Novel evidence for a PIWI-interacting RNA (piRNA) as an oncogenic mediator of disease progression, and a potential prognostic biomarker in colorectal cancer.
ABSTRACT: Emerging evidence suggests that PIWI-interacting RNAs (piRNAs) may be important epigenetic regulators of gene expression in human cancers; however, their functional and clinical significance in colorectal cancer (CRC) remains unknown.We performed piRNA expression profiling in paired cancer and normal tissues through small RNA-sequencing. The clinical significance of candidate piRNAs was investigated, and independently validated in 771 CRC patients from three independent cohorts. The biological function of piRNAs was characterized in cell lines, followed by identification and validation of downstream target genes in CRC tissues.We identified piR-1245 as a novel and frequently overexpressed noncoding RNA in CRC, and its expression significantly correlated with advanced and metastatic disease. Patients with high piR-1245 expression experienced significantly shorter overall survival, and multivariate analysis identified its expression to serve as an independent prognostic biomarker in CRC. Functionally, piR-1245 acts as an oncogene and promotes tumor progression, and gene expression profiling results identified a panel of downstream target-genes involved in regulating cell survival pathway. Based upon piRNA:mRNA sequence complementarity, we identified a panel of tumor suppressor genes (ATF3, BTG1, DUSP1, FAS,NFKBIA, UPP1, SESN2, TP53INP1 and MDX1) as direct targets of piR-1245, and successfully validated an inverse correlation between their expression and piR-1245 in CRC.We for the first time have identified the role for a PIWI-interacting noncoding RNA, piR-1245, as a novel oncogene and a potential prognostic biomarker in colorectal cancer.
Project description:Piwi-interacting RNAs (piRNAs) represent a novel class of small non-coding RNAs (ncRNAs) that have been shown to have a deregulated expression in several cancers, although their clinical significance in colorectal cancer (CRC) remains unclear. With an aim of delineating the piRNA distribution in CRC, we conducted a systematic discovery and validation of piRNAs within two clinical cohorts. In the discovery phase, we profiled tumor and adjacent normal tissues from 18 CRC patients by deep sequencing and identified a global piRNA downregulation in CRC. Moreover, we identified piR-24000 as an unexplored piRNA that was significantly overexpressed in CRC. Using qPCR, we validated the overexpression of piR-24000 in 87 CRC patients. Additionally, we identified a significant association between a high expression of piR-24000 and an aggressive CRC phenotype including poor differentiation, presence of distant metastases, and a higher stage. Lastly, ROC analysis demonstrated a strong diagnostic power of piR-24000 in discriminating CRC patients from normal subjects. Taken together, this study provides one of the earliest large-scale reports of the global distribution of piRNAs in CRC. In addition, piR-24000 was identified as a likely oncogene in CRC that can serve as a biomarker or a therapeutic target.
Project description:RASSF1C up-regulates important genes involved in lung cancer cell growth, including a stem cell self-renewal gene, piwil1. In this article, we report the identification of small noncoding PIWI-interacting RNAs (piRNAs) in lung cancer cells over-expressing RASSF1C. A piRNA microarray screen was performed using RNA isolated from the lung cancer cell line H1299 stably over-expressing RASSF1C and corresponding control. The piRNA microarray screen identified several piRNAs that are regulated by RASSF1C and we have validated the expression of two up-regulated piRNAs (piR-34871 and piR-52200) and two down-regulated piRNAs (piR-35127 and piR-46545) in lung cancer cells with silenced and over-expressed RASSF1C using RT-PCR. We also assessed the expression of these four piRNAs in lung tumor and matched normal tissues (n = 12). We found that piR-34871 and piR-52200 were up-regulated in 58% and 50%, respectively; while piR-35127 and piR-46545 were down-regulated in 50% in lung tumor tissues tested. The expression of piR-35127 was inversely correlated with RASSF1C expression in 10/12 tumor tissues. Over-expression of piR-35127 and piR-46545 and knock-down of piR-34871 and piR-52200 significantly reduced normal lung and breast epithelial cell proliferation and cell colony formation as well as proliferation of lung cancer cell lines (A549 and H1299) and breast cancer cell lines (Hs578T and MDA-MB-231). This suggests that these novel piRNAs may potentially be involved in regulating lung cell transformation and tumorigenesis. RASSF1C may potentially modulate the expression of its piRNA target genes through attenuation of the AMPK pathway, as over-expression of RASSF1C resulted in reduction of p-AMPK, p21, and p27 protein levels.
Project description:PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that partner with PIWI proteins to protect germline tissues from destabilizing transposon activity. While the aberrant expression of PIWI proteins has been linked with poor outcomes for many cancers, less is known about the expression or function of piRNAs in cancer. We performed array-based piRNA expression profiling in seven pairs of normal brain and glioblastoma multiforme (GBM) tissue specimens, and identified expression of ~350 piRNAs in both tissues and a subset with dysregulated expression in GBM. Over-expression of the most down-regulated piRNA in GBM tissue, piR-8041, was found to reduce glioma cell line proliferation, induce cell cycle arrest and apoptosis, and inhibit cell survival pathways. Furthermore, pre-treatment with piR-8041 significantly reduced the volume of intracranial mouse xenograft tumors. Taken together, our study reveals reduced expression in GBM of piR-8041 and other piRNAs with tumor suppressive properties, and suggests that restoration of such piRNAs may be a potential strategy for GBM therapy.
Project description:Piwi-interacting RNAs (piRNAs) are a distinct group of small noncoding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Because of their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that had at least two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found three piRNAs (piR-32051, piR-39894 and piR-43607) to be derived from the same piRNA cluster at chromosome 17. We confirmed the three selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We further validated the aberrant expression of the three piRNAs in a 68-case formalin-fixed and paraffin-embedded (FFPE) ccRCC tissue cohort and showed the up-regulation of the three piRNAs to be highly associated with ccRCC metastasis, late clinical stage and poor cancer-specific survival.
Project description:Emerging studies demonstrate that PIWI-interacting RNAs (piRNAs) participate in the development of cancers. 75 pairs of papillary thyroid carcinoma (PTC) samples and 31 benign thyroid nodule samples were included in this three-phase biomarker identifying study. First, piRNA expression profiles of five pairs of PTC samples were acquired piRNA sequencing. The expression of all upregulated piRNAs were further validated by RT-qPCR. Paired t and nonparametric test were used to evaluate the association between all upregulated piRNAs and clinic stage. The expression levels of key piRNAs were corrected by demographic data to construct a multivariate model to distinguish malignant nodules from benign. Additionally, the intersection between target genes of key piRNAs and differentially expressed genes in The Cancer Genome Atlas (TCGA) PTC samples were used to perform enrichment analysis. Only piR-13643 and piR-21238 were significantly upregulated in PTC and associated with clinic stage. Moreover, both piR-13643 (Area Under Curve (AUC): 0.821) and piR-21238 (AUC: 0.823) showed better performance in distinguishing malignant nodules from benign than currently used biomarkers HBME1 (AUC: 0.590). Based on our findings, piR-13643 and piR-21238 were observed to be significantly upregulated in human PTC. PIWI-interacting RNAs could serve as promising novel biomarkers for accurate detection of PTC.
Project description:PIWI-interacting RNAs (piRNAs) are thought to silence transposon and gene expression during development. However, the roles of piRNAs in somatic tissues are largely unknown. Here we report the identification of 555 piRNAs in human lung bronchial epithelial (HBE) and non-small cell lung cancer (NSCLC) cell lines, including 295 that do not exist in databases termed as piRNA-like sncRNAs or piRNA-Ls. Distinctive piRNA/piRNA-L expression patterns are observed between HBE and NSCLC cells. piRNA-like-163 (piR-L-163), the top downregulated piRNA-L in NSCLC cells, binds directly to phosphorylated ERM proteins (p-ERM), which is dependent on the central part of UUNNUUUNNUU motif in piR-L-163 and the RRRKPDT element in ERM. The piR-L-163/p-ERM interaction is critical for p-ERM's binding capability to filamentous actin (F-actin) and ERM-binding phosphoprotein 50 (EBP50). Thus, piRNA/piRNA-L may play a regulatory role through direct interaction with proteins in physiological and pathophysiological conditions.
Project description:The piwi interacting RNAs (piRNAs) are small non-coding RNAs that specifically bind to the PIWI proteins, a functional requirement. The piRNAs regulate germline development, transposons control, and gene expression. However, piRNA-mediated post-transcriptional gene regulation in human somatic cells is not well understood. We discovered a human piRNA (piR-FTH1) which has a complementary sequence in the ferritin heavy chain 1 (Fth1) mRNA. We demonstrated that expression of piR-FTH1 and Fth1 are inversely correlated in the tested tumor cell lines. We found that piR-FTH1 negatively regulates the Fth1 expression at post-transcriptional level in triple negative breast cancer (TNBC) cells. Additionally, we confirmed that transfected piR-FTH1 knocks down the Fth1 mRNA via the HIWI2 and HILI mediated mechanism. piR-FTH1 mediated Fth1 repression also increased doxorubicin sensitivity by a remarkable 20-fold in TNBC cells. Since the current piRNA-mediated knockdowns of target mRNA are mostly reported in germ line cells, piRNA-mediated post-transcriptional gene regulation in somatic cells is rather unique in its application and mechanistically uses an alternative pathway to siRNA and miRNA. This work begins to lay the groundwork with a broader impact on treatment of various diseases that are linked to elevated levels of specific mRNAs which have a piRNA target.
Project description:PURPOSE:PIWI-interacting RNA (piRNA) is a sub-group of small RNAs about 30 nucleotides length which specifically expressed in mammalian germ cells. Although piRNAs play pivotal roles in spermatogenesis regulation, little is known in the testicular tissues of infertile men. To explore whether piRNA profile could serve as a biomarker for male infertility diagnosis in a clinic, in this study, we systematically investigated the expression profile of piRNAs in testicular tissues from the patients with non-obstructive azoospermia (NOA) between successful and unsuccessful sperm retrieval before micro-dissection testicular sperm extraction (micro-TESE). METHODS:The differential expression levels of piRNAs were evaluated using small RNA-Seq method. Ontologic analyses were performed to determine the presence of enriched biological processes. RESULTS:A total of 18,324 Homo sapiens piRNAs were identified by small RNA-Seq from NOA patient testicular tissues; among them, 959 piRNAs were significantly altered between successful and unsuccessful sperm retrieval groups, of which 951 testicular piRNAs were significantly downregulated and 8 piRNAs were upregulated in NOA patients with unsuccessful sperm retrieval (USR) groups compared to those with successful sperm retrieval (SSR) groups, respectively. Unexpectedly, 553 testicular piRNAs were found completely absent in USR but showing abundant in SSR, which suggests that those piRNAs might serve as a biomarker for micro-TESE application. A total of 20 significantly differential piRNAs (hsa-piR-20830, hsa-piR-4731, hsa-piR-6254, hsa-piR-419, hsa-piR-7152, hsa-piR-7548, hsa-piR-14195, hsa-piR-5026, hsa-piR-11482, hsa-piR-17765, hsa-piR-17102, hsa-piR-4484, hsa-piR-17260, hsa-piR-17098, hsa-piR-20511, hsa-piR-5802, hsa-piR-19121, hsa-piR-2510, hsa-piR-4745, hsa-piR-11873) were selected to further validate the RNA-Seq data by quantitative real-time polymerase chain reaction. In addition, bioinformatic analyses revealed that those altered piRNAs were involved in many important biological pathways, including apoptosis, cell proliferation, and differentiation. CONCLUSIONS:Our results demonstrate that testicular tissues from NOA patients with successful and unsuccessful spermatozoa retrieval exhibit differential piRNA profiles. This study provides a useful resource to further elucidate the regulatory role of piRNAs in spermatogenesis and provides a profound clue to identify useful biomarkers for predicting residual spermatogenic loci in NOA patients during assisted reproductive treatment.
Project description:The blood-tumor barrier (BTB) restricts the efficient delivery of anti-glioma drugs to cranial glioma tissues. Increased BTB permeability may allow greater delivery of the therapeutic agents. Increasing evidence has revealed that PIWI proteins and PIWI-interacting RNAs (piRNAs) play an important role in tumor progression. However, whether PIWI proteins and piRNAs regulate BTB permeability remains unclear. In the present study, we demonstrated that the PIWIL1/piRNA-DQ593109 (piR-DQ593109) complex was the predominant regulator of BTB permeability. Briefly, PIWIL1 was upregulated in glioma endothelial cells (GECs). Furthermore, piR-DQ593109 was also overexpressed in GECs, as revealed via a piRNA microarray. Downregulation of PIWIL1 or piR-DQ593109 increased the permeability of the BTB. Moreover, PIWIL1 and piR-DQ593109, which formed a piRNA-induced silencing complex, degraded the long non-coding RNA maternally expressed 3 (MEG3) in a sequenced-dependent manner. Furthermore, restoring MEG3 released post-transcriptional inhibition of Runt related transcription factor 3 (RUNX3) by sponging miR-330-5p. In addition, RUNX3 bounded to the promoter regions and reduced the promoter activities of ZO-1, occludin, and claudin-5, which significantly impaired the expression levels of ZO-1, occludin, and claudin-5. In conclusion, downregulating PIWIL1 and piR-DQ593109 increased BTB permeability through the MEG3/miR-330-5p/RUNX3 axis. These data may provide insight into glioma treatment.
Project description:Piwi-interacting RNAs (piRNAs), a novel class of small non-coding RNAs, were first discovered in germline cells and are thought to silence transposons in spermatogenesis. Recently, piRNAs have also been identified in somatic tissues, and aberrant expression of piRNAs in tumor tissues may be implicated in carcinogenesis. However, the function of piR-823 in colorectal cancer (CRC) remains unclear. Here, we first found that piR-823 was significantly upregulated in CRC tissues compared with its expression in the adjacent tissues. Inhibition of piR-823 suppressed cell proliferation, arrested the cell cycle in the G1 phase and induced cell apoptosis in CRC cell lines HCT116 and DLD-1, whereas overexpression of piR-823 promoted cell proliferation in normal colonic epithelial cell line FHC. Interestingly, Inhibition of piR-823 repressed the expression of heat shock protein (HSP) 27, 60, 70. Furthermore, elevated HSPs expression partially abolished the effect of piR-823 on cell proliferation and apoptosis. In addition, we further demonstrated that piR-823 increased the transcriptional activity of HSF1, the common transcription factor of HSPs, by binding to HSF1 and promoting its phosphorylation at Ser326. Our study reveals that piR-823 plays a tumor-promoting role by upregulating phosphorylation and transcriptional activity of HSF1 and suggests piR-823 as a potential therapeutic target for CRC.