Interplay of spermatogonial subpopulations during initial stages of spermatogenesis in adult primates.

The spermatogonial compartment maintains spermatogenesis throughout the reproductive lifespan. Single-cell RNA sequencing (scRNA-seq) has revealed the presence of several spermatogonial clusters characterized by specific molecular signatures. However, it is unknown whether the presence of such clusters can be confirmed in terms of protein expression and whether protein expression in the subsets overlaps. To investigate this, we analyzed the expression profile of spermatogonial markers during the seminiferous epithelial cycle in cynomolgus monkeys and compared the results with human data. We found that in cynomolgus monkeys, as in humans, undifferentiated spermatogonia are largely quiescent, and the few engaged in the cell cycle were immunoreactive to GFRA1 antibodies. Moreover, we showed that PIWIL4+ spermatogonia, considered the most primitive undifferentiated spermatogonia in scRNA-seq studies, are quiescent in primates. We also described a novel subset of early differentiating spermatogonia, detectable from stage III to stage VII of the seminiferous epithelial cycle, that were transitioning from undifferentiated to differentiating spermatogonia, suggesting that the first generation of differentiating spermatogonia arises early during the epithelial cycle. Our study makes key advances in the current understanding of male germline premeiotic expansion in primates.

PLIC11 drives lung cancer progression through regulating the YY1/PIWIL4 axis.

Lung cancer is the leading cause of cancer related deaths worldwide. Nonsmall cell lung cancers (NSCLC), the most common histological type of lung cancer, are known to be less well characterized. Long noncoding RNAs are a new class of cancer regulators. Here, we aimed to investigate the effect of lncRNA PLIC11 in NSCLC progression. In our study, we found that PLIC11 was upregulated in lung cancer, particularly in metastatic lung cancer tissues. Overexpression of PLIC11 enhanced cell proliferation, migration, and metastasis in vitro and in vivo. Mechanically, PLIC11 could interact with YY1 and promote PIWIL4 expression by transcription activation. Therefore, PLIC11 upregulation is a potential indicator of aggressive lung cancer, Silencing of PLIC11 has great potential therapeutic strategy in NSCLC.

PIWIL4 Maintains HIV-1 Latency by Enforcing Epigenetically Suppressive Modifications on the 5' Long Terminal Repeat.

Although substantial progress has been made in depicting the molecular pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection, the comprehensive mechanism of HIV-1 latency and the most promising therapeutic strategies to effectively reactivate the HIV-1 latent reservoir to achieve a functional cure for AIDS remain to be systematically illuminated. Here, we demonstrated that piwi (P element-induced Wimpy)-like RNA-mediated gene silencing 4 (PIWIL4) played an important role in suppressing HIV-1 transcription and contributed to the latency state in HIV-1-infected cells through its recruitment of various suppressive factors, including heterochromatin protein 1α/ß/γ, SETDB1, and HDAC4. The knockdown of PIWIL4 enhanced HIV-1 transcription and reversed HIV-1 latency in both HIV-1 latently infected Jurkat T cells and primary CD4+ T lymphocytes and resting CD4+ T lymphocytes from HIV-1-infected individuals on suppressive combined antiretroviral therapy (cART). Furthermore, in the absence of PIWIL4, HIV-1 latently infected Jurkat T cells were more sensitive to reactivation with vorinostat (suberoylanilide hydroxamic acid, or SAHA), JQ1, or prostratin. These findings indicated that PIWIL4 promotes HIV-1 latency by imposing repressive marks at the HIV-1 5' long terminal repeat. Thus, the manipulation of PIWIL4 could be a novel strategy for developing promising latency-reversing agents (LRAs).IMPORTANCE HIV-1 latency is systematically modulated by host factors and viral proteins. During this process, the suppression of HIV-1 transcription plays an essential role in promoting HIV-1 latency. In this study, we found that PIWIL4 repressed HIV-1 promoter activity and maintained HIV-1 latency. In particular, we report that PIWIL4 can regulate gene expression through its association with the suppressive activity of HDAC4. Therefore, we have identified a new function for PIWIL4: it is not only a suppressor of endogenous retrotransposons but also plays an important role in inhibiting transcription and leading to latent infection of HIV-1, a well-known exogenous retrovirus. Our results also indicate a novel therapeutic target to reactivate the HIV-1 latent reservoir.

Role of PIWI-like 4 in modulating neuronal differentiation from human embryonal carcinoma cells.

PIWI homologs constitute a subclass of the Argonaute family. Traditionally, they have been shown to associate with a specific class of small RNAs, piRNAs, to suppress transposable elements and protect genomic integrity in germ cells. Recent studies imply that PIWI proteins may also exert important biological functions in somatic contexts, including the brain. However, their exact role in neural development remains unknown. Hence we investigated whether PIWI proteins are involved in neuronal differentiation. By using an established cell model for studying neurogenesis, NTera2/D1 (NT2) cells, we found that a particular PIWI homolog, PIWIL4 was increasingly upregulated throughout the course of all-trans retinoic acid (RA)-mediated neuronal differentiation. During this process, PIWIL4 knockdown led to partial recovery of embryonic stem cell markers, while suppressing RA-induced expression of neuronal markers. Consistently, PIWIL4 overexpression further elevated their expression levels. Furthermore, co-immunoprecipitation revealed an RA-induced interaction between PIWIL4 and the H3K27me3 demethylase UTX. Chromatin immunoprecipitation showed that this interaction could be essential for the removal of H3K27me3 from the promoters of RA-inducible genes. By a similar mechanism, PIWIL4 knockdown also suppressed the expression of PTN and NLGN3, two important neuronal factors secreted to regulate glioma activity. We further noted that the conditioned medium collected from PIWIL4-silenced NT2 cells significantly reduced the proliferation of glioma cells. Thus, our data suggest a novel somatic role of PIWIL4 in modulating the expression of neuronal genes that can be further characterized to promote neuronal differentiation and to modulate the activity of glioma cells.

tRNA-derived fragments from the Sus scrofa tissues provide evidence of their conserved role in mammalian development.

The recently discovered group of noncoding RNAs, which are fragments of tRNA molecules (tRFs), has not been fully characterized and its potential functions still require investigation. Porcine tRFs were characterized and compared to mouse and human tRFs. Two tRFs, 5' 32-33 nt and 3' 41-42 nt that are derived from the mature tRNAVal(CAC) and tRNAGly(GCC) were detected with the use of bioinformatics and the Northern blot method. The abundance of these tRFs in the case of Sus scrofa is restricted to the ovary and the kidney. The same tRFs were found in human cancer cells and in mouse sperm, circulating blood and its serum. The binding of selected sncRNAs (piRNA, 5'tRFVal(CAC) and miRNA) to the overexpressed PAZ domain of the PIWIL4 protein was also studied. It is noteworthy that porcine 5'tRFVal(CAC) and human 5'tRFVal(CAC)as well as 5'tRFGly(GCC) are bound to the PIWIL4 protein. The potential role of the analyzed tRFs in the development of mammals is also discussed.

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.

A Novel PiRNA Enhances CA19-9 Sensitivity for Pancreatic Cancer Identification by Liquid Biopsy.

Pancreatic cancer is one of the deadliest tumours worldwide, and its poor prognosis is due to an inability to detect the disease at the early stages, thereby creating an urgent need to develop non-invasive biomarkers. P-element-induced wimpy testis (PIWI) proteins work together with piwi-interacting RNAs (piRNAs) to perform epigenetic regulation and as such hold great potential as biomarkers for pancreatic cancer. PIWIL2 and PIWIL4 are associated with better prognosis, while PIWIL1 and PIWIL3 involvement appears to be associated with carcinogenesis. We aimed to discover PIWIL3- and PIWIL4-modulated piRNAs and determine their potential mechanisms in pancreatic cancer and the clinical implications. PIWIL3 or PIWIL4 was downregulated in pancreatic cancer-derived cell lines or in a non-tumour cell line. Differentially expressed piRNAs were analysed by next generation sequencing of small RNA. Nine fresh-frozen samples from solid human pancreases (three healthy pancreases, three intraductal papillary mucinous neoplasms, and three early-stage pancreatic cancers) were included in the sequencing analysis. Two piRNAs associated with PIWIL3 (piR-168112 and piR-162725) were identified in the neoplastic cells; in untransformed samples, we identified one piRNA associated with PIWIL4 (pir-366845). After validation in pancreatic cancer-derived cell lines and one untransformed pancreatic cell line, these piRNAs were evaluated in plasma samples from healthy donors (n = 27) or patients with pancreatic cancer (n = 45). Interestingly, piR-162725 expression identified pancreatic cancer patients versus healthy donors in liquid biopsies. Moreover, the potential of the serum carbohydrate antigen 19-9 (CA19-9) biomarker to identify pancreatic cancer patients was greatly enhanced when combined with piR-162725 detection. The enhanced diagnostic potential for the early detection of pancreatic cancer in liquid biopsies of these new small non-coding RNAs will likely improve the prognosis and management of this deadly cancer.

piR-31470 epigenetically suppresses the expression of glutathione S-transferase pi 1 in prostate cancer via DNA methylation.

Inactivation of glutathione S-transferase pi 1 (GSTP1) via hypermethylation is an early and common event in prostate carcinogenesis. Functional inactivation of GSTP1 increases the susceptibility to oxidative stress and enhance progression risk of the prostatic carcinoma. In this study, we hypothesized that the Piwi-interacting RNA (piRNA) could be a sequence-recognition and guidance molecule for induction of promoter methylation of GSTP1 facilitating prostate carcinogenesis. We found that piR-31470 was highly expressed in prostate cancer cells, and piR-31470 could bind to piwi-like RNA-mediated gene silencing 4 (PIWIL4) to form the PIWIL4/piR-31470 complex. This complex could bind to the nascent RNA transcripts of GSTP1, and recruit DNA methyltransferase 1, DNA methyltransferase 3 alpha and methyl-CpG binding domain protein 2 to initiate and maintain the hypermethylation and inactivation of GSTP1. Our data demonstrated that the overexpression of piR-31470 inhibited the levels of GSTP1 and increased vulnerability to oxidative stress and DNA damage in human prostate epithelial RWPE1 cells. In conclusion, this study characterized the roles of the PIWIL4/piR-31470 complex in the regulation of the transcription of GSTP1 by methylating the CpG island of GSTP1. This discovery may provide a novel therapeutic strategy by targeting piRNAs for the epigenetic treatment of prostate cancer.

The Clinical Significance of PIWIL3 and PIWIL4 Expression in Pancreatic Cancer.

P-element-induced wimpy testis (PIWI) proteins have been described in several cancers. PIWIL1 and PIWIL2 have been recently evaluated in pancreatic cancer, and elevated expression of PIWIL2 conferred longer survival to patients. However, PIWIL3's and PIWIL4's role in carcinogenesis is rather controversial, and their clinical implication in pancreatic cancer has not yet been investigated. In the present study, we evaluated PIWIL1, PIWIL2, PIWIL3 and PIWIL4 expression in pancreatic cancer-derived cell lines and in one non-tumor cell line as healthy control. Here, we show a differential expression in tumor and non-tumor cell lines of PIWIL3 and PIWIL4. Subsequently, functional experiments with PIWIL3 and/or PIWIL4 knockdown revealed a decrease in the motility ratio of tumor and non-tumor cell lines through downregulation of mesenchymal factors in pro of epithelial factors. We also observed that PIWIL3 and/or PIWIL4 silencing impaired undifferentiated phenotype and enhanced drug toxicity in both tumor- and non-tumor-derived cell lines. Finally, PIWIL3 and PIWIL4 evaluation in human pancreatic cancer samples showed that patients with low levels of PIWIL4 protein expression presented poor prognosis. Therefore, PIWIL3 and PIWIL4 proteins may play crucial roles to keep pancreatic cell homeostasis not only in tumors but also in healthy tissues.

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

PURPOSE: This 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. RESULTS: Piwil2 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. METHODS: HCC 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. CONCLUSIONS: The 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.

The significance of PIWI family expression in human lung embryogenesis and non-small cell lung cancer.

The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1-positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) (p = 0.006) and overall survival (OS) (p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue (p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR (p = 0.048) and OS (p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression.

Expression of piR-9994 in gastric carcinoma and its correlation with PIWIL4 / 临床与实验病理学杂志

Purpose To explore the relationship between the expression level of piR-9994 in gastric cancer and its clinical pathological features,and to analyze its correlation with PIWIL4 expression. Methods Express of piR-9994 and PIWIL4 in 76 cases of human gastric cancer tissue with different clinical stage and differentiated degree and matching adjacent tissue were detected by qRT-PCR and immunohistochemistry,respectively. Results The expression of piR-9994 in gastric cancer was 2. 3 times higher than that in paracancerous tissues (P = 0. 002 2) . The expression of piR-9994 in stage Ⅲ + Ⅳ gastric cancer was 3. 5 times higher than that in stage Ⅰ + Ⅱ (P = 0. 002) ,and the expression of piR-9994 in cancers with nerve invasion was 2. 5 times higher than that in cancers without invasion (P = 0. 036) . The positive expression of PIWIL4 in gastric cancer tissues was significantly higher than that in adjacent tissues (χ2 = 18. 346,P < 0. 001) ,and the expression of PIWIL 4 in stage Ⅲ +Ⅳ gastric cancer group was higher than that stage Ⅰ + Ⅱ gastric cancer group (χ2 = 8. 60,P = 0. 003) . There was a positive correlation between piR-9994 expression and PIWIL4 expression in gastric carcinoma (r = 0. 231,P < 0. 05) . Conclusion piR-9994 overexpression in gastric cancer tissues is closely related to tumor staging and nerve invasion,and piR-9994 may promote the occurrence and progression of gastric cancer by regulating PIWIL4 expression. piR-9994 may be a molecular markers for judging malignant degree and prognosis of gastric cancer.

The Expression and Functional Research of PIWIL4 in Human Ovarian Cancer / 生物化学与生物物理进展

To explore the expressional information of PIWIL4 in human ovarian cancer, semi-quantitative reverse transcription polymerase chain reaction(semi-qRT-PCR) was used to detect the mRNA expression level of PIWIL1, PIWIL2, PIWIL3 and PIWIL4 in human ovarian clear cell carcinoma ES-2 cells.Meanwhile, in human ovarian cancer and adjacent normal tissues, the mRNA expression of PIWIL4 was determined.Then, PIWIL4-siRNA, which was designed to target PIWIL4 and chemical synthesized, was transfected into ES-2 cells with lipofectamine.MTT assay and clone formation assay were used to investigate the effects of PIWIL4-siRNA on the cell growth activity and proliferation capacity of ES-2 cells.Experimental results showed that the mRNA expression level of PIWIL4 was the highest compared to PIWIL1, PIWIL2 and PIWIL3 in ES-2 cells(P