Novel evidence for a PIWI-interacting RNA (piRNA) as an oncogenic mediator of disease progression, and a potential prognostic biomarker in colorectal cancer
- Wenhao Weng†1, 2, 3,
- Na Liu†4,
- Yuji Toiyama5,
- Masato Kusunoki5,
- Takeshi Nagasaka6,
- Toshiyoshi Fujiwara6,
- Qing Wei7,
- Huanlong Qin8,
- Haifan Lin4,
- Yanlei Ma9, 10Email author and
- Ajay Goel1Email author
© The Author(s). 2018
Received: 26 July 2017
Accepted: 15 January 2018
Published: 30 January 2018
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.
Colorectal cancer (CRC) constitutes a major public health burden , being the third most commonly diagnosed cancer, and the fourth leading cause of cancer-related deaths worldwide. Interestingly, recent reports have shown that the incidence of colorectal cancer in Asian countries, which historically was relatively low, has increased dramatically during the last two decades [2, 3]. Considering the high disease burden and mortality associated with this global disease, it is imperative to develop effective prevention and treatment strategies for the management of patients suffering from this malignancy.
CRC develops as a consequence of stepwise accumulation of multiple genetic and epigenetic alterations, which occur with tumor initiation and ensue during disease progression. In view of tumor heterogeneity, the prognosis and response to chemotherapy between individual patients can vary significantly. However, current guidelines for risk stratification of patients predominantly rely on the clinicopathological factors, which are inadequate and often result in under or over-treatment for CRC patients [4, 5]. In view of this clinical challenge, identification of novel molecular targets that more robustly typify and represent disease biology would be of great value in improving prognosis and allowing precision therapeutic targeting in CRC patients.
Data gathered during the last decade has revealed that microRNAs play a crucial role in cancer pathogenesis, and may serve as promising disease biomarkers and potential therapeutic targets [6, 7]. Meanwhile, emerging data from RNA-Sequencing efforts of cancer specimens have led to the discovery of additional classes of novel, small noncoding RNAs (ncRNAs), which may also significantly contribute to cancer pathogenesis [8–12]; however, such data remain in their infancy at this time in point. Among these, PIWI-interacting RNAs (piRNAs), represent the most diversified group, but currently remain the least characterized class of small ncRNAs. The piRNA pathway consists of piRNAs that interact with PIWI proteins, in which the precursor piRNAs are transcribed from their clusters, cleaved by PIWI proteins, and subsequently amplified in the cytoplasm through sequence-complementary-dependent cycle. The piRNA-PIWI protein pathway was initially found associated with safeguarding of the germline genome against transposon-induced insertional mutations [13–15]. However, emerging evidence indicates that piRNAs may also function on a somatic level, whereby they regulate gene expression; through histone modifications and DNA methylation [16–19]. In other words, although such recent evidence suggests the role of PIWI-piRNA pathway in controlling epigenetic function, our understanding for the biological involvement of piRNAs in human cancers currently remains in its infancy, but presents an exciting new area of basic and translational research worthy of exploration.
Although piRNA-mediated gene expression regulation may have a broader implication for cancer research, recent studies have largely been limited to expression profiling of a handful or selected, small subset of piRNAs in different cancer types. [9, 12] Furthermore, the role of piRNAs in CRC is poorly understood. Hence, we envisaged this first of its kind of study to systematically and comprehensively interrogate the molecular contributions of piRNAs in CRC, with a goal to identify novel, differentially expressed piRNAs that promote colorectal carcinogenesis, and decipher whether these piRNAs may have translational relevance as clinically relevant disease biomarkers. Accordingly, we performed a discovery step by performing small RNA-Sequencing-based expression profiling for piRNAs between cancer and normal tissues. Using a series of bioinformatic approaches, we identified candidate, CRC-specific piRNAs, followed by their validation in multiple CRC patient cohorts. We subsequently supported these findings by performing a series of functional assays and investigated downstream pathways and target genes of candidate piRNAs, which contribute to the neoplastic progression in colorectal cancer.
Patients and study design
Clinicopathological characteristic and piR-1245 expression in training and validation cohort
Pathological T category
Lymph node metastasis
Small RNA-sequencing, piRNA quantification and gene expression analysis
For RNA-sequencing, 1 μg of total RNA was used for library preparation with Illumina’s TruSeq small RNA sample preparation Kit using manufacturer’s recommended protocols and the previously published articles mirBase [21, 22]. Expression of identified piRNAs was analyzed using Custom TaqMan small RNA assays as described previously [23–25]. The average expression levels of tissue piRNAs was normalized against U6 using the 2-ΔCt method. The relative expression of target genes was determined by 2-Δct method using GAPDH as a normalizer as described in details in the Additional file 1: Supplementary Methods and primer sequences shown in Additional file 2: Table S1.
Cell lines, RNA oligos, antisense and transfection
HCT116 and SW480 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in Iscove’s modified Dulbecco’s medium (Invitrogen, Carlsbad, CA). For the overexpression of piR-1245, both cells lines were transfected in triplicates with either single-stranded RNA oligos or scrambled RNA controls. For the inhibition of piR-1245 in CRC cell lines, we designed antisense oligos as described previously  and as described in the Additional file 1: Supplementary Methods.
MTT, colony formation, cell invasion, migration and apoptosis assays
MTT, colony formation, invasion, migration and apoptosis assays were performed in CRC cell lines at different time points using standard approaches, and according to the manufacturer’s instructions, as described in the Additional file 1: Supplementary Methods.
Immunofluorescence (IF) staining
For IF, cells were fixed by 4% paraformaldehyde for 15 min, washed with PBS and blocking buffer (3% FBS, 1% heat-inactivated sheep serum, 0.1% Triton X-100), and thereafter incubated overnight at 4 °C with primary antibodies against Ki-67 (Santa Cruz, Dallas, TX), and fluorescent Alexa Fluor 488- conjugated secondary antibodies (Thermo Scientific, Rockford, IL) were subsequently used for fluorescence detection. The Ki-67 staining intensity was semi-quantified as follows: – for negative staining, ± for very weak staining, + for weak staining and ++ for strong staining.
Gene expression microarray analysis
To investigate the regulatory role of piR-1245 on genome-wide target mRNAs, we treated HCT116 cells with or without piR-1245 antisense, and subsequently performed Affymetrix GeneChip Human gene 2.0 ST arrays and subsequent bioinformatic analysis as described in the Additional file 1: Supplementary Methods.
piRNA target prediction
Based upon piRNA:mRNA sequence complementarity, we used Miranda v3.3a and RNA22 program to search for targets of piR-1245 against all human transcripts. The candidate piRNAs were selected based on prediction scores and binding energy. The whole transcript region of human transcripts were used for piRNA target prediction.
All statistical analyses were performed using the GraphPad Prism version 6.0 or MedCalc version 12.3 programs. Statistical differences between groups were determined by Wilcoxon’s signed rank test, the χ2 test or Mann-Whitney U test. Kaplan-Meier analysis and log-rank test was used to estimate and compare overall survival (OS) rates of CRC patients with high and low piR-1245 expression. The optimal cutoff values were determined by ROC curves to discriminate patients with or without death. The Cox’s proportional hazards models were used to estimate hazard ratios (HRs) for death. All P values were 2-sided, and those less than 0.05 were considered statistically significant.
Identification of cancer-related piRNAs in CRC
High expression of piR-1245 correlates with advanced disease and metastatic in colorectal cancer
We next examined the expression pattern of piR-1245 in the context of its clinical significance in the training cohort (Shanghai cohort, n = 195). The piR-1245 was overexpressed in all CRCs, and this phenomenon occurred in a stage-dependent manner (P = 0.006, Table 1). The tumors in the distal colon or rectum showed a much higher expression level of piR-1245 compared to the proximal neoplasms (P = 0.0123). Furthermore, higher expression of piR-1245 was significantly more pronounced in cancer tissues with poor differentiation (P = 0.0566), advanced T stage (P = 0.0008), lymph node metastasis (P = 0.025) and distant metastasis (P = 0.0319).
To further validate the correlation between piR-1245 expression and clinicopathological variables, we interrogated these associations in an independent patient cohort (Okayama cohort, n = 189). We were able to successfully validate our findings in the training cohort, as the upregulatedpiR-1245 was also associated with advanced T-stage (P = 0.0434), lymph node (P = 0.0025) and distant metastasis (P = 0.0027) in this second patient cohort as well. Collectively, our analyses provided evidence that expression ofpiR-1245 is not only upregulated, but also associates with specific clinicopathological features, suggesting that piR-1245plays a crucial role in the cancer pathogenesis within the colon.
High expression of piR-1245 associates with poor prognosis in colorectal cancer patients
Univariate and multivariate analysis for predictors of overall survival in training and validation cohort
Univariate survival analysis
Multivariate survival analysis
Age (> 69)
Tumor location (Proximal)
Histological type (Poor)
T classification (pT4)
Node involvement (Present)
Distant metastasis (Present)
piR-1245 expression level (High)
Age (> 69)
Tumor location (Proximal)
Histological type (Poor)
1.2996 - 9.3799
T classification (pT4)
** < 0.0001
1.1551 - 4.2673
Node involvement (Present)
0.5790 - 2.8776
Distant metastasis (Present)
** < 0.0001
2.3622 - 9.5220
** < 0.0001
piR-1245 expression level (High)
1.4584 - 5.9057
The piR-1245 has multiple functional roles in promoting tumor progression in colorectal cancer cells
Since high expression of piR-1245 associated with lymph node and distant metastasis in CRC patients, we next interrogated whether it may regulate cell migration and invasion in colorectal cancer cells. As illustrated in Fig. 2d, inhibition of piR-1245 significantly suppressed cell migration and invasion in both HCT116 and SW480 cells compared to the control cells, confirming our findings in the clinical patient cohorts.
Resistance to programmed cell death is recognized as one of the cancer hallmarks that contributes to disease progression and eventual tumor metastasis . Based on our clinical data, we hypothesized that piR-1245 also plays a key role in resistance to apoptosis in colorectal cancer cells. In line with our hypothesis, inhibition of piR-1245 significantly induced apoptosis in HCT116 and SW480 cells (Fig. 2e). Collectively, our data showed newly discovered piR-1245 exerts oncogenic function in CRC through promotion of cell survival, migration and invasion as well as suppression of apoptosis.
The piR-1245 affects multiple cancer-related pathways involved in cell proliferation, cell death and apoptosis
Strikingly, the top 10 GO term enrichment analysis for upregulated genes favored cell death or apoptosis, cell proliferation, protein metabolic process and protein, while the downregulated genes were enriched for chromatin assembly and catalytic activity (Additional file 7: Figure S5).
In order to obtain further insights into disease and functional networks, we performed Ingenuity Pathway Analysis (IPA) based on our gene expression profiling results. The results confirmed the putative model that activated p53 pathway, which was induced by piR-1245 inhibition, led to cell apoptosis, necrosis, cell death, contact growth inhibition, senescence of cells, and inhibited cell proliferation, colony formation. Furthermore, IPA showed the piR-1245 acts as important regulator of cell death and survival (Fig. 3b). Based on these findings, these biological processes and molecular functions could contribute to the development of CRC.
Identification of piR-1245 target genes in CRC
Colorectal cancer is one of the most common cancers worldwide. Consequently, elucidation of the molecular mechanisms underlying its progression is critical for the development of new diagnostic and prognostic biomarkers, as well as identification of better therapeutic targets for the management of patients with this deadly malignancy. Herein, we for the first time performed systematic piRNA expression profiling, and identified piR-1245, as a novel oncogenic piRNA mediating CRC pathogenesis. We have made several novel observations in this study. First, we discovered that piR-1245 is frequently overexpressed in CRC tissues from different cohorts, and its overexpression associated with several known risk clinicopathological factors including tumor differentiation and metastasis. Second, our data revealed that patients with high expression of piR-1245 had shorter overall survival, highlighting its applicability as a promising prognostic biomarker in CRC. Third, ours is the first study to demonstrate the biological relevance of this piR-1245 as a tumor-promoting noncoding RNA in CRC. Fourth, microarray analysis revealed thatpiR-1245 regulates several key cancer pathways, supporting its oncogenic role in CRC. Finally, we discovered several important tumor suppressors as direct downstream gene targets, and their expression was inversely correlated with the piR-1245, suggesting this small noncoding RNA promotes CRC development through inhibition of these target genes at the transcriptional level.
In contrast to the growing body of studies that underpin the miRNA-cancer connection, knowledge of piRNAs in tumorigenesis, particularly in CRC, remains currently in its infancy. PiRNAs are roughly 26-30 nucleotides in length and associate specifically with Argonaute proteins that belong to the PIWI subfamily [40, 41]. Although previously considered to be germline specific and guardians for protecting the integrity of the genome against transposon-induced insertional mutations , mounting evidence now point towards novel active role of piRNAs in somatic gene regulation, through other mechanisms such as transcriptional gene silencing and sequence-specific DNA methylation [16, 40, 41]. Interestingly, recent studies have demonstrated that piRNAs are widely expressed and play important roles in somatic cells . Furthermore, few studies have begun to investigate the differentially expressed piRNAs in human cancers and their benign counterparts. It is noteworthy that piR-651 and piR-823 were found to be dysregulated in gastric cancer [43, 44], and moreover, recent study showed a panel of piRNAs are associated with prognosis in breast cancer , suggesting that piRNAs which previously considered as “junk” RNAs, are indeed involved in tumor progression and could be used as clinically-relevant biomarkers.
Until now, there are limited studies reporting the functional or clinical significance of piRNAs in CRC. We noted Cheng, et al. revealed piR-651 was overexpressed in several types of cancers including CRC . However, the clinical and biologic significance of this piRNA in CRC remains unknown. In this study, through small RNA-seq analysis, we identified another specific piRNA, the piR-1245, which was consistently overexpressed in colorectal cancer tissues across different cohorts, highlighting its important role in CRC development. Notwithstanding its overexpression in cancer, we discovered that piR-1245 is a promising cancer biomarker, since its overexpression correlated with known risk clinicopathological features such as tumor depth, tumor differentiation and metastasis. Furthermore, another major finding of our study was that piR-1245 was a robust prognostic biomarker for survival prediction in CRC patients. These findings may help provide a better understanding of the mechanisms of piRNA in cancer progression and metastasis in CRC, and suggest that this novel small RNA may be an important disease biomarker and a potential therapeutic target in this disease.
To fully appreciate the clinical significance of piR-1245 in CRC, its biological significance as a contributor to colorectal pathogenesis should also be considered. Our functional experiments provide convincing evidence to support for the associations of piR-1245 with an aggressive clinical phenotype, where piR-1245 promotes CRC cells survival, migration and invasion as well as suppression of apoptosis. Consistent with this paradigm, our gene expression profiling results revealed that piR-1245 affects cancer-related pathways and functions as an oncogenic regulator. Accordingly, our results successfully proved our hypothesis, whereby overexpression of piR-1245 affected gene regulatory network for CRC and resulted in an aggressive phenotype, both biologically and clinically.
To further decipher the mechanic role of piR-1245 in CRC, we interrogated its potential downstream gene targets. By using bioinformatics approach, we identified nine ‘functionally relevant’ cancer-related genes. Interestingly, these nine candidates are involved in key tumor suppressive pathways and their expression inversely correlated with piR-1245 expression, supporting the oncogenic role of piR-1245 in CRC. Surprisingly, piR-1245 was found to not only bind to the exonic regions but also within the intronic regions. A recent study reported that piRNAs are able to bind to pre-mRNA introns and subsequently lead to the decay of targeted pre-mRNA through nuclear exosomes , suggesting that piR-1245 may use a similar mechanism to downregulate the expression of target genes. Furthermore, Watanabe, et al. suggested that piRNAs may suppress expression level of mRNAs harboring transposon sequence in 3’UTR or 5’UTR region . Besides, piRNAs may also serve as natural antisense molecules that target genes by binding to their CDS regions or function as siRNAs to target 3’UTR [39–42]. In our study, we observed that piR-1245 could target 3’UTR, CDS or 5’UTR region via perfect or imperfect base-pairing between the two types of RNAs, by a mechanism that closely resembles that of antisense or siRNA. Although a number of possible scenarios could account for the interaction between piR-1245 and its target mRNAs, our data clearly demonstrated that the expression of these targets was significantly altered following gain or loss of piR-1245 expression in CRC cell lines.
Our findings implicate piR-1245 as a potential modulator of colorectal carcinogenesis; a function possibly linked to piRNA-dependent mRNA degradation of its downstream targets. However, the precise mechanisms for the interaction between piR-1245 and its targets merit further investigation. To the best of our knowledge, these data represents first evidence for the role of piRNAs as prognostic biomarkers in CRC. Since piRNAs are abundant in cancer tissues, with improved profiling platforms and availability of tumor samples with extensive clinical annotations, it will be helpful to identify novel CRC-related piRNAs, which will further enhance our understanding of their mechanistic and prognostic contribution to this disease.
The present work was supported by the grants R01 CA72851, CA181572, CA184792 and CA202797 from the National Cancer Institute, National Institute of Health, a grant (RP140784) from the Cancer Prevention Research Institute of Texas (CPRIT), pilot grants from the Baylor Sammons Cancer Center and Foundation, as well as funds from the Baylor Research Institute. In addition, the present work was supported by the grants from the National Natural Science Foundation of China (No.81672826, No. 81372615), the Shanghai Health System Outstanding Young Talent Training Plan (No. XYQ2013118).
Availability of data and materials
Study concept and design: WW, AG; Specimen providers: YT, MK, TN, TF, QW, HQ, YM; Acquisition of clinical data: YT, TN, QW, YM; In vitro experiment: WW; Analysis and interpretation of data and statistical analysis: WW, AG; Sequencing and microarray data and pathway analysis: NL, HL; Drafting of the manuscript: WW and AG. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Written informed consent was obtained from all patients, and the study was approved by the institutional review boards of all participating institutions (Shanghai Tenth People’s Hospital and Okayama University Medical Hospital).
Consent for publication
The authors declare that they have no competing interests.
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