Small RNA Sequencing Identifies PIWI-Interacting RNAs Deregulated in Glioblastoma-piR-9491 and piR-12488 Reduce Tumor Cell Colonies In Vitro.
ABSTRACT: Glioblastoma (GBM) is the most frequently occurring primary malignant brain tumor of astrocytic origin. To change poor prognosis, it is necessary to deeply understand the molecular mechanisms of gliomagenesis and identify new potential biomarkers and therapeutic targets. PIWI-interacting RNAs (piRNAs) help in maintaining genome stability, and their deregulation has already been observed in many tumors. Recent studies suggest that these molecules could also play an important role in the glioma biology. To determine GBM-associated piRNAs, we performed small RNA sequencing analysis in the discovery set of 19 GBM and 11 non-tumor brain samples followed by TaqMan qRT-PCR analyses in the independent set of 77 GBM and 23 non-tumor patients. Obtained data were subsequently bioinformatically analyzed. Small RNA sequencing revealed 58 significantly deregulated piRNA molecules in GBM samples in comparison with non-tumor brain tissues. Deregulation of piR-1849, piR-9491, piR-12487, and piR-12488 was successfully confirmed in the independent groups of patients and controls (all p < 0.0001), and piR-9491 and piR-12488 reduced GBM cells' ability to form colonies in vitro. In addition, piR-23231 was significantly associated with the overall survival of the GBM patients treated with Stupp regimen (p = 0.007). Our results suggest that piRNAs could be a novel promising diagnostic and prognostic biomarker in GBM potentially playing important roles in gliomagenesis.
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: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), 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.
Project description:Although the importance of PIWI-interacting RNAs (piRNAs) in cancer has recently been recognized, studies on the role and functional mechanism of piRNAs in lung adenocarcinoma (LUAD) development and progression are limited. In this study, we identified 10 differently expressed piRNAs in LUAD tissues compared to normal tissues, among which, piR-hsa-211106 expression levels were downregulated in LUAD tissues and cell lines. Furthermore, the effects of piR-hsa-211106 on the malignant phenotypes and chemosensitivity of LUAD cells were detected by gain- and loss-of-function analyses <i>in vitro</i> and <i>in vivo</i>, which showed that piR-hsa-211106 inhibited LUAD cell proliferation, tumor growth, and migration, but promoted apoptosis. Moreover, our finding indicated that piR-hsa-211106 is a potential therapeutic target that synergistically imparts anticancer effects with a chemotherapeutic agent for LUAD-cisplatin. Further mechanistic investigation indicated that piR-hsa-211106 could bind to pyruvate carboxylase (PC) by RNA pull down and RNA immunoprecipitation assays and inhibited <i>PC</i> mRNA and protein expression. Our study demonstrates that piR-hsa-211106 inhibits LUAD progression by hindering the expression and function of PC and enhances chemotherapy sensitivity, suggesting that piR-hsa-211106 is a novel diagnostic and therapeutic target for LUAD.
Project description:Resistance to doxorubicin (DOX) is an obstacle to successful sarcoma treatment and a cause of tumor relapse, with the underlying molecular mechanism still unknown. PIWI-interacting RNAs (piRNAs) have been shown to enhance patient outcomes in cancers. However, there are few or no reports on piRNAs affecting chemotherapy in cancers, including fibrosarcoma. The current study aims to investigate the relationship between piR-39980 and DOX resistance and the underlying mechanisms. We reveal that piR-39980 is less expressed in DOX-resistant HT1080 (HT1080/DOX) fibrosarcoma cells. Our results show that inhibition of piR-39980 in parental HT1080 cells induces DOX resistance by attenuating intracellular DOX accumulation, DOX-induced apoptosis, and anti-proliferative effects. Its overexpression in HT1080/DOX cells, on the other hand, increases DOX sensitivity by promoting intracellular DOX accumulation, DNA damage, and apoptosis. The dual-luciferase reporter assay indicates that piR-39980 negatively regulates RRM2 and CYP1A2 via direct binding to their 3'UTRs. Furthermore, overexpressing RRM2 induces DOX resistance of HT1080 cells by rescuing DOX-induced DNA damage by promoting DNA repair, whereas CYP1A2 confers resistance by decreasing intracellular DOX accumulation, which piR-39980 restores. This study reveals that piR-39980 could reduce fibrosarcoma resistance to DOX by modulating RRM2 and CYP1A2, implying that piRNA can be used in combination with DOX.
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: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 and glioblastoma multiforme (GBM) tissue specimens and identified expression of ~350 piRNAs in both tissues and a subset with dysregulated expression in GBM. Several down-regulated piRNAs inhibited proliferation when transfected into glioma cell lines while those equivalently expressed in tumor and normal tissues did not, consistent with piRNA-specific tumor-suppressive roles. Upregulation of the most underexpressed piRNA, piR-8041, was found to induce cell cycle arrest and apoptosis and to alter transcriptional levels of several genes involved in stress and cell survival pathways. Additionally, the volume of intracranial mouse xenograft tumors was significantly reduced for approximately ten days after pre-implantation transfection with piR-8041. Taken together, our study reveals a previously unidentified functional role for piRNAs as tumor suppressors in gliomagenesis, and suggests that restoration of piRNA levels may be a potential strategy for GBM therapy.
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:<h4>Background</h4>Lung adenocarcinoma (LUAD) is the main subtype of primary lung cancer and is a leading cause of cancer-related death worldwide. PIWI-interacting RNAs (piRNAs) are a type of small non-coding RNAs that may play crucial roles in cancer progression and serve as biomarkers for tumor detection. This study aimed to explore the expression profiles and diagnostic values of piRNAs in LUAD.<h4>Methods</h4>Small RNA sequencing was performed to investigate tissue piRNA profiles of LUAD. The expression of selected upregulated piRNAs were detected in tissues and serum exosome samples by quantitative real-time polymerase chain reaction (qRT-PCR). Serum exosomes were identified by transmission electron microscope, nanoparticle tracking analysis, and western blot analysis. Receiver operating characteristic (ROC) curve was adopted to quantify the diagnostic potentials of piRNAs in LUAD. Finally, a piRNA panel was developed by multivariate logistic regression model.<h4>Results</h4>We identified that 76 piRNAs were overexpressed and 9 piRNAs were underexpressed in LUAD tissues compared with adjacent non-tumor tissues. Among the top 10 overexpressed piRNAs, 4 piRNAs (piR-hsa-26925, piR-hsa-5444, piR-hsa-30636, and piR-hsa-8757) were verified by qRT-PCR to be significantly upregulated in LUAD tissues. Moreover, piR-hsa-26925 and piR-hsa-5444 had a significantly higher level in serum exosome samples of LUAD patients than those of healthy controls. We finally established a 2-piRNA panel composed of piR-hsa-26925 and piR-hsa-5444, which showed higher diagnostic performance for LUAD with an AUC of 0.833.<h4>Conclusions</h4>Our finding revealed the abnormally expressed piRNAs in LUAD, and serum exosomal piR-hsa-26925 and piR-hsa-5444 could serve as potential biomarkers for LUAD diagnosis.
Project description:P-element-induced wimpy testis (Piwi)-interacting RNAs (piRNAs) are a class of germline-enriched small non-coding RNA that associate with Piwi family proteins and mostly induce transposon silencing and epigenetic regulation. Emerging evidence indicated the aberrant expression of Piwil proteins and associated piRNAs in multiple types of human cancer including breast cancer. Although the majority of piRNAs in breast cancer remains unclear of the function mainly due to the variety of regulatory mechanisms, the potential of piRNAs serving as biomarkers for cancer diagnosis and prognosis or therapeutic targets for cancer treatment has been demonstrated by <i>in vitro</i> and <i>in vivo</i> studies. Herein we summarized the research progress of oncogenic or tumor suppressing piRNAs and their regulatory mechanisms in regulating human breast cancer, including piR-021285, piR-823, piR-932, piR-36712, piR-016658, piR-016975 and piR-4987. The challenges and perspectives of piRNAs in the field of human cancer were discussed.