PIWIL2 Expression Research Articles

Co-expression of Piwil2/Piwil4 in nucleus indicates poor prognosis of hepatocellular carcinoma.

PurposeThis study aimed to explore the localization and expression of P-element-induced wimpy testis-like 2 (piwil2)/Piwil4 in hepatocellular carcinoma (HCC) tissues, and analyze the correlation between co-expression pattern and prognosis of HCC.ResultsPiwil2 showed 100% positive expression in the cell nucleus, with the intensity higher than in the cytoplasm. Piwil4 showed a lower intensity of expression in the cell nucleus than in the cytoplasm. The molecular chaperone Piwil2/Piwil4 had four co-expression patterns: nuclear co-expression, nuclear and cytoplasmic co-expression, cytoplasmic co-expression, and non-coexpression. The survival rate and the overall survival sequentially increased. The prognostic phenotype of the nuclear co-expression of Piwil2/Piwil4 was worse than that of non-coexpression, and the intracellular localization and expression of Piwil2 and Piwil4 were not significantly different.MethodsHCC pathological tissue samples with follow-up information (90 cases) and 2 normal control liver tissues were collected and made into a 92-site microarray. The expression of Piwil2 and Piwil4 was detected using the immunofluorescence double staining method. The differences in the expression and location of Piwil2 and Piwil4 in tumor cells were explored, and the influence of such differences on the long-term survival rate of HCC was studied using Kaplan-Meier survival curve and log-rank test. The clinical staging was analyzed according to the HCC international TNM staging criteria.ConclusionsThe nuclear co-expression of Piwil2/Piwil4 indicated that patients with HCC had a worse prognostic phenotype. The molecular chaperone Piwil2/Piwil4 seems promising as a molecular marker for prognosis judgment; a single marker (Piwil2/Piwil4) cannot be used for prognosis judgment.

Amniotic membrane mesenchymal stem cells can differentiate into germ cells in vitro.

This is the first report on differentiation of mouse amniotic membrane mesenchymal stem cells (AM-MSCs) into male germ cells (GCs). AM-MSCs have the multipotent differentiation capacity and can be differentiated into various cell types. In the present study, AM-MSCs were induced for differentiation into GCs. AM-MSCs were isolated from mouse embryonic membrane by enzymatic digestion. AM-MSCs were characterized with osteogenic and adipogenic differentiation test and flow cytometric analysis of some CD-markers. AM-MSCs were induced to differentiate into GCs using a creative two-step method. Passage-3 AM-MSCs were firstly treated with 25ng/ml bone morphogenetic protein 4 (BMP4) for 5d and in continuing with 1μM retinoic acid (RA) for 12d (total treatment time was 17d). At the end of the treatment period, real-time reverse transcription (RT)-PCR was performed to evaluate the expression of GC-specific markers-Itgb1, Dazl, Stra8, Piwil2, Mvh, Oct4, and c-Kit- in the cells. Moreover, flow cytometry and immunofluorescence staining were performed to evaluate the expression of Mvh and Dazl at protein level. Real-time RT-PCR showed that most of the tested markers were upregulated in the treated AM-MSCs. Furthermore, flow cytometric and immunofluorescence analyses both revealed that a considerable part of the treated cells expressed GC-specific markers. The percentage of positive cells for Mvh and Dazl was about 23 and 46%, respectively. Our results indicated that a number of AM-MSCs successfully differentiated into the GCs. Finally, it seems that AM-MSCs would be a potential source of adult pluripotent stem cells for in vitro generation of GCs and cell-based therapies for treatment of infertility.

Genomic identification, rapid evolution, and expression of Argonaute genes in the tilapia, Oreochromis niloticus.

Argonaute proteins are key components of the small RNA-induced silencing complex and have multiple roles in RNA-directed regulatory pathways. Argonaute genes can be divided into two subfamilies: the Ago (interacting with microRNA/small interfering RNA) and Piwi subfamilies (interacting with piwi-interacting RNAs (piRNAs)). In the present study, genome-wide analyses firstly yielded the identification of different members of Agos and Piwis in the tilapia, coelacanth, spotted gar, and elephant shark. The additional teleost Ago3b was generated following the fish-specific genome duplication event. Selective pressure analysis on Agos and Piwis between cichlids and other teleosts showed an accelerated evolution of Piwil1 in the cichlid lineages, and the positive selected sites were located in the region of PIWI domain, suggesting that these amino acid substitutions are adapt to targeted cleavage of messenger RNA (mRNA) in cichlids. Ago1 and Ago4 were detected at higher levels at 5days after hatching (dah) in both ovaries and testes compared with other stages, supporting the previously reported requirement of Ago-mediated pathways to clear the maternal mRNAs during the early embryogenesis. The Piwis were abundantly expressed in tilapia testes, indicating their essential roles in male germline, especially in spermatogenesis. Notable expression of Piwis was also detected in skeletal muscle, indicating that piRNA pathway may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. The expression of Piwil1 and Piwil2 was examined by quantitative PCR (qPCR) and in situ hybridization (ISH) to validate the spatial and temporal expression profiles. Taken together, these results present a thorough overview of tilapia Argonaute family and provide a new perspective on the evolution and function of this family in teleosts.

The Role of PIWIL4, an Argonaute Family Protein, in Breast Cancer

P-element-induced wimpy testis (PIWI) proteins bind to PIWI-interacting RNAs and play key roles in the biogenesis and functions of PIWI-interacting RNAs. It has been reported that PIWI proteins are essential for stem cell self-renewal and germline development in diverse organisms and that they are ectopically expressed in multiple forms of cancer. However, the role of PIWI in cancer remains elusive. Here we report that one of the four PIWI proteins in humans, PIWIL4, is highly expressed in both breast cancer tissues and the cytoplasm of MDA-MB-231 cells derived from breast cancer. Reducing PIWIL4 expression drastically impairs the migration ability of MDA-MB-231 cells, significantly increases their apoptosis, and mildly affects their proliferation. Our transcriptome and proteome analysis reveal that these functions are at least partially achieved via the PIWIL4 regulation of TGF-β and FGF signaling pathways and MHC class II proteins. These findings suggest that PIWIL4 may serve as a potential therapeutic target for breast cancer.

Identification of PIWIL1 Isoforms and Their Expression in Bovine Testes, Oocytes, and Early Embryos.

PIWI proteins are members of the larger Argonaute family and bind to specific 24-32 nucleotide RNAs called PIWI-interacting RNAs (piRNAs). PIWI-interacting RNAs direct PIWI-mediated suppression of retrotransposon expression in the male germline in humans and mice, but their roles in bovine reproduction and embryogenesis are unknown. Although the majority of research in mammals has focused on the functions of PIWI proteins during spermatogenesis, this family of proteins and their associated piRNAs have recently been identified in early embryos. The goals of this study were to characterize the expression of PIWIL1 in bovine testis, oocytes, and early embryos. A full-lengthPIWIL1transcript and protein was found in the testis, specifically in the germs cells of mature seminiferous tubules. RNA-immunoprecipitation demonstrated the presence of putative piRNAs with a mean length of 30 nucleotides bound to PIWIL1 in testes. 3'-Rapid amplification of cDNA ends analysis ofPIWIL1transcripts in testes and oocytes revealed two shorter isoforms in addition to the full-length transcript that was only present in testes. TruncatedPIWIL1isoforms in oocytes and testes were confirmed through amplification of their unique intronic fragments. Expression profiling ofPIWIL1through early embryogenesis demonstrated peak mRNA expression at the 2-cell stage with decreasing levels through to the blastocyst. PIWIL1-YFP fusion plasmids were produced for each isoform and expressed in HEK 293 cells, demonstrating nuclear exclusion and size-specific banding of the different isoforms. These data represent the first comprehensive characterization of PIWIL1 in bovine, revealing functional similarities with PIWIL1 in other species and suggest tissue-specific expression of several isoforms.

Piwil1 mediates meiosis during spermatogenesis in chicken

Piwil1 mediates spermatogenesis and ensures stable cell division rates in germline cells in mammals. However, the involvement of Piwil1 in poultry spermatogenesis and meiosis is poorly understood. In the present study, we used TaqMan RT-qPCR to characterize Piwil1 mRNA expression in different types of spermatogenic cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia cells (Sa), tetraploid cells (Tp), round sperm cells (Rs), mature sperm, and in PGCs treated with retinoic acid. Our results revealed that Piwil1 is differentially expressed during spermatogenesis in chicken. Compared to PGCs, SSCs, Tp, and Sa, Rs cells presented the highest Piwil1 mRNA expression levels. Retinoic acid significantly upregulated Piwil1 and Stra8 mRNA expression as well as Piwil1 levels in chicken PGCs. In addition, retinoic acid induced PGCs to progress through all the meiotic stages, eventually leading to haploid cell formation, which was determined using flow cytometry and western blot analysis. Taken together, our results showed that during spermatogenesis, Piwil1 was first expressed at low levels in germ stem cells, PGCs, and SSCs. Its expression levels increased during later meiosis stages. Finally, no expression was detected in mature sperm after meiosis. Treatment of PGCs with retinoic acid further demonstrated that Piwil1 plays a key role in meiosis during chicken spermatogenesis.

Expression Levels of MMP9 and PIWIL2 in Prostate Cancer: a Case-Control Study

Prostate cancer is the second most prevalent cancer in humans. Also, this is the most common malignancy and the sixth most important cause of death in men worldwide. The most routine diagnostic test for prostate cancer is PSA test which is associated with some limitations like too many false positive results. This study intends to investigate the role of MMP9 and PIWIL2 expression levels as different biomarkers in prostate cancer biopsy specimens. This study is one of the most brilliant studies in the field of prostate cancer research for the first time focusing on the investigation of the role of two different genes in prostate cancer in biopsy specimens and on formalin-fixed paraffin-embedded tissue types in order to detect the progression and prognosis of prostate cancer patients in early stages. Seventy formalin-fixed paraffin embedded samples (35 normal and 35 cancerous cases) were selected. Expression levels of PIWIL2 and MMP9 genes were evaluated, using real-time PCR. MMP9 and PIWIL2 expression levels in cancerous tissues were significantly higher than the adjacent normal tissues (p < 0.05). The survival analysis showed a significant correlation between expression level of PIWIL2 and survival rate (p < 0.05), but such correlation was not observed in case of MMP9 (p > 0.05). Higher levels of MMP9 and PIWIL2 expression were strongly related to the Gleason score and age, using Pearson's correlation co- efficient test; however, this kind of association was not evident between prostate specific antigen (PSA) and expression levels of the genes of interest. The expression level of PIWIL2 had a significant correlation with metastasis rate, but this relationship was not seen in the case of MMP9. The results confirmed the validity of PIWIL2 expression as a valuable prognostic biomarker for early diagnosis of prostate cancer.

Decreased expression levels of PIWIL1, PIWIL2, and PIWIL4 are associated with worse survival in renal cell carcinoma patients

Piwi-interacting RNAs (piRNAs) are a newly discovered class of small non-coding RNAs involved in silencing of transposable elements and in sequence-specific chromatin modifications. PIWI proteins (PIWIL), which belong to the family of Argonaute genes/proteins, bind to piRNAs and function mainly in germ line cells, but more recently were described to be functional also in stem cells and cancer cells. To date, there have been four PIWI proteins discovered in humans: PIWIL1, PIWIL2, PIWIL3, and PIWIL4. Recent studies suggested that deregulated expression of PIWI proteins and selected piRNAs is common to many types of cancers. We found significantly lower expression of PIWIL1 (P<0.0001) and piR-823 (P=0.0001) in tumor tissue in comparison to paired renal parenchyma. Further, we observed a progressive decrease in PIWIL1 (P=0.0228), PIWIL2 (P=0.0015), and PIWIL4 (P=0.0028) expression levels together with increasing clinical stage. PIWIL2 (P=0.0073) and PIWIL4 (P=0.0001) expression also progressively decreased with increasing Fuhrman grade. Most importantly, low-expression levels of PIWIL1 (P=0.009), PIWIL2 (P<0.0001), and PIWIL4 (P=0.0065) were significantly associated with worse overall survival in renal cell carcinoma (RCC) patients. Our results suggest the involvement of PIWIL genes and piR-823 in RCC pathogenesis, and indicate PIWIL1, PIWIL2, and PIWIL4 as potential prognostic biomarkers in patients with RCC.

DNA methylation-mediated transcription factors regulate Piwil1 expression during chicken spermatogenesis.

The P-element induced wimpy testis (Piwi) protein family is responsible for initiating spermatogenesis and maintaining the integrity of germ cells and stem cells, but little is known regarding its transcriptional regulation in poultry. Here, we characterized the methylation status of the Piwil1 promoter in five different spermatogenic cell lines using direct bisulfite pyrosequencing and determined that methylation correlates negatively with germ cell type-specific expression patterns of piwil1. We demonstrated that methylation of the −148 CpG site, which is the predicted binding site for the transcription factors TCF3 and NRF1, was differentially methylated in different spermatogenic cells. This site was completely methylated in PGCs (primordial germ cells), but was unmethylated in round spermatids. A similar result was obtained in the region from +121 to +139 CpG sites of the Piwil1 promoter CpG island, which was predicted to contain SOX2 binding sites. In addition, demethylation assays further demonstrated that DNA methylation indeed regulates Piwil1 expression during chicken spermatogenesis. Combined with transcription factor binding site prediction, we speculate that methylation influences the recruitment of corresponding transcription factors. Collectively, we show the negative correlation between promoter methylation and piwil1 expression and that the spatiotemporal expression of chicken Piwil1 from the PGC stage to the round spermatid stage is influenced by methylation-mediated transcription factor regulation.

The Evaluation of Cancer Testis Gene PIWIL2 Expression Levels as a New Prognostic Biomarker for Breast Cancer.

Breast cancer is the most prevalent cancer and foremost reason of death resulting from malignancy among women worldwide. In recent years, it has been reported that a group of genes named cancer testis genes (CTg) express in adult immune privileged sites and some embryonic tissues. Likewise, it has been demonstrated that CTgs express aberrantly in various tumors and play essential roles in both initiation and development of cancers. In this study, PIWIL2, one member of CTgs, which has an indispensable role in spermatogenesis and tumorigenesis, was examined as an efficient prognostic and diagnostic biomarker in breast cancer. It is worth mentioning that the expression study of PIWIL2 by qPCR on breast cancer samples was performed for the first time, since this approach is much more sensitive than western blot, RT-PCR, and immunohistochemistry. To investigate the expression status of PIWIL2 in breast cancer, 72 fresh cancerous and normal adjacent specimens from 44 patients who had undergone surgery in Shahid Faghihi Hospital were used. None of the patients had received chemotherapy. The relationships between the PIWIL2 status and the clinicopathological parameters were ultimately determined. It was ascertained that the expression rates of PIWIL2 in cancerous breast tissues were related to patients' age, tumor stage, tumor size, tumor grade, and lymph node involvement (p < 0.05). The PIWIL2 gene could be considered as an efficient prognostic biomarker in breast cancer.

PIWIL1 Is Expressed in the Canine Testis, Increases with Sexual Maturity, and Binds Small RNAs.

Spermatogenesis is a highly regulated process leading to the development of functional spermatozoa through meiotic division and subsequent maturation. Recent studies have suggested that a novel class of Argonaute proteins, known as the PIWI clade, plays important roles in multiple stages of spermatogenesis. PIWI proteins bind specific small noncoding RNAs, called PIWI-interacting RNAs (piRNAs). These piRNAs guide the PIWI-piRNA complex to retrotransposon targets that become expressed during meiosis. Retrotransposons are subsequently silenced, either through PIWI "slicer" activity or through PIWI-directed methylation of the retrotransposon locus. Most mammalian studies have employed mouse models where sterility follows PIWI inactivation. The goal of this study was to characterize canine PIWIL1 to determine whether expression pattern and functional characteristics support a similar function in that species. Canine PIWIL1 cDNA is a 2.6-kb transcript that encodes an 861-amino acid protein showing high homology to other mammalian PIWIL1 proteins and containing features consistent with PIWI family members (PAZ, PIWI domains). Analysis of PIWIL1 protein and transcript levels revealed that PIWIL1 expression is limited to the testes and is associated with sexual maturity, with mature dogs showing higher levels of PIWIL1 expression. Immunohistochemistry demonstrated expression primarily in seminiferous tubules and confirmed higher levels of PIWIL1 in mature dogs. Functional characterization by RNA immunoprecipitation demonstrated that canine PIWIL1 binds short RNAs consistent in size with piRNAs (27-32 nucleotides). Together, these studies represent the first characterization of a PIWI protein in the dog and suggest that it is a functional piRNA-binding protein most highly expressed in the mature testes.

Sodium orthovanadate induces the apoptosis of SH-SY5Y cells by inhibiting PIWIL2.

PIWIs have been shown to be abnormally expressed in a variety of cancers and may be important in the maintenance and invasion of cancer cells. The high expression of PIWIL2 contributed to the resistance effect of cisplatin in colon cancer cells, and the knockout of the PIWIL2 gene reduced the aggressive nature and malignant degree of colon cancer cells. Sodium orthovanadate (SOV) is a vanadium compound, and exhibited antineoplastic activity in certain types of human cancer cells, including lung, kidney and prostate cancer cells. However, its effects in human neuroblastoma (NB) cells have not yet been reported. The objective of this study was to investigate the effect of SOV on the apoptosis of NB cells and to explore how PIWIL2 is involved in the mechanism underlying this effect. In the present study, SH‑SY5Y cells were treated with SOV and the optimal concentration was determined for further assays. Cell apoptosis, cell count, viability, the cell cycle, and the expression of PIWIL2 mRNA and protein were then determined. The results showed that SOV could induce cell apoptosis, reduce the percentage of viable cells, induce accumulation of SH‑SY5Y cells at the G2/M and S phase of the cell cycle, and inhibit the expression of PIWIL2 and Bcl‑2 mRNA and protein. The results suggested that the underlying mechanisms may be, at least in part, due to SOV inhibiting the expression of PIWIL2. These findings demonstrated the effect of SOV and supported its further evaluation as a treatment for human NB.

Stem cell protein Piwil1 endowed endometrial cancer cells with stem-like properties via inducing epithelial-mesenchymal transition.

BackgroundStem cell protein Piwil1 functions as an oncogene in various tumor types. However, the exact function and mechanism of Piwil1 in endometrial cancer remains unclear.MethodsThe expression of Piwil1 and its relationships with clinicopathological factors were investigated using immunohistochemistry. Up- or down-regulation of Piwil1 were achieved by stable or transient transfection with plasmids or short hairpin RNA (shRNA). Effects of Piwil1 on cancer cells viability, invasion and migration were evaluated by MTT, plate colony formation, transwell assay and nude mouse tumor xenograft assay. The stem-like properties of endometrial cancer cells was detected by spheroid formation assay. Effects of Piwil1 on expression levels of target genes were detected by qRT-PCR, western blotting and Immunofluorescence.ResultsCompared with atypical hyperplasia and normal tissues, Piwil1 was much higher in endometrial carcinoma tissues. We found that Piwil1 expression was significantly correlated with FIGO stage, lymphovascular space involvement, lymph node metastasis and level of myometrial invasion. Overexpression of Piwil1 functioned to maintain stem-like characteristics, including enhancing tumor cell viability, migration, invasion and sphere-forming activity. Conversely, Piwil1 knockdown inhibited cell viability, migration, invasion, sphere-forming activity in vitro and tumor formation in xenograft model in vivo. Furthermore, study of the expression of epithelial and mesenchymal markers showed that Piwil1 was responsible for an EMT-like phenotype associated with an increase in mesenchymal markers and suppression of E-cadherin. Moreover, Piwil1 augmented expression levels of CD44 and ALDH1 expression, two known endometrial CSC markers, as well as other stemness-associated genes.ConclusionsOur results suggested that stem cell protein Piwil1 play important roles in regulating EMT and the acquisition of stem-like properties of endometrial cancer cells. Therefore, it indicated that Piwil1 may represent a promising target for developing a novel treatment strategy for endometrial cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1794-8) contains supplementary material, which is available to authorized users.

In vitro differentiation process of human Wharton's jelly mesenchymal stem cells to male germ cells in the presence of gonadal and non-gonadal conditioned media with retinoic acid.

Human umbilical Wharton's jelly-derived mesenchymal stem cells (HWJMSCs) are the best candidate to get plentiful stem cells and differentiate them to germ cells under appropriate conditions to treat infertility. We sought to determine under which conditions HWJMSCs could form male germ cells in vitro. So, HWJMSCs were differentiated to male germ cells under a mixture of bone morphogenetic protein-4 (BMP-4) and testicular and placental culture condition (TCC and PCC) medium followed by retinoic acid for 21d. In the present study, the HWJMSCs were obtained from Wharton's jelly of umbilical cords of male neonates delivered by cesarean section. At the third passage, mesenchymal stem cell markers and differentiation to osteocytes and adipocytes were investigated. Then, HWJMSCs were induced to differentiate into male germ cells in the presence of BMP-4, all-trans retinoic acid, PCC, and TCC for 21d. The profile of c-Kit, DDX4, Piwil2, and Dazl gene expression was evaluated by qPCR and ICC. Data was analyzed by ANOVA test. After 3wk of treatment with different reagents, the morphology of these spindle-like cells changed to shiny clusters and germ cell-specific markers in mRNA were upregulated in both TCC + retinoic acid (RA) and BMP-4 + RA. Induction of HWJMSCs with TCC in the presence of RA resulted in significant upregulation (P ≤ 0.05) of all germ cell-specific genes (c-Kit 2.6795 ± 0.75, DDX4 4.3188 ± 1.18, Piwil2 4.9962 ± 1.55, Dazl 6.1199 ± 0.78) compared to control and PCC + RA. Our results indicated that TCC and RA are involved in human germ cell development. Moreover, BMP signaling also induced differentiation. Our findings provide a novel effective approach for generation of germ cells in vitro and studying the interaction of germ cells with their niche. Our work represents an essential step toward gaining knowledge of the molecular properties of HWJMSCs in the field of cell therapy. We demonstrated that under a suitable situation, HWJMSCs have the ability to differentiate into germ cells and this provides an excellent pattern to study infertility cause and cure.

DNA methylation and NF-Y regulate Piwil1 expression during chicken spermatogenesis

The P-element induced wimpy testis (Piwi) protein family, a subfamily of the Argonaute protein family, is involved in gene silencing and shows specific expression in spermatogenic cells. To reveal the transcriptional regulatory mechanisms of Piwil1 in chickens, we cloned sequences of the chicken Piwil1 promoter region and performed luciferase reporter and electrophoretic mobility shift assays to analyze the transcriptional activity and identify important transcriptional regulatory elements. The results showed that the region from −90 to −43 in the 5′-flanking region of Piwil1 contains a transcriptional regulatory CCAAT box that was necessary for the transcriptional activity of the Piwil1 promoter. Moreover, the transcription factor nuclear factor Y (NF-Y) was bound to the Piwil1 promoter CCAAT box specifically in germ cells. In addition, bisulfite sequencing to determine the methylation profile of the Piwil1 promoter CpG island in different spermatogenic and non-germ cell populations was performed. Compared with germ cells, non-germ cells showed increased methylation of the promoter region containing the CCAAT box, loss of NF-Y binding, and silencing of the Piwil1 locus. It is demonstrated that the specific expression of Piwil1 in chicken germ cells is regulated by the transcription factor NF-Y and differential CpG island methylation.

PIWIL2 promotes progression of non-small cell lung cancer by inducing CDK2 and Cyclin A expression

BackgroundPIWI proteins have important roles in tumorigenesis due to their interaction with piRNAs. Recent studies suggest that PIWI proteins affect prognosis of various cancers.MethodsIn the present study, PIWI genes expression was assayed in non-small cell lung cancer (NSCLC). To determine the effects of PIWIL2 on NSCLC cells, overexpression and interference assays were performed using the A549 and H460 cell lines. The tumor formation model was performed to demonstrate the effects of PIWIL2 on tumor formation in vivo.ResultsPIWIL2 was increased both at the RNA and protein level in malignant cancer tissues compared with adjacent normal tissue. Moreover, increased PIWIL2 gene expression was negatively correlated with prognosis in NSCLC patients. Overexpression and interference of PIWIL2 promoted and depressed cell proliferation, respectively. Meanwhile, PIWIL2 interference arrested cells at the G2/M stage. In addition, we found that CDK2 and Cyclin A expression were correlated with PIWIL2 expression. Moreover, transfection of PIWIL2 promoted tumor growth in nude mice.ConclusionOur findings shed light on the function of PIWIL2 in NSCLC and suggest potential prognostic and therapeutic value.

Abstract 237: Piwi genes and tissue/serum piR-651 are related to clinicopathologic features of renal cell carcinoma

Abstract Introduction: Piwi-interacting RNAs (piRNAs) consists a newly discovered class of non-coding RNA. PiRNAs were first identified in the germ cells of various animal species. They bind to Piwi proteins which are a subfamily of Argonaut proteins. In humans were identified four Ago genes and four Piwi genes. Human Piwi genes include PIWIL1, PIWIL2, PIWIL3 and PIWIL4. Recent studies suggest that deregulated expression of Piwi proteins is common to many types of tumors and also correlate with clinicopathologic features and worse prognosis in patients with breast, cervical, ovarian, colorectal and other cancers. PiRNAs are short single-stranded RNAs with 26-31 nucleotides in length. They are involved in silencing of transposable elements and it is assumed that also participate in sequence-specific chromatin modifications. The differential expression of piRNAs was found in gastric and breast cancer. In our pilot study we analyzed expression of Piwi genes and piR-651, which was previously found to be deregulated in various types of tumors. Patients and Methods: In our study, we have used the tumor tissue and the paired renal parenchyma tissue of 56 patients with renal cell carcinoma (RCC). From the tissue was isolated total RNA and by RT-qPCR were examined the expression of Piwi genes and piR-651. For the analysis of circulating piR-651, blood serum samples of 75 patients with RCC and 75 age, gender-matched healthy donors were used. We have compared expression levels of the studied genes in tumor and non-tumor tissues, serum samples, and also correlated them with clinicopathologic features of RCC patients (stage, grade, RFS, OS). Results: We found a significant down-regulation of PIWIL1 (p&amp;lt;0,0001) and piR-651 (p&amp;lt;0,0001) expression levels in tumor tissue when compared to paired renal parenchyma samples. The expression levels of PIWIL2, PIWIL3 and PIWIL4 were not significantly deregulated in tumor tissue. Interestingly, levels of PIWIL2 and PIWIL4 correlated significantly with the stage (p = 0,002 and p = 0,003, resp.) and grade (p = 0.007 and p&amp;lt;0.0001, resp.) of RCC. We found also a correlation between higher levels of PIWIL1 and overall survival (p&amp;lt;0.05). We have observed significantly decreased expression levels of piR-651 in blood serum samples of RCC patients when compared to healthy donors and accordingly to ROC analysis we were able to distinguish blood serum of RCC patients and control subjects with the sensitivity of 77.33% and a specificity of 72.37%. Expression levels of serum piR-651 were not in correlation with the stage and grade of RCC. Conclusion: Accordingly to our pilot data expression of Piwi genes is altered in tumor tissue and is correlated to selected clinicopathologic features of RCC. We also suggest the potential of piR-651 in blood serum as novel non-invasive diagnostic biomarker in RCC. Acknowledgments: IGA MZCR No: NT/13860-4/2012, NT/13549-4/2012, NT/13547-4/2012, NT/13514-4/2012 Citation Format: Robert Iliev, Petra Vychytilova-Faltejskova, Jaroslav Juracek, Hana Mlcochova, Michal Stanik, Jan Dolezel, Michal Fedorko, Dalibor Pacik, Marek Svoboda, Ondrej Slaby. Piwi genes and tissue/serum piR-651 are related to clinicopathologic features of renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2015-237

Piwi proteins and piRNAs in mammalian oocytes and early embryos: From sample to sequence.

The role of the Piwi/piRNA pathway during mammalian oogenesis has remained enigmatic thus far, especially since experiments with Piwi knockout mice did not reveal any phenotypic defects in female individuals. This is in striking contrast with results obtained from other species including flies and zebrafish. In mouse oocytes, however, only low levels of piRNAs are found and they are not required for their function. We recently demonstrated dynamic expression of PIWIL1, PIWIL2, and PIWIL3 during mammalian oogenesis and early embryogenesis. In addition, small RNA analysis of human, crab-eating macaque and cattle revealed that piRNAs are also expressed in the female germline and closely resemble piRNAs from testis. Here, we thoroughly describe the experimental and computational methods that we applied for the generation, processing and analyses of next generation sequencing (NGS) data associated with our study on Piwi proteins and piRNAs in mammalian oocytes and embryos (Roovers et al., 2015). The complete sequence data is available at NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession GSE64942.

Piwil2 modulates the invasion and metastasis of prostate cancer by regulating the expression of matrix metalloproteinase-9 and epithelial-mesenchymal transitions.

Piwi-like RNA-mediated gene silencing 2 (Piwil2) is an oncogene that is highly expressed in breast, gastric, colorectal and papillary thyroid cancer. As a candidate oncogene, its role in prostrate cancer has yet to be elucidated. In the present study, 30 tumor specimens and four prostate cancer cell lines were analyzed. The total RNA and protein from the specimens and the cells were analyzed using quantitative polymerase chain reaction and western blotting, respectively. The expression of Piwil2 in PC-3 cells was knocked down using specific small hairpin RNA. Transwell assays and wound-healing models were used to assess cell invasion and migration. In addition, the expression of several factors associated with epithelial-mesenchymal transitions (EMT) were evaluated by western blotting. The results revealed that the Piwil2 gene was associated with the Gleason score and the tumor-node-metastasis stage of the tumor tissues. Cell invasion and migration decreased significantly in PC-3 cells with knocked-down Piwil2. In addition, silencing Piwil2 downregulated the expression of N-cadherin, Twist and vimentin and upregulated the expression of E-cadherin, factors associated with EMT, and also reduced the expression of matrix metalloproteinase-9. Piwil2 was demonstrated to possess an important role in the invasive ability of prostate cancer, and therefore, may be a potential therapeutic target for the treatment of this cancer.

The clinical significances of the abnormal expressions of Piwil1 and Piwil2 in colonic adenoma and adenocarcinoma.

ObjectiveThe objective of the present investigation was to study the clinical significances of the abnormal expressions of Piwil1 and Piwil2 protein in colonic adenoma and adenocarcinoma.MethodsThis study had applied immunohistochemical method to detect 45 cases of tissues adjacent to carcinoma (distance to cancerous tissue was above 5 cm), 41 cases of colonic adenoma and 92 cases of colon cancer tissues, and their Piwil1 and Piwil2 protein expression levels.AnalysisThe correlation of both expression and its relationship with clinicopathological features of colon cancer was analyzed.ResultsPositive expression rates of Piwil1 in tissues adjacent to carcinoma, colonic adenoma, and colon cancer were 11.1% (5/45), 53.7% (22/41), and 80.4% (74/92), respectively; the expression rates increased, and the comparisons between each two groups were statistically significant (P<0.05). In each group, the positive expression rates of Piwil2 were 24.4% (11/45 cases), 75.6% (31/41 cases), and 92.4% (85/92 cases); expression rates increased, and the comparisons between each two groups were statistically significant (P<0.05). Piwil1 expression and the correlation of the degree of differentiation, TNM stage, and lymph node metastasis were statistically significant (P<0.05). Piwil2 expression and the correlation of the degree of differentiation, tumor node metastasis (TNM) stage, and lymph node metastasis had no statistical significance (P>0.05). In colon cancer tissue, Piwil1 and Piwil2 expressions were positively correlated (r=0.262, P<0.05).ConclusionThe results showed that the abnormal expression of Piwil1 and Piwil2 might play an important role in the process of colon cancer development.