Novel evidence for a PIWI-interacting RNA (piRNA) as an oncogenic mediator of disease progression, and a potential prognostic biomarker in colorectal cancer

To identify CRC-associated piRNAs, we performed small RNA-sequencing on a subset of frozen cancer tissues and paired normal mucosa (NM) specimens (4 each), which were collected at the Mie University, Japan. To confirm the expression levels of candidate piRNAs between cancer and normal tissues, we measured their expression in matched cancer and normal tissues in three independent patient cohorts from the Mie University, Japan (n = 20), Shanghai Tenth People’s Hospital, China (n = 20) and Okayama University Medical Hospital, Japan (n = 18). To investigate the prognostic potential of candidate piRNAs in CRC, we analyzed piR-1245 expression pattern in three different patient cohorts with a combined total of 771 CRC patients from the TCGA dataset, a clinical testing cohort and an independent validation cohort. The expression profiles of piRNAs from the TCGA (The Cancer Genome Atlas) dataset (n = 387) was characterized by Martinez, et al. [20]. We thereafter analyzed expression of candidate piRNAs in a clinical testing cohort (n = 195, Shanghai Tenth People’s Hospital) and an independent validation cohort (n = 189, Okayama University Medical Hospital). Both testing and validation cohort were formalin fixed paraffin embedded tissue specimens, which allowed us to perform micro-dissection for enriching the RNA content from the neoplastic cells. The baseline characteristic of these patient cohorts is described in Table 1. To further understand the mechanistic correlation of piRNAs with its downstream target genes, we evaluated their expression in a cohort of 159, fresh frozen tissues. Written informed consent was obtained from all patients, and the study was approved by the institutional review boards of all participating institutions. All CRC patients were followed up for survival for at least 5 years from their date of surgery. Patients treated with radiotherapy or chemotherapy before surgery were excluded from the study.

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.

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 [26] and as described in the Additional file 1: Supplementary Methods.

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.

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.

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.

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.

PIWI proteins, central to piRNA biogenesis, have recently been found to be frequently overexpressed in different cancer types [27‐31]. Interestingly, through a systematic query of the Oncomine [32] and Protein Atlas database [33‐36], we noted that the mRNA and protein levels of PIWIL1 and PIWIL4, two of the major PIWI proteins, are significantly overexpressed in CRC vs. normal tissues. The implications of these findings are that piRNAs may also be consequently dysregulated and involved in CRC development- a question that has not been interrogated previously (Additional file 3: Figure S1). Accordingly, we firstly performed small RNA-seq analysis to identify CRC-associated piRNAs, by analyzing a subset of CRC and paired normal mucosa (NM) specimens in the Mie cohort. Our results revealed that 3 piRNAs (piR-23,619, piR-24,000 and piR-1245) were up-regulated, while piR-26,525 was down-regulated in cancer tissues (with ≥ 2 fold change and a P value ≤ 0.01; Fig. 1a). To reduce sampling error, we subsequently measured expression level of these candidates in a subset of 20 cancer and paired NM specimens from the same cohort. Unfortunately, we only found piR-1245 and piR-26,525 had significant differential expression between cancer and normal tissues (Fig. 1b). Furthermore, in order to account for patient cohort differences and tumor heterogeneity, we also validated the expression of these two piRNAs in two independent patient cohorts (Shanghai and Okayama cohorts). These validation experiments confirmed that the expression of piR-1245, but not piR-26,525, was higher in CRC tissues, that its expression levels demonstrated a 5.49-fold (P < 0.01) and a 7.0 fold (P < 0.01) increase in CRC vs. NM tissue expression in the two cohorts (Additional file 4: Figure S2). These data suggested that piRNA piR-1245 was a potential oncogenic piRNA in colorectal cancer. In particular, we noticed that piR-1245 is not only overexpressed in CRC but also exhibits a pan-cancer expression pattern. In the TCGA datasets, we found that the expression of piR-1245 was upregulated in other types of cancer including lung, breast, stomach, bladder, kidney and prostate cancer, highlighting its important key role in carcinogenesis (Additional file 5: Figure S3).

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.

To further interrogate the impact of overexpression of piR-1245 and its potential impact on prognosis in CRC patients, we analyzed its expression pattern in three different cohorts with a combined total of 771 CRC patients from the TCGA dataset [20], clinical training cohort and the validation cohort. In the TCGA dataset, piR-1245-high expression group showed a strong tendency to be associated with poor OS (P = 0.0802, HR = 1.604; Fig. 1c). Therefore, we examined prognostic potency of piR-1245 in a training cohort that comprised of high quality tissues with complete follow-up clinical data. As expected, piR-1245-high expression group significantly correlated with poor OS (P = 0.0026, HR = 2.387; Fig. 1d). To further confirm the prognostic value of piR-1245 in CRC patient survival, we investigated these associations in another validation cohort, and successfully once again confirmed that piR-1245-high expression group demonstrated shorter OS (P = 0.0002, HR = 3.208; Fig. 1e), highlighting its clinical significance as independent prognostic biomarker in CRC patients. Furthermore, multivariate cox’s regression analysis revealed that high piR-1245 expression was an independent predictor for poor prognosis in both clinical testing and validation cohorts (HR: 1.965, 95%CI: 1.0683 to 3.6144, P = 0.0298, HR: 2.9347, 95%CI: 1.4584 to 5.9057, P = 0.0025, respectively, Table 2). Taken together, these findings elucidate that overexpression of piR-1245 has clinical significance, and can serve as a potential prognostic biomarker in CRC patients.

Since high expression of piR-1245 indicates aggressive clinical behavior in CRC patients, we questioned whether piR-1245 plays a functional role in this malignancy. To this effect, we performed several functional assays to determine phenotypic alterations following overexpression or inhibition of piR-1245in colon cancer cells. We employed MTT assays to determine the proliferation rates of colon cancer cells transfected with piR-1245 mimics or antisense oligonucleotides. Our results revealed that inhibition of piR-1245 had a pronounced effect on suppressing growth proliferation of HCT116 and SW480 cells, while in contrast, overexpression of piR-1245 resulted in enhanced cell proliferation (Fig. 2a). Meanwhile, we also performed colony formation assays to evaluate the effect of piR-1245 on the colony-forming ability of single cells. As shown in Fig. 2b, inhibition of piR-1245 in HCT116 and SW480 cells demonstrated significantly reduced number of colonies compared to control cells, while up-regulation of piR-1245 markedly increased the total number of colonies. In line with above findings, inhibition of piR-1245 significantly reduces the percent of Ki-67 positive colon cancer cells, suggesting that piR-1245 functions as a positive regulator of cell survival (Fig. 2c and Additional file 6: Figure S4).

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 [37]. 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.

To investigate the oncogenic mechanism of piR-1245 in CRC, we investigated its impact on transcriptomic alterations in CRC cell lines. We treated HCT116 cells with or without piR-1245 antisense and subsequently performed gene expression microarray analysis. As shown in Fig. 3a, a total of 244 mRNAs were detected to be differentially expressed with a fold change of ≥ 1.5 and a corresponding P < 0.01. Notably, 168 genes were found to be upregulated, while 76 genes were downregulated in piR-1245-inhibited cells compared to control cells.

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.

A growing body of studies have shown that piRNAs have the capability to bind a diverse spectrum of downstream target genes by forming specific piRNA silencing complexes (pi-RISC), leading to RNA repression via imperfect base-pairing between the two types of RNAs [38‐40]. We therefore examined potential target sites of piR-1245 interaction and the impacted downstream genes. We used miRANDA and RNA22 tool, applied stringent thermodynamic parameters and binding energy thresholds, to predict biologically relevant piRNA:mRNA interactions. We found there were at least 9 potential targets complementary to piR-1245including ATF3, BTG1, DUSP1, FAS, NFKBIA, UPP1, SESN2, TP53INP1 and MDX1. The examples of piRNA:mRNA complementarities identified by this approach are shown in Fig. 3c and Additional file 8: Figure S6 and Additional file 9: Figure S7.These genes have been reported to be involved in key cellular processes in CRC, including cell death and survival, cell cycle, DNA replication and repair or cell-cell communication (Additional file 10: Table S2). We further performed qPCR to confirm the expression change of target genes following piR-1245 overexpression or knockdown in HCT116 and SW480 cells and we are able to successively validate these findings (Fig. 4a). To further validate our in vitro results that piR-1245 regulated those tumor suppressors, we investigated the expression correlation between piR-1245 and its target genes in colorectal cancer tissues. Our results indicated that the expression of these targets were all negatively associated with piR-1245 expression in CRC (P < 0.05; Fig. 4b). Moreover, several genes demonstrated a strong inverse correlation with piR-1245 including MXD1, BTG1 and FAS, suggesting their expression levels are tightly synchronized with the piR-1245 function, and highlighting that piR-1245 serves as a potential key oncogenic regulator in CRC.