Identification of a novel mutation in chibby family member 2 in a non-obstructive azoospermic patient.

Azoospermia constitutes a significant factor in male infertility, defined by the absence of spermatozoa in the ejaculate, afflicting 15% of infertile men. However, a subset of azoospermic cases remains unattributed to known genetic variants. Prior investigations have identified the chibby family member 2 (CBY2) as prominently and specifically expressed in the testes of both humans and mice, implicating its potential involvement in spermatogenesis. In this study, we conducted whole exome sequencing (WES) on an infertile family to uncover novel genetic factors contributing to azoospermia. Our analysis revealed a homozygous c .355 C>A variant of CBY2 in a non-obstructive azoospermic patient. This deleterious variant significantly diminished the protein expression of CBY2 both in vivo and in vitro, leading to a pronounced disruption of spermatogenesis at the early round spermatid stage post-meiosis. This disruption was characterized by a nearly complete loss of elongating and elongated spermatids. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation assays demonstrated the interaction between CBY2 and Piwi-like protein 1 (PIWIL1). Immunofluorescence staining further confirmed the co-localization of CBY2 and PIWIL1 in the testes during the spermatogenic process in both humans and mice. Additionally, diminished PIWIL1 expression was observed in the testicular tissue from the affected patient. Our findings suggest that the homozygous c .355 C>A variant of CBY2 compromises CBY2 function, contributing to defective spermatogenesis at the round spermiogenic stage and implicating its role in the pathogenesis of azoospermia.

PIWIL1 Promotes Malignant Progression of Papillary Thyroid Carcinoma by Inducing EVA1A Expression.

INTRODUCTION: Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid cancer. Previous studies have reported on the ectopic expression of P-element-induced wimpy testis ligand 1 (PIWIL1) in various human cancers, but its role in PTC progression has not been investigated. METHODS: In this study, we measured the expression levels of PIWIL1 and Eva-1 homolog A (EVA1A) in PTC using qPCR and WB. We performed a viability assay to evaluate PTC cell proliferation and used flow cytometry to investigate apoptosis. Moreover, we conducted a Transwell invasion assay to quantify cell invasion and assessed PTC growth in vivo using xenograft tumor models. RESULTS: Our findings showed PIWIL1 to be highly expressed in PTC and promote cell proliferation, cell cycle activity, and cell invasion, while suppressing apoptosis. Additionally, PIWIL1 accelerated tumor growth in PTC xenografts by modulating the EVA1A expression. CONCLUSION: Our study suggests that PIWIL1 contributes to the progression of PTC through EVA1A signaling, indicating its potential role as a therapeutic target for PTC. These results provide valuable insights into PIWIL1 function and may lead to more effective treatments for PTC.

Elevated expression patterns of P-element Induced Wimpy Testis (PIWI) transcripts are potential candidate markers for Hepatocellular Carcinoma.

BACKGROUND: P-Element-induced wimpy testis (PIWI) proteins, when in combination with PIWI-interacting RNA (piRNA), are engaged in the epigenetic regulation of gene expression in germline cells. Different types of tumour cells have been found to exhibit abnormal expression of piRNA, PIWIL-mRNAs, and proteins. We aimed to determine the mRNA expression profiles of PIWIL1, PIWIL2, PIWIL3, & PIWIL4, in hepatocellular carcinoma patients, and to associate their expression patterns with clinicopathological features. METHODS: The expression patterns of PIWIL1, PIWIL2, PIWIL3, PIWIL4 mRNA, was assessed via real-time quantitative polymerase chain reaction (RT-QPCR), on tissue and serum samples from HCC patients, their impact for diagnosis was evaluated by ROC curves, prognostic utility was determined, and In Silico analysis was conducted for predicted variant detection, association with HCC microRNAs and Network Analysis. RESULTS: Expression levels were significantly higher in both HCC tissue and serum samples than in their respective controls (p< 0.001). Additionally, the diagnostic performance was assessed, Risk determination was found to be statistically significant. CONCLUSION: PIWIL mRNAs are overexpressed in HCC tissue and serum samples, the expression patterns could be valuable molecular markers for HCC, due to their association with age, tumour grade and pattern. To the best of our knowledge, our study is the first to report the expression levels of all PIWIL mRNA and to suggest their remarkable values as diagnostic and prognostic biomarkers, in addition to their correlation to HCC development. Additionally, a therapeutic opportunity might be also suggested through in silico miRNA prediction for HCC and PIWIL genes through DDX4 and miR-124-3p.

Relationship between PIWIL1 gene polymorphisms and epithelial ovarian cancer susceptibility among southern Chinese woman: a three-center case-control study.

OBJECTIVE: To investigate the potential correlation between piwi-like RNA-mediated gene silencing 1 (PIWIL1) polymorphisms and susceptibility to epithelial ovarian cancer (EOC). METHODS: A case-control study was conducted to evaluate the susceptibility of EOC using multinomial logistic regression analysis. The study analyzed the relationship between five functional single nucleotide polymorphisms (SNPs) in the PIWIL1 gene and EOC risk. Genotyping of 288 cases and 361 healthy samples from South China was identified using a TaqMan assay. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the relationship between the five selected SNPs and EOC susceptibility. RESULTS: Among the five SNPs analyzed, the rs10848087 G > A and rs7957349 G > C variants significantly increased the susceptibility of EOC, rs10773771 C > T was associated with a decreased risk of EOC, while the rs35997018 and rs1106042 variants were not in Hardy-Weinberg equilibrium (p < 0.05). The rs10848087 G > A was significantly associated with increased risk of EOC in individuals with metastasis, FIGO stage I and III, low and high pathological grade, tumor numbers ≤ 3 and > 3, tumor size > 3 cm and ≤ 3 cm, pregnant more than 3 times, pre-menopausal status, and strong positive expression of ER (estrogen receptor), PR (progesterone receptor), PAX8 (paired-box 8), wild-type p53 (tumor protein 53), WT1 (Wilm's tumor gene), P16 (cyclin-dependent kinase inhibitor 2A). In addition, rs10848087 G > A enhanced the EOC risk of cases with negative/mild positive expression of wild p53 and Ki67, and with or without mutant p53 expression. The rs7957349 G > C variant was linked to an increased risk of EOC in subgroups with certain characteristics, including age equal or less than 53 years, metastasis, clinical stage I, low pathological grade, tumor number, tumor size, pregnant times, post-menopause, pre-menopause, and strong positive expression of wild p53 and Ki67 (Antigen identified by monoclonal antibody Ki-67), as well as without mutant p53 expression. The rs10773771 CT/TT alleles were identified to have a protective effect on EOC in women aged 53 years or older, as well as in cases with metastasis, advanced clinical stage, high pathological grade, multiple tumors, tumor size equal to or less than 3 cm, history of pregnancy, post-menopausal status, and strong positive expression of ER, PR, wild-type p53, PAX8, WT1, P16, and Ki67. Furthermore, rs10773771 CT/TT also showed a protective effect in patients with negative or mildly positive expression of PR, PAX8, wild-type p53, WT1, and P16, as well as positive expression of mutant p53. Compared to the reference haplotype GCG, individuals harboring haplotypes GTG were found to have a significantly decreased susceptibility to EOC. PIWIL1 was significantly expressed in the thyroid, pituitary, and adrenal glands with rs7957349 CC alleles. CONCLUSIONS: PIWIL1 rs10848087 and rs7957349 were associated with increased risk of EOC, while rs10773771 may have a protective effect against EOC. These genetic variants may serve as potential biomarkers for EOC susceptibility in the South China population.

PIWIL1 gene polymorphism and pediatric acute lymphoblastic leukemia relapse susceptibility among Chinese children: a five-center case-control study.

Objective: PIWIL1 polymorphisms' role in pediatric acute lymphoblastic leukemia (ALL) relapse susceptibility remains undiscovered. Methods: A case-control designed and multiple logistic regression model was performed to evaluate the overall risk of pediatric ALL and five single-nucleotide polymorphisms (SNPs) of PIWIL1 gene (rs35997018 C>T, rs1106042 A>G, rs7957349 C>G, rs10773771 C>T, and rs10848087 A>G) in 785 cases and 1,323 controls, which were genotyped by TaqMan assay. The odds ratio (OR) and its 95% confidence interval (CI) were used to estimate the relationship. Stratified analysis was used to investigate the correlation of rs1106042 and rs10773771 genotypes and pediatric ALL relapse susceptibility in terms of age, sex, number of white blood cells (WBC), immunophenotyping, gene fusion type, karyotype, primitive/naïve lymphocytes, and minimal residual disease (MRD) in bone marrow. Finally, the haplotype analysis was performed to appraise the relationship between inferred haplotypes of PIWIL1 and pediatric ALL risk. Results: Among the five analyzed SNPs, rs1106042 A>G was related to increased ALL risk, and rs10773771 C>T was related to decreased ALL risk. Compared to the GG genotype, the rs1106042 GA/AA had a deleterious effect on children of age <120 months, who were female and male, had high or average number of WBC, pro-B ALL, pre-B ALL, T-ALL, low- and middle-risk ALL, E2A-PBX fusion gene, non-gene fusion, abnormal diploid, high hyperdiploid, hypodiploid, and normal diploid. Moreover, rs1106042 A>G harmfully affected primitive/naïve lymphocytes and MRD on days 15-19, day 33, and week 12. On the contrary, rs10773771 TC/CC exhibited a protective effect on ALL children with the TEL-AML fusion gene. Haplotype analysis demonstrated that haplotypes CAGT, TACC, TACT, and TAGT were significantly associated with increased pediatric ALL relapse susceptibility. Conclusion: PIWIL1 rs1106042 A>G was related to increased ALL risk, and rs10773771 C>T was linked to decreased ALL risk in eastern Chinese children. Rs1106042 GA/AA may predict poor prognosis.

HPV16 E6/E7-mediated regulation of PiwiL1 expression induces tumorigenesis in cervical cancer cells.

PURPOSE: PiwiL1 has been reported to be over-expressed in many cancers. However, the molecular mechanism by which these proteins contribute to tumorigenesis and their regulation in cancer cells is still unclear. We intend to understand the role of PiwiL1 in tumorigenesis and also its regulation in cervical cells. METHODS: We studied the effect of loss of PiwiL1 function on tumor properties of cervical cancer cells in vitro and in vivo. Also we have looked into the effect of PiwiL1 overexpression in the malignant transformation of normal cells both in vitro and in vivo. Further RNA-seq and RIP-seq analyses were done to get insight of the direct and indirect targets of PiwiL1 in the cervical cancer cells. RESULTS: Here, we report that PiwiL1 is not only over-expressed, but also play a major role in tumor induction and progression. Abolition of PiwiL1 in CaSki cells led to a decrease in the tumor-associated properties, whereas, its upregulation conferred malignant transformation of normal HaCaT cells. Our study delineates a new link between HPV oncogenes, E6 and E7 with PiwiL1. p53 and E2F1 directly bind and differentially regulate PiwiL1 promoter in a context-dependant manner. Further, RNA-seq together with RIP-RNA-seq suggested a strong and direct role for PiwiL1 in promoting metastasis in cervical cancer cells. CONCLUSION: Our study demonstrates that PiwiL1 act as an oncogene in cervical cancer by inducing tumor-associated properties and EMT pathway. The finding that HPV oncogenes, E6/E7 can positively regulate PiwiL1 suggests a possible mechanism behind HPV-mediated tumorigenesis in cervical cancer.

PIWI interacting RNA-13643 contributes to papillary thyroid cancer development through acting as a novel oncogene by facilitating PRMT1 mediated GLI1 methylation.

BACKGROUND: Recently, aberrant expression of PIWI-interacting RNAs (piRNAs) has been discovered in a variety of cancer cells. However, the roles of PIWI proteins and piRNAs in papillary thyroid carcinoma (PTC) are still elusive. METHODS: RT-qPCR and Northern blotting were used to evaluate piR-13643 levels in PTC and para-carcinoma tissues, as well as in PTC cell lines. piR-13643 mimic and piR-13643 inhibitor were transfected into K-1 and B-CPAP cells. CCK-8, Transwell, annexin V-FITC/PI, flow cytometry and Western blot assays were performed to measure cell proliferation, invasion, apoptosis, cell cycle and E-cadherin and Vimentin proteins, respectively. Total RNA from B-CPAP cells was pulled down with PIWIL1, PIWIL2, or PIWIL3 specific antibodies or IgG as a control, respectively, followed by detection of piR-13643 expression with RT-qPCR. Immunoblotting of PRMT1 was detected in piR-13643 / PIWIL1 complex immune-precipitates by Co-IP assay. Subsequently, PRMT1 protein expression was detected by stably transfection of Flag tagged GLI1 (Flag-GLI1) into B-CPAP cells. Methylation assay with PRMT1 and wild-type or R597 lysine (R597K)-mutant GLI1. Then rescue experiments were applied to explore effects of piR-13643 and GLI1 on the malignant behavior of PTC cells. B-CPAP cells transfected with piR-13643 inhibitor were subcutaneously injected into nude mice to evaluate the effect of piR-13643 knockdown on the xenograft tumor growth of PTC. RESULTS: piR-13643 was elevated in PTC patient specimens and cell lines. piR-13643 overexpression facilitated cell proliferation, invasion and Vimentin level, and restrained apoptosis and E-cadherin expression, whereas piR-13643 knockdown showed the opposite results. Mechanically, piR-13643 could bind to PIWIL1 to form the PIWIL1/piR-13643 complex, and PRMT1 enhanced GLI1 transcription by methylating GLI1 at R597. Further, PIWIL1/piR-13643 promoted PRMT1-mediated GLI1 methylation. GLI1 knockdown countered the effects of piR-13643 mimic on cell malignant behaviors. piR-13643 knockdown preeminently prevented the xenograft tumor growth of PTC in vivo. CONCLUSIONS: This study confirmed that piR-13643 facilitates PTC malignant behaviors in vitro and in vivo by promoting PRMT1-mediated GLI1 methylation via forming a complex with PIWIL1, which may provide a novel insight for PTC treatment.

Identification of fish spermatogenic cells through high-throughput immunofluorescence against testis with an antibody set.

Image-based identification and quantification of different types of spermatogenic cells is of great importance, not only for reproductive studies but also for genetic breeding. Here, we have developed antibodies against spermatogenesis-related proteins in zebrafish (Danio rerio), including Ddx4, Piwil1, Sycp3, and Pcna, and a high-throughput method for immunofluorescence analysis of zebrafish testicular sections. By immunofluorescence analysis of zebrafish testes, our results demonstrate that the expression of Ddx4 decreases progressively during spermatogenesis, Piwil1 is strongly expressed in type A spermatogonia and moderately expressed in type B spermatogonia, and Sycp3 has distinct expression patterns in different subtypes of spermatocytes. Additionally, we observed polar expression of Sycp3 and Pcna in primary spermatocytes at the leptotene stage. By a triple staining of Ddx4, Sycp3, and Pcna, different types/subtypes of spermatogenic cells were easily characterized. We further demonstrated the practicality of our antibodies in other fish species, including Chinese rare minnow (Gobiocypris rarus), common carp (Cyprinus carpio), blunt snout bream (Megalobrama amblycephala), rice field eel (Monopterus albus) and grass carp (Ctenopharyngodon idella). Finally, we proposed an integrated criterion for identifying different types/subtypes of spermatogenic cells in zebrafish and other fishes using this high-throughput immunofluorescence approach based on these antibodies. Therefore, our study provides a simple, practical, and efficient tool for the study of spermatogenesis in fish species.

The Role of RASSF1C in the Tumor Microenvironment.

The tumor microenvironment (TME) plays a vital role in tumor invasion and metastasis and provides a rich environment for identifying novel therapeutic targets. The TME landscape consists of an extracellular matrix (ECM) and stromal cells. ECM is a major component of TME that mediates the interaction between cancer cells and stromal cells to promote invasion and metastasis. We have shown in published work that RASSF1C promotes cancer stem cell development, migration, and drug resistance, in part, by promoting EMT through a mechanism that involves up-regulation of the PIWIL1-piRNA axis. Consistent with this, in this study, we demonstrate that RASSF1C promotes lung cancer metastasis in vivo using an orthotopic mouse model. Interestingly, two target genes identified in a previously conducted microarray study to be up-regulated by RASSF1C in breast and non-small cell lung cancer (NSCLC) cells are prolyl 4-hydroxylase alpha-2 (P4HA2) and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). In cancer, P4H2A and PLOD2 are vital for collagen posttranslational modification and folding leading to the formation of a stiff ECM and induction of EMT and cancer stem cell marker gene expression, resulting in metastatic dissemination. Here, we also show that overexpression of RASSF1C up-regulates Collagen I, P4HA2, and PLOD2 in vitro. Up-regulation of P4HA2 and PLOD2 by RASSF1C was also confirmed in lung and breast cancer cells in vivo using mouse models. Further, we found that treatment of wildtype lung cancer cells or lung cancer cells overexpressing RASSF1C or PIWIL1 with piR-35127 and 46545 (both down-regulated by RASSF1C) decreased lung cancer cell invasion/migration. Taken together, our findings suggest that RASSF1C may promote lung cancer cell ECM remodeling to induce lung cancer cell stemness, invasion, and metastasis, in part, by up-regulating a previously unknown PIWIL1-P4HA2-PLOD2 pathway. Furthermore, piR-35127 and piR-46545 could potentially be important anti-metastatic tools.

PAZ domain is critical for spermatogenesis in chicken.

Piwi-like protein 1 (PIWIL1) plays a crucial role in stem cell proliferation, embryogenesis, growth, and development. We aimed to unravel the function of PIWIL1 and its Piwi/Argonaute/Zwille (PAZ) domain in chicken embryogenesis. The expression of PIWI1 at different stages of spermatogenesis was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the PAZ domain was mutated based on its 3D structure model using the clustered regularly interspaced short palindromic repeats Cas9 (CRISPR/Cas9) technology. The results indicated that PIWIL1 mRNA was specifically expressed in spermatogonium cells undergoing meiosis. After targeting the PAZ domain (300-370 amino acid residues), we obtained two mutant DF-1 cell clones with 23-bp and 8-bp deletions. Injection of the pCMV-Cas9-puro-sgRNA-2 construct into 2.5-day embryos resulted in generation of 19 different PAZ mutants (13 males and 6 females), which showed delayed hatching, reduced quality of semen, and decreased expression of PIWIL1 and SOX2 at embryonic days 5 and 18. However, we could not obtain PAZ double knockout (KO) chickens by crossing of the F0 generation, suggesting that PAZ double KO may halt embryonic development. Our results indicate that PIWIL1 plays an important role in meiosis and that PAZ mutations can lead to decreased sperm quality, whereas its double KO may arrest embryogenesis in chicken.

P-element-Induced Wimpy-Testis-Like Protein 1 Regulates the Activation of Pancreatic Stellate Cells Through the PI3K/AKT/mTOR Signaling Pathway.

AIM: Pancreatic fibrosis is the main pathological characteristic of chronic pancreatitis (CP) and pancreatic cancer. Pancreatic stellate cells (PSCs) play a critical role in pancreatic fibrosis. Any targets that may have an impact on the activation of PSCs could become potential treatment candidates for CP and pancreatic cancer. Our goal was to investigate the effect of P-element-induced wimpy-testis (PIWI) protein 1 (PIWIL1) on PSC activation. METHODS: Lentivirus-based RNA interference (RNAi) and overexpression vector construction were used to knock down and over-express the PIWIL1 protein. Immunocytofluorescent staining, western blotting, wound healing assay, transwell assay, and phalloidin staining were used to investigate the effects of PIWIL1 on the secretion of extracellular matrix components (EMC), actin cytoskeleton, and on the invasion and migration abilities of primary PSCs isolated from C57BL/6 mice. Moreover, pancreatic fibrosis was induced by L-arginine in C57BL/6 mice. The expression of PIWIL1 and collagen deposition in vivo were tested by western blotting and Sirius red staining. RESULTS: Expression levels of collagen I, collagen III, and α-smooth muscle actin were significantly decreased in the LV-PIWIL1 group. Compared with the si-PIWIL1 group, significant differences were observed in the expression of desmin, p-PI3K, p-AKT, and p-mTOR in the LV-PIWIL1 group. Furthermore, PIWIL1 suppressed the PSCs' invasion and migration abilities. In a rescue experiment, the PI3K/AKT/mTOR signaling pathway was found to be the underlying mechanism in PSCs activation mediated by PIWIL1. CONCLUSIONS: Our findings suggest that PIWIL1 inhibits the activation of PSCs via the PI3K/AKT/mTOR signaling pathway. PIWIL1 is a potential therapeutic target for pancreatic fibrosis.

Noncanonical functions of PIWIL1/piRNAs in animal male germ cells and human diseases†.

PIWI proteins and PIWI-interacting RNAs (piRNAs) are specifically expressed in animal germlines and play essential roles during gametogenesis in animals. The primary function of PIWI/piRNAs is known to silence transposable elements for protecting genome integrity in animal germlines, while their roles beyond silencing transposons are also documented by us and others. In particular, we show that mouse PIWIL1 (MIWI)/piRNAs play a dual role in regulating protein-coding genes in mouse spermatids through interacting with different protein factors in a developmental stage-dependent manner, including translationally activating a subset of AU-rich element-containing mRNAs in round spermatids and inducing massive mRNA degradation in late spermatids. We further show that MIWI is eliminated through the ubiquitin-26S proteasome pathway during late spermiogenesis. By exploring the biological function of MIWI ubiquitination by APC/C, we identified ubiquitination-deficient mutations in human PIWIL1 of infertile men and further established their causative role in male infertility in mouse model, supporting PIWIL1 as a human male infertility-relevant gene. Additionally, we reported that PIWIL1, aberrantly induced in human tumors, functions as an oncoprotein in a piRNA-independent manner in cancer cells. In the current review, we summarize our latest findings regarding the roles and mechanisms of PIWIL1 and piRNAs in mouse spermatids and human diseases, and discuss the related works in the field.

Germ cell-specific expression of Venus by Tol2-mediated transgenesis in endangered endemic cyprinid Honmoroko (Gnathopogon caerulescens).

Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.

Critical Roles of PIWIL1 in Human Tumors: Expression, Functions, Mechanisms, and Potential Clinical Implications.

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a class of small non-coding RNA molecules that are 24-31 nucleotides in length. PiRNAs are thought to bind to PIWI proteins (PIWL1-4, a subfamily of Argonaute proteins), forming piRNA/PIWI complexes that influence gene expression at the transcriptional or post-transcriptional levels. However, it has been recently reported that the interaction of PIWI proteins with piRNAs does not encompass the entire function of PIWI proteins in human tumor cells. PIWIL1 (also called HIWI) is specifically expressed in the testis but not in other normal tissues. In tumor tissues, PIWIL1 is frequently overexpressed in tumor tissues compared with normal tissues. Its high expression is closely correlated with adverse clinicopathological features and shorter patient survival. Upregulation of PIWIL1 drastically induces tumor cell proliferation, epithelial-mesenchymal transition (EMT), invasion, cancer stem-like properties, tumorigenesis, metastasis and chemoresistance, probably via piRNA-independent mechanisms. In this article, we summarize the current existing literature on PIWIL1 in human tumors, including its expression, biological functions and regulatory mechanisms, providing new insights into how the dysregulation of PIWIL1 contributes to tumor initiation, development and chemoresistance through diverse signaling pathways. We also discuss the most recent findings on the potential clinical applications of PIWIL1 in cancer diagnosis and treatment.

PIWIL1 interacting RNA piR-017061 inhibits pancreatic cancer growth via regulating EFNA5.

PIWI (P element induced wimpy testis) integrating RNAs (piRNAs) are small non-coding RNAs with the length of approximately 30 nucleotides that plays crucial roles in germ cells and adult stem cells. Recently, accumulating data have shown that piRNA and PIWI proteins are involved in tumorigenesis. However, the roles of PIWI proteins and piRNAs in pancreatic cancer are still elusive. Here, we showed that piR-017061 is significantly downregulated in pancreatic cancer patients' samples and pancreatic cancer cell lines. Furthermore, we studied the function of piR-017061 in pancreatic cancer and our data revealed that piR-017061 inhibits pancreatic cancer cell growth in vitro and in vivo. Moreover, we analyzed the genomic loci around piR-017061 and identified EFNA5 as a novel target of piR-017061. Importantly, our data further revealed a direct binding between piR-017061 and EFNA5 mRNA mediated by PIWIL1. Mechanically, piR-017061 cooperates with PIWIL1 to facilitate EFNA5 mRNA degradation and loss of piR-017061 results in accumulation of EFNA5 which facilitates pancreatic cancer development. Hence, our data provided novel insights into PIWI/piRNA-mediated gene regulation and their function in pancreatic cancer. Since PIWI proteins and piRNA predominately express in germline and cancer cells, our study provided novel therapeutic strategy for pancreatic cancer treatment.

PIWIL1 Drives Chemoresistance in Multiple Myeloma by Modulating Mitophagy and the Myeloma Stem Cell Population.

As an important member of the Argonaute protein family, PIWI-like protein 1 (PIWIL1) plays a key role in tumor cell viability. However, the exact function of PIWIL1 in multiple myeloma (MM) and the underlying mechanism remain unclear. Here, we revealed that PIWIL1 was highly expressed in myeloma cell lines and newly diagnosed MM patients, and that its expression was notably higher in refractory/relapsed MM patients. PIWIL1 promoted the proliferation of MM cells and conferred resistance to chemotherapeutic agents both in vitro and in vivo. More importantly, PIWIL1 enhanced the formation of autophagosomes, especially mitophagosomes, by disrupting mitochondrial calcium signaling and modulating mitophagy-related canonical PINK1/Parkin pathway protein components. Mitophagy/autophagy inhibitors overcome PIWIL1-induced chemoresistance. In addition, PIWIL1 overexpression increased the proportion of side population (SP) cells and upregulated the expression of the stem cell-associated genes Nanog, OCT4, and SOX2, while its inhibition resulted in opposite effects. Taken together, our findings demonstrated that PIWIL1 induced drug resistance by activating mitophagy and regulating the MM stem cell population. PIWIL1 depletion significantly overcame drug resistance and could be used as a novel therapeutic target for reversing resistance in MM patients.

The impact of the RASSF1C and PIWIL1 on DNA methylation: the identification of GMIP as a tumor suppressor.

INTRODUCTION: Recently we have identified a novel RASSF1C-PIWIL1-piRNA pathway that promotes lung cancer cell progression and migration. PIWI-like proteins interact with piRNAs to form complexes that regulate gene expression at the transcriptional and translational levels. We have illustrated in previous work that RASSF1C modulates the expression of the PIWIL1-piRNA gene axis, suggesting the hypothesis that the RASSF1C-PIWI-piRNA pathway could potentially contribute to lung cancer stem cell development and progression, in part, through modulation of gene methylation of both oncogenic and tumor suppressor genes. Therefore, we tested this hypothesis using a non-small cell lung cancer (NSCLC) cell model to identify Candidate Differentially Methylated Regions (DMRs) modulated by the RASSF1C-PIWIL1-piRNA pathway. MATERIALS AND METHODS: We studied the impact of over-expressing RASSF1C and knocking down RASSF1C and PIWIL1 expression on global gene DNA methylation in the NSCLC cell line H1299 using the Reduced Representation Bisulfite Sequencing (RRBS) method. RESULTS: DMRs were identified by comparing DNA methylation profiles of experimental and control cells. Over-expression of RASSF1C and knocking down RASSF1C and PIWIL1 modulated DNA methylation of genomic regions; and statistically significant candidate genes residing DMR regions in lung cancer cells were identified, including oncogenes and tumor suppressors. One of the hypermethylated genes, Gem Interacting Protein (GMIP), displays tumor suppressor properties. GMIP expression attenuates lung cancer cell migration, and its over-expression is associated with longer survival of lung cancer patients. CONCLUSIONS: The RASSF1C-PIWI-piRNA pathway modulates key oncogenes and tumor suppressor genes. GMIP is hypermethylated by this pathway and has tumor suppressor properties.

[Single nucleotide polymorphism of rs28416520 in Piwil1 gene promoter region is associated with an increased risk of gastric cancer].

OBJECTIVE: To analyze the correlation between the single nucleotide polymorphisms (SNPs) in the promoter of Piwil1 gene and gastric cancer. METHODS: The expression of Piwil1 mRNA in the tumor tissues of 3 patients with gastric cancer was detected by RT-qPCR, and RNA-Sequencing data from the Cancer RNA-Seq Nexus were analyzed for Piwil1 mRNA expression in gastric patients. Blood samples were collected from 24 gastric cancer patients and 29 healthy control subjects for PCR amplification of Piwil1 gene promoter region. The SNP loci in the promoter region of Piwil1 gene were determined by direct sequencing, and the results were analyzed by SnapGene software. RESULTS: Analysis of the data from Cancer RNA-Seq Nexus and the results of RT-qPCR in 3 gastric cancer patients all showed significantly increased Piwil1 expression in gastric cancer tissues compared with the adjacent tissues. Seven SNP loci in two CpG regions of the Piwil1 gene promoter were genotyped, and only one SNP locus was found to be related to gastric cancer. The frequencies of GG, GA, and AA genotypes at the rs28416520 locus in CpG 67 region were 79.2%, 16.7%, and 4.1% in the gastric cancer group, and were 37.9%, 55.2%, and 6.9% in the control group, respectively, showing a significantly higher frequency of the GG genotype in gastric cancer group (OR=0.144, 95%CI: 0.045-0.564, χ2=9.071, P < 0.01). The frequency of allele G of the rs28416520 locus was significantly higher in gastric cancer group than in the control group (87.5% vs 65.5%; OR=0.271, 95%CI: 0.099-0.766, χ2=6.856, P < 0.01). The genotype or allele frequencies of the other 6 SNPs locus did not differ significantly between gastric cancer group and control group. CONCLUSIONS: The expression of Piwil1 is increased in gastric cancer tissues as compared with the adjacent tissues. The GG genotype and G allele of rs28416520 within CpG 67 region are associated with an increased risk of gastric cancer.

Long non-coding RNA FALEC promotes colorectal cancer progression via regulating miR-2116-3p-targeted PIWIL1.

BACKGROUND: Colorectal cancer (CRC) is one of the most common digestive malignant tumors globally. Focally amplified lncRNA on chromosome 1 (FALEC) is a novel lncRNA that has been reported to be involved in many biological processes during carcinogenesis. However, its role in CRC remains poorly understood. METHODS: Gene expression at mRNA or protein level was measured by qRT-PCR or western blot, respectively. In vitro experiments including EdU, colony formation, flow cytometry, wound-healing and transwell assays, as well as in vivo xenograft experiment, were utilized to determine the functional role of FALEC in CRC. Relevant mechanical assays were performed to investigate the underlying molecular mechanism. RESULTS: FALEC was aberrantly up-regulated in CRC. FALEC knockdown could impair CRC cell proliferation, migration and invasion, whereas facilitate cell apoptosis. MiR-2116-3p was revealed to be sponged by FALEC. PIWIL1 was identified as the target of miR-2116-3p. Mechanically, FALEC restored the expression of PIWIL1 via absorbing miR-2116-3p. MiR-2116-3p inhibition and PIWIL1 enrichment could counteract the anti-tumor impact induced by silenced FALEC on the oncogenic behaviors of CRC cells. CONCLUSION: Our study revealed that FALEC promoted CRC progression via restoring the expression of miR-2116-3p-targeted PIWIL1, suggesting the potential application of targeting FALEC in the treatment of CRC.