TGS), which primarily occurs in PIWI proteins with low catalytic activity[38,39]. In TGS, the PiRNA/PIWI complex mediates chromatin silencing by collaborating with histone-modifying enzymes or DNA methyltransferases, thereby suppressing mRNA transcription[40,41] (Figure 2A). The PiRNA/PIWI complex mediates target gene silencing at the transcriptome level, a mechanism that has been demonstrated in multiple types of cancer. Studies demonstrated that the piR-31470/PIWI-like 4 (piR-31470/PIWIL4) complex recruits multiple DNA methyltransferases to regulate the methylation level of the target gene glutathione S-transferase pi 1 (GSTP1), thereby mediating GSTP1 silencing and driving prostate cancer progression. In multiple myeloma, PiRNA-823 induces abnormal DNA hypermethylation by activating DNA methyltransferase 3 beta (DNMT3B), ultimately mediating target gene silencing and exerting carcinogenic effects. In esophageal squamous cell carcinoma tissues, upregulated PiRNA-823 may activate DNMT3B through epigenetic inheritance pathways, triggering abnormal DNA methylation and participating in tumorigenic processes via transcriptional-level target gene silencing[42-44]. The second mechanism is post-transcriptional gene silencing (PTGS), a core regulatory process mediated by piRNAs/PIWI complexes. PTGS can be triggered by the introduction of transgenes or double-stranded RNA into diverse hosts, leading to silencing of all homologous endogenous genes and/or transgenes. This mechanism depends on the RNA cleavage activity of PIWI proteins and their target transcribed RNA fragments[26,45] (Figure 2B). Through sequence complementarity, PiRNA interacts with various RNA molecules (including mRNA, transcriptional pseudogenes, and long ncRNA), thereby regulating post-transcriptional control networks. Its mechanism of action is similar to that of miRNA, achieving gene silencing through RNA degradation or translation inhibition[46,47]. Peng et al[47] conclusively demonstrated that piR-55490 specifically binds to the 3' untranslated region (3'UTR) of the mechanistic target of rapamycin kinase (mTOR) gene, inducing mRNA degradation. This mechanism suppresses mTOR signaling pathway activity, thereby promoting lung cancer development. The findings provided evidence for the post-transcriptional regulatory role of PiRNA in tumor progression[26].

PiRNA/PIWI complex interactions with proteins

The PiRNA/PIWI complex can assist the PIWI protein in directly binding certain proteins through sequence complementarity and conformational fitting, thereby promoting diverse protein interactions[14]. On one hand, this complex can recruit epigenetic modifiers, such as DNA methyltransferases, in a PiRNA-dependent manner, leading to hypermethylation of tumor suppressor gene promoters and subsequent transcriptional silencing. Concurrently, it can interact with N⁶-methyladenosine RNA methylation-associated proteins to influence the stability and translation efficiency of cancer-related mRNAs[48,49]. On the other hand, PIWI proteins can also function independently of PiRNAs. These PiRNA-independent activities are involved in regulating mRNA stability, cell cycle, signaling pathways, and chromatin state. In gastric cancer, PIWIL1 promotes cell proliferation and progression via a piRNA-independent mechanism by modulating oncogene and tumor suppressor expression[50,51]. This broadens the function of PIWI beyond germline transposon silencing to somatic cell proliferation, tumor progression, and immune regulation. Furthermore, PIWI proteins are capable of functioning in a PiRNA-independent manner. For example, the piR-54265/PIWIL2 complex recruits signal transducer and activator of transcription 3 (STAT3) and phosphorylated-SRC (P-SRC) via the PAZ domain of PIWIL2, forming a PIWIL2/STAT3/P-SRC complex. This complex promotes STAT3 phosphorylation and activates signaling pathways, ultimately contributing to colorectal cancer development[52]. Together, these multi-faceted mechanisms drive tumor proliferation, invasion, and the maintenance of stem cell properties.

THE ROLE OF PIRNAS IN CANCER

PiRNAs are recognized as epigenetic regulators involved in tumor angiogenesis, invasiveness, growth, and metastasis[53]. They play a dual role in cancer; they promote tumor progression and suppress malignant phenotypes, depending on the functional attributes of the target genes involved in tumor development and the specific regulatory effects of PiRNAs on their expression levels or functional states of these target genes.

Perilipin 3 (PLIN3), as a key regulator of lipid droplet metabolism, exhibits impaired expression that disrupts lipid droplet metabolism and subsequently promotes hepatocellular carcinoma cell proliferation. The piR-017724 targets PLIN3 mRNA, inducing PTGS that enhances the capacity of liver cancer for invasion and metastasis, thereby promoting tumor progression[54]. Pinin (PNN) proteins, as crucial molecules for maintaining cell adhesion, exhibit impaired intercellular adhesion when their expression is reduced. PIWIL1 activates the expression of the anaphase promoting complex/cyclosome (APC/C) E3 complex in the mitotic APC/C complex, thereby promoting ubiquitin-dependent degradation of PNN protein. This process results in PTGS of the PNN gene, thus enhancing the metastatic capacity of pancreatic cancer cells[55]. The ephrin A5 (EFNA5) gene plays a key role in pancreatic cancer cell proliferation and metastasis. The piR-017061 complex synergizes with PIWIL1 to degrade EFNA5 mRNA, achieving gene silencing and downregulating EFNA5 expression, thereby inhibiting cancer cell proliferation and metastasis[56]. The piR-823 promotes DNMT3B-mediated DNA methylation, suppresses target gene transcription, and enhances the proliferation, metastasis, and invasion of esophageal squamous cell carcinoma[44]. In colorectal cancer, piR-823 specifically associates with PTEN induced kinase 1 (PINK1) to promote the ubiquitination and proteasome-mediated degradation of PINK1, thereby regulating mitochondrial autophagic processes and inhibiting apoptotic pathways in colorectal cancer cells[57]. Moreover, piR-36712 suppresses cell proliferation, invasion, and migration by binding to selenoprotein W pseudogene 1 RNA[58].

Recent studies have further revealed the pivotal role of PiRNA in cancer stem cells. For instance, myeloid-derived suppressor cells originating from neutrophils enhance their stem cell-like properties and promote angiogenesis through the PiRNA823 pathway and DNA methylation[43]. Furthermore, PiRNAs can be delivered via exosomes within the tumor microenvironment, influencing the phenotypes of neighboring cells. For example, cancer cells and retinal fibroblasts interact through the exosome piR-25783, activating the transforming growth factor-beta/SMAD2/SMAD3 (TGFB/SMAD2/SMAD3) pathway in fibroblasts. This process leads to the secretion of various cytokines and enhances tumor proliferation, migration, and invasiveness[59]. Therefore, exploring PiRNAs and their molecular mechanisms in cancer development can provide new approaches for cancer diagnosis and prognosis research.

PIRNAS AS BIOMARKERS FOR GASTRIC CANCER DIAGNOSIS AND PROGNOSIS

Gastric cancer is widely acknowledged as one of the top two causes of cancer-associated deaths globally. Its early clinical manifestations are often subtle, with most patients being diagnosed at advanced stages. This significantly increases treatment difficulty and yields poor outcomes. Early diagnosis to identify lesions and implement effective interventions, combined with dynamic monitoring of disease progression, holds crucial clinical significance for improving patient outcomes and enhancing quality of life. However, the usefulness of current clinical biomarkers, such as carcinoembryonic antigen (CEA), carbohydrate antigen 72-4 (CA72-4), CA19-9, and alpha-fetoprotein (AFP), is limited by their insufficient specificity and low early detection sensitivity. Therefore, there is an urgent need for highly sensitive and specific biomarkers to enable early diagnosis and prognostic monitoring.

PiRNAs exhibit high stability in body fluids by resisting nuclease degradation through vesicle encapsulation and protein binding protection, owing to their inherent structural features of 5'-end phosphorylation and 3'-end methylation. Their expression patterns show characteristic changes during cancer development, allowing the non-invasive detection of tumor-associated biological information through fluid samples. These characteristics make PiRNAs potential biomarkers for cancer prediction, and their expression levels closely correlate with the malignancy degree of gastric cancer[60-62]. Research indicates that PiRNA-1245 is overexpressed in gastric fluid of patients with gastric cancer compared to healthy individuals, and its levels significantly correlate with tumor size and tumor-node-metastasis (TNM) staging. Due to its short fragment size and resistance to degradation, PiRNA-1245 maintains relatively stable levels in body fluid samples. Its detection and isolation from body fluids demonstrate higher specificity and sensitivity than CEA and CA72-4, making it a promising new biomarker for gastric cancer screening[8,63].

Comparison of the serum exosome levels of piR-019308, piR-004918, and piR-018569 in patients with gastric cancer and healthy controls revealed significant upregulation of these biomarkers in cancer. The area under the curve values for distinguishing patients with gastric cancer from healthy controls were 0.820, 0.754, and 0.732, respectively; these values were significantly higher than those of CEA, CA199, and AFP. This finding suggests that combining these exosomes with serum tumor markers, such as CEA and CA199, could further enhance the diagnostic value of piR-019308, piR-004918, or piR-018569 for gastric cancer, identifying them as promising new biomarkers for screening gastric cancer[64]. As a proto-oncogene overexpressed in gastric cancer, piR-651 inhibits cell growth, indicating that PiRNAs play a crucial role in tumor development. Research demonstrated that the peripheral blood levels of piR-651 and piR-823 were significantly lower in patients with gastric cancer than in healthy subjects, and piR-651 levels were higher in gastric adenocarcinoma than in gastric signet ring cell carcinoma[65].

Moreover, commonly used biomarkers, such as CA19-9, CEA, piR-823, and piR-651, exhibit higher sensitivity for gastric cancer detection. This is attributed to the 2'-O-methylation modification of PiRNAs, which protects them from 3'-uridylation and truncation. This stability allows the detection and isolation of piR-651 and piR-823 in bodily fluids, enabling early diagnosis of gastric cancer. Beyond blood samples, piR-823 expression levels are significantly lower in gastric cancer tissues than in adjacent non-cancerous tissues and normal tissues, correlating with tumor suppressive effects. This makes piR-823 a viable diagnostic marker for gastric cancer. PiRNA serves as a screening tool for early-stage gastric cancer and functions as a potential prognostic biomarker for this disease. Jiang et al[66] employed high-throughput sequencing to investigate PiRNA expression patterns in gastric cancer and normal gastric mucosa. Their analysis revealed that four PiRNAs (DQ570956, DQ575659, DQ594126, and DQ597128) were significantly underexpressed in gastric cancer tissues compared to normal mucosa.

Furthermore, transcriptomic profiling of 358 non-malignant gastric and gastric adenocarcinoma samples showed that nearly half of the PiRNAs were overexpressed in tumors. Notably, a PiRNA cluster (FR290353, FR064000, FR387750/FR157678) effectively distinguished low-risk and high-risk recurrence groups in patients with gastric cancer. Additionally, PiRNA (FR222326) demonstrated a positive correlation with overall survival[66,67] (Table 1). However, current studies comparing the diagnostic sensitivity between piRNAs and traditional tumor markers are constrained by small sample sizes and a paucity of cross-sectional multi-center data. Moving forward, large-scale, multi-center studies are warranted to further validate the diagnostic sensitivity and specificity of PiRNAs, which can promote their clinical translation.

Table 1 P-element-induced wimpy testis-interacting RNA as biomarker for gastric cancer diagnosis and prognosis.

Biomarker	Biological process	Target area	Ref.
PiRNA-1245	Upregulated	Succus gastricus	[60]
FR290353	Upregulated	Gastric mucosa	[64]
FR064000	Upregulated	Gastric mucosa
FR387750	Upregulated	Gastric mucosa
piR-019308	Upregulated	Gastric mucosa	[61]
piR-004918	Upregulated	Gastric mucosa
piR-018569	Upregulated	Gastric mucosa
piR-651	Upregulated	Peripheral blood	[62]
piR-823	Upregulated	Peripheral blood
DQ570956	Downregulated	Gastric mucosa	[65]
DQ575659	Downregulated	Gastric mucosa
DQ597128	Downregulated	Gastric mucosa

PiRNA: P-element-induced wimpy testis-interacting RNA.

PIRNA AND GASTRIC CANCER METASTASIS

The high mortality rate of gastric cancer is closely associated with the aggressive invasiveness and tendency of tumors to metastasize early. Approximately 30%-40% of patients are diagnosed with lymph node or distant metastases at initial presentation. Moreover, the formation of metastatic lesions often leads to treatment failure for conventional surgery and chemoradiotherapy, posing a major challenge in clinical treatment. Research on metastatic mechanisms has identified epithelial-mesenchymal transition, extracellular matrix remodeling, and tumor microenvironment regulation as key mechanisms. However, the core signaling network driving gastric cancer invasion and metastasis remains incompletely elucidated. The dual functions of PiRNA in epigenetic regulation and post-transcriptional modification may contribute to the regulation of gastric cancer invasion and metastasis.

Abnormal PiRNA expression induces DNA modifications and activates genomic regions (including tumor-promoting genes), generating an abnormal “stem cell-like” state and driving tumorigenesis. Cui et al[65] observed that PiR-823 expression in peripheral blood of patients with gastric cancer positively correlated with tumor-lymph node metastasis stages and distant metastasis. PiR-823 enhances glucose-6-phosphate dehydrogenase (G6PD) expression, increases glucose uptake by cancer cells, reduces intracellular reactive oxygen species levels, and inhibits hypoxia-inducible factor 1α (HIF-1α) ubiquitination. Therefore, by regulating the G6PD/HIF-1α pathway, piR-823 enhances the growth, invasion, and anti-apoptotic capacity of cancer cells[68]. Subsequently, Cheng et al[69] found that piR-823 overexpression inhibits cell growth. Furthermore, research involving xenograft nude mouse models demonstrated dose-dependent tumor suppression, indicating that piR-823 plays a pivotal role in gastric cancer by regulating tumor proliferation and metastasis[70]. RNA sequencing analysis of PiRNA expression profiles in patients with gastric cancer revealed significant differences in the expression of piR-48966, piR-49145, and piR-31335 across gastric cancer tissues, adjacent tissues, and non-tumor tissues. These findings suggest that PiRNAs play a critical role in the early development, progression, and metastasis of gastric cancer[71].

Functional analysis indicated that piR-48966, piR-49145, and piR-31335 could modulate the TGFB signaling pathway by targeting bone morphogenetic protein 5, bone morphogenetic protein receptor type 1A, SMAD2, and TGFB3, thereby regulating Janus kinase and extracellular signal-regulated kinase signaling pathways to promote tumor progression, cell invasion, and metastasis[72]. They also enhance epithelial-mesenchymal transition in advanced gastric cancer, increasing tumor cell stemness and invasiveness[73]. Zhu et al[74] discovered that piR-47851 is highly expressed in gastric cancer tissues, and its expression levels closely correlate with tumor size, differentiation degree, and survival prognosis. Inhibiting piR-47851 activity significantly reduces the proliferation and invasion of gastric cancer cells, likely through its binding to the 3'UTR of mitogen-activated protein kinase 1 (MAPK1) to downregulate MAPK1 protein expression[74]. Given that MAPK1 is a central node in cell proliferation and survival signaling, its downregulation by piR-47851 may suppress metastatic capacity and has been associated with improved overall survival in gastric cancer cohorts, highlighting the prognostic relevance of this regulatory axis.

The precise mechanism by which PiRNA promotes the proliferation and metastasis of gastric cancer remains unclear. Nevertheless, the abnormal expression of PiRNA and PIWI proteins in gastric cancer tissues compared to normal tissues suggests promising prospects[69] (Table 2).

Table 2 P-element-induced wimpy testis-interacting RNA and gastric cancer metastasis.

PiRNA	Biological process	Target area	Function	Ref.
piR-823	Upregulated	Peripheral blood	Regulating PINK1/Parkin-mediated mitochondrial autophagy	[66-68]
piR-48966	Upregulated	Gastric mucosa	Regulating JNK and ERK signaling pathways to promote EMT transformation	[70]
piR-49145	Upregulated	Gastric mucosa
piR-31335	Upregulated	Gastric mucosa
piR-47851	Upregulated	Gastric mucosa	Regulating MAPK1 protein levels to inhibit gastric cancer cell proliferation	[72]

EMT: Epithelial–mesenchymal transition; ERK: Extracellular signal-regulated kinase; JNK: Janus kinase; MAPK1: Mitogen-activated protein kinase 1; PINK1: PTEN induced kinase 1; PiRNA: P-element-induced wimpy testis-interacting RNA; PIWI: P-element-induced wimpy testis.

PIWI AND DEVELOPMENT OF GASTRIC CANCER

Although PiRNAs play a crucial role in cancer initiation and progression, their biological functions are not achieved independently. They must specifically bind to members of the PIWI protein family to form PiRNA/PIWI complexes. Subsequently, these complexes participate in cancer-related processes by regulating target gene expression and maintaining genomic stability. Moreover, PIWI proteins have independent and critical functions in tumorigenesis. Four PIWI proteins with expression activity have been identified in humans, namely PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2[75]. These proteins frequently exhibit abnormal expression in various cancer types, influencing tumor progression by regulating cellular proliferation, apoptosis, invasion, and metastasis. They serve as key molecules bridging PiRNA functions and cancer mechanisms.

Liu et al[76] identified high expression of HIWI in gastric cancer tissues through gene profiling and subcellular localization studies. The protein was significantly upregulated in most cases, surpassing its expression levels in normal gastric mucosa (10%), atrophic gastritis (36%), and intestinal metaplasia (36%). Notably, HIWI expression levels showed a positive correlation with the progression of precancerous lesions. Functional experiments demonstrated that delivering antisense HIWI or short-hairpin RNA (hivi106) via adenovirus vectors could significantly inhibit AGS gastric cancer cell proliferation and induce G2/M phase cell cycle arrest. This indicates that HIWI drives gastric cancer cell proliferation by regulating the cell cycle process, and its expression levels are closely associated with malignant progression. The findings provide a novel molecular biomarker for gastric cancer diagnosis and reveal the biological function and clinical significance of the human PIWI family gene HIWI in gastric cancer. Xia et al[77] reported that PIWIL4 expression was significantly higher in gastric cancer tissues than in adjacent normal tissues, particularly in patients with stage III/IV gastric cancer compared to those with stage I/II disease. This evidence suggests that PIWIL4 could serve as a molecular marker for assessing gastric cancer malignancy and prognosis[77].

Wang et al[78] conducted immunohistochemical analysis on tissue microarrays from 182 patients with gastric cancer and found that PIWIL1-4 expression levels were significantly higher in gastric cancer tissues than in adjacent non-neoplastic tissues. Patients with high PIWIL1 and PIWIL2 expression showed significantly lower 5-year overall survival rates compared to those with low expression levels. Cox regression analysis confirmed PIWIL1 as an independent prognostic risk factor for gastric cancer. Additionally, high PIWIL2 and PIWIL4 levels were significantly associated with T stage, lymph node metastasis, and clinical TNM staging. The mechanism may involve mediating DNA hypermethylation and regulating tumor invasion/metastasis-related pathways (such as STAT3/B-cell lymphoma-extra large and STAT3/cyclin D1) that promote tumor progression. However, the specific molecular pathways and clinical applications require further validation.

The abnormal expression of PIWIL1, a member of the PIWI protein family, in gastric cancer and its pro-cancer mechanisms have attracted extensive attention in recent years. Wang et al[78] demonstrated that PIWIL1 is highly expressed in gastric cancer tissues and cell lines, and its expression levels show significant positive correlations with tumor lymph node metastasis, TNM staging, and low differentiation status. Inhibiting PIWIL1 expression can reduce cancer cell proliferation and migration, tumor development, and metastasis. This mechanism may be related to the non-PiRNA-dependent function of PIWI proteins in collaboration with the nonsense-mediated mRNA decay mechanism. Jiang et al[79] demonstrated that PIWIL3 expression is elevated in gastric cancer tissues. In diffuse gastric cancer, PIWIL3 upregulation correlates with tumor infiltration depth, lymph node metastasis, and clinical stage. Conversely, PIWIL3 downregulation inhibits gastric cancer progression and cell proliferation via the JAK2/STAT3 signaling pathway, induces G0/G1 phase arrest in vitro and in vivo, and reduces the expression of genes associated with metastasis[80] (Table 3).

Table 3 P-element-induced wimpy testis-interacting RNA and development of gastric cancer.

PiRNA	Biological process	Target area	Ref.
PIWIL1	Upregulated	Gastric mucosa	[74]
PIWIL2	Upregulated	Gastric mucosa	[74]
PIWIL1	Upregulated	Gastric mucosa	[75]
PIWIL3	Upregulated	Gastric mucosa	[76]

PiRNA: P-element-induced wimpy testis-interacting RNA; PIWIL: P-element-induced wimpy testis-like.

PIWI/PIRNAS AS THERAPEUTIC TARGETS FOR GASTRIC CANCER

Multiple studies have investigated therapeutic applications of PiRNA beyond its role as a biomarker. Despite limited understanding of the molecular pathways underlying the activity of PiRNA and PIWI proteins in carcinogenesis, their functional properties make them excellent therapeutic candidates[81]. Synthetic PiRNAs can be used to block the synthesis of cancer-associated proteins by binding to mRNA; this process requires no enzymatic processing and offers enhanced target specificity. Furthermore, designing synthetic PiRNAs to bind PIWI proteins and induce genomic silencing of PIWI genes at the transcriptional level may contribute to combination therapies for multiple types of cancer. Previous studies have demonstrated that PIWI protein expression is inversely correlated with patient survival rates. Inhibiting PIWI protein reduces the number of cells in G2/M phase and enhances p53 protein expression, thereby suppressing proliferation and promoting apoptosis[82]. Furthermore, PIWI protein increases resistance to chemotherapy drugs such as cisplatin, which is widely used for treating malignant tumors but carries the drawback of dose-dependent nephrotoxicity[83].

Consequently, reducing PIWI protein expression may enhance cancer cell sensitivity to cisplatin, providing a potential strategy for overcoming chemotherapy resistance in patients undergoing treatment[84]. For instance, in their study of cisplatin resistance in lung adenocarcinoma cells, Liu and Qi[80] discovered that piR-hsa-211106 expression was downregulated in all malignant tissues and interacted with pyruvate carboxylase (PC), thereby inhibiting PC mRNA expression and preventing cisplatin resistance in lung cancer cells[85]. The functions and interactions of PiRNA and PIWI proteins remain incompletely understood. However, the clinical translation of PiRNAs-targeted therapeutics still faces major obstacles, such as off-target effects of synthetic piRNAs, inefficient in vivo delivery, and limited understanding of the molecular pathways. Nevertheless, as our understanding of PiRNA mechanisms and functions in cancer continues to deepen, an increasing number of treatments will be developed and applied in the future[83].

CONCLUSION

PiRNAs, as a novel class of ncRNA, form functional complexes with PIWI proteins to participate in critical biological processes including gene silencing, transposon suppression, and genomic stability maintenance through epigenetic regulation, transcription, and post-transcriptional mechanisms.

This review systematically elucidates the multifaceted roles of the PiRNA/PIWI system in gastric cancer initiation, progression, and metastasis. Key contributions include its dual functions as both an oncogenic and tumor suppressor factor, its role in maintaining cancer stem cell properties and regulating the tumor microenvironment, and its inherent value in serving as a biomarker for both diagnosis and prognosis assessment, and therapeutic target. Abnormal PiRNA expression has been detected in body fluids or gastric mucosal tissues of patients with gastric cancer, demonstrating superior sensitivity and specificity compared to traditional tumor markers and exhibiting promising translational potential.

Despite demonstrating significant biological relevance and clinical application potential in malignant tumors such as gastric cancer, current PiRNA detection methods are characterized by numerous limitations, including low PiRNA concentrations in bodily fluids, insufficient standardization in extraction and quantification, and insufficient large-scale clinical validation. Although existing technologies such as quantitative reverse transcription-polymerase chain reaction and high-throughput sequencing enable qualitative and quantitative analysis, they are also characterized by limitations in terms of sensitivity, reproducibility, and cost-effectiveness.

Future researches should focus on developing novel detection technologies with enhanced sensitivity and specificity, such as piRNAs detection platforms based on nanomaterials or digital polymerase chain reaction. Additionally, we will employ gastric cancer organoid models to dissect the precise mechanisms by which piRNAs mitigate chemotherapy resistance, and conduct large-scale, multi-center clinical trials to validate and translate piRNAs-based biomarkers into routine clinical practice, ultimately addressing the high mortality burden of gastric cancer as a critical public health challenge.

Furthermore, elucidating the mechanisms of PiRNA in gastric cancer heterogeneity, metastasis, and drug resistance, while exploring their potential roles in targeted therapy and immune regulation, will provide novel strategies for achieving personalized diagnosis and treatment with improved prognosis in gastric cancer.