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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Biomarker; Colorectal cancer; Noncoding RNA; Oncogene; Prognosis; Tumor suppressor; piR-1245; piRNA.

Fig. 1

The clinical significance of cancer-related piRNAs in CRC. a Small RNA-seq revealed differentially expressed piRNAs between cancer and paired normal tissues (4 each) from the Mie cohort. The heatmaps maps illustrate the Z-score of each candidate piRNAs. b The expression of candidate piRNAs were validated in a subset of 20 cancer and paired normal mucosa (NM) specimens in Mie cohort. **P < 0.01, Wilcoxon paired test. The prognostic significance of piR-1245 was evaluated in colorectal cancer patients from TCGA datasets (c) and the clinical training cohort (Shanghai) (d) and the validation cohort (Okayama). e The OS (overall survival) analysis was performed by Kaplan–Meier test and the log-rank method (**P < 0.05, HR: Hazard Ratio)

Fig. 2

The piR-1245 promotes cell growth, colony formation, migration and invasion and inhibits apoptosis in colorectal cancer cells. HCT116 and SW480 cells were transfected with either piR-1245 RNA oligonucleotides, antisense or scrambled controls. The treated cells or control cells were subsequently used for MTT assay (a), colony formation assay (b), Ki-67 staining (c), Migration and invasion assay (d) and apoptosis assay (e). All the experiments were performed biological triplicate. (*P < 0.05, **P < 0.01; independent t-test was used to compare control and treated cells)

Fig. 3

Identification of piR-1245 downstream target genes. a HCT116 cells were treated with or without piR-1245 antisense and subsequently performed gene expression microarray analysis. 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 up-regulated, while 76 genes were down-regulated in piR-1245-inhibited cells compared to control cells. b IPA analysis was performed for the upregulated genes to interrogate the function of piR-1245 in CRC. 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 (c) miRANDA and RNA22 tool was used to predict the binding of piR-1245 to potential targets

Fig. 4

The correlation between piR-1245 and its target genes in CRC tissues. a qPCR was performed to confirm the expression change of target genes after piR-1245 overexpression or knockdown in HCT116 and SW480 cells. (n = 3, *P < 0.05, **P < 0.01, independent t-test was used to compare control and treated cells). b qPCR was performed to evaluate the expression correlation between piR-1245 and its targets in CRC tissues. (n = 159, *P < 0.05, **P < 0.01; Spearman’s rank correlation (ρ) was used for the correlation analysis)