| Part one Screening for colorectal cancer-related pi RNAsBackground & Objective: Colorectal cancer remains a serious threat to human health with high incidence and mortality worldwide.Piwi-interacting RNA(piRNA)is a novel class of non-coding RNAs(ncRNAs),which are expressed aberrantly in many types of tumors.However,Changes in piRNA expression profiling of colorectal cancer have not been investigated.This part of the study aimed to understand the changes in piRNA expression profiling of colorectal cancer and screen out colorectal cancer-related piRNAs.Methods: Deep sequencing was used to investigate the differences in piRNA expression profiling between colorectal cancer tissues and adjacent tissues,as well as screen for colorectal cancer-related piRNAs.Then,the expression of these colorectal cancer-related piRNAs was verified by real-time PCR,and further analyzed for any correlation with patients’ clinicopathological characteristics.Results: Deep sequencing showed that global pi RNAs of colorectal cancer tissues and adjacent tissues accounted for 0.17‰ and 0.23‰ of small ncRNAs in the species,respectively,and for 0.11‰ and 0.08‰ of small ncRNAs in the expression,respectively.Although the expression of piRNAs in colorectal cancer tissues and adjacent tissues was extremely low,it was markedly different between the two tissues,and the global pi RNAs expression was up-regulated in colorectal cancer.We identified 33 up-regulated piRNAs and 2 down-regulated piRNAs in colorectal cancer,among which piR-18849,piR-19521 and piR-17724 are the top three up-regulated piRNAs in colorectal cancer.Real-time PCR further confirmed that the expression of pi R-18849,piR-19521 and piR-17724 was higher in 80 colorectal cancer tissues than that in paired adjacent tissues.Moreover,the expression of piR-18849 was associated with the differentiation degree of tumors and lymph node metastasis,and piR-19521 expression only associated with the differentiation degree of tumors.However,there were no any correlation between the expression of pi R-17724 and the patient’s clinicopathological characteristics.Conclusions: These data reveals that the global expression of piRNAs is up-regulated in colorectal cancer,suggesting that piRNAs may be involved in colorectal tumorigenesis.piR-18849 and piR-19521 could be prognostic biomarkers for patients with colorectal cancer.Part two Colorectal cancer-related piR-19521 drives stem cell-like properties of colorectal cancerBackground & Objective: Recurrence and metastasis are the major causes of high mortality in patients with colorectal cancer.Cancer stem cells(CSCs)CSCs are believed to be the root causes of the initiation,growth,recurrence and metastasis of tumors.Therefore,eradication of CSCs will lead to complete tumor cure.pi RNAs are involved in the stemness maintenance of germ stem cells,and CSCs share similar characteristics with germ stem cells,implying the role of piRNAs in CSCs.Whether piRNAs are involved in the regulation of colorectal cancer stem cell(CRCSC)-like properties remains unclear.The aim of this part was to assess the regulatory effect of colorectal cancer-related pi RNA pi R-19521 on CRCSC-like properties.Methods: The correlation between piR-1952 expression and CRCSC markers expression in colorectal cancer tissues was analyzed by real-time PCR.Then,serum-free culture method was used to enrich CRCSCs from three colorectal cancer cell lines,and subsequently the differences of piR-19521 expression in CRCSCs and corresponding parental cells were evaluated.Finally,the effect of piR-19521 on CRCSC-like properties was determined both in vitro and in vivo by overexpressing piR-19521 in colorectal cancer cell lines and inhibiting piR-19521 in CRCSCs.Results: We found that piR-19521 expression was positively correlated with the expression of CRCSC markers CD29,CD133,CD166 and Lgr5 in colorectal cancer tissues.piR-19521 expression in CRCSCs was significantly higher than that in corresponding parental colorectal cancer cells.Overexpression of piR-19521 promoted the expression of CRCSC markers and embryonic stem cell marker SOX2 and increased the proportion of CRCSC populations.Overexpression of piR-19521 promoted colony formation,migration and invasion of colorectal cancer cells as well as tumorigenicity and liver metastasis in vivo.In contrast,inhibition of piR-19521 suppressed the expression of CRCSC markers and SOX2 and reduced the proportion of CRCSC populations.In addition,inhibition of piR-19521 attenuated the capacity of tumor initiation and self-renewal,as well as restrained tumorigenicity and liver metastasis of CRCSCs in vivo.Conclusions: Our results demonstrate that colorectal cancer-associated piR-19521 is up-regulated in CRCSCs and increases CRCSC-like properties,thereby promoting tumor cell invasion and metastasis.piR-19521 may serve as a novel therapeutic target for colorectal cancer.Part three piR-19521 drives stem cell-like properties of colorectal cancer by inducing ALX4 expressionBackground & Objective: The results from the previous section suggest that piR-19521 regulates CRCSC-like properties.Nevertheless,the molecular mechanisms underlying this process remain poorly understood.This part of the study aimed to elucidate the mechanism by which piR-19521 regulates CRCSC-like properties.Methods: Digital gene expression profiling(DGE)technology was performed to screen piR-19521 target genes involved in regulation of CRCSC-like properties.The candidate target gene was confirmed in HCT116 cells.The molecular mechanism underlying regulation of target gene expression by pi R-19521 was explored using bioinformatics and molecular biology techniques.Results: DGE sequencing revealed that 899 genes were up-regulated and 662 genes were down-regulated following piR-19521 overexpression in colorectal cancer cell lines.GO enrichment analysis and Reactome pathway analysis showed that these differentially expressed genes are involved in a variety of biological processes in cells,including those associated with tumorigenesis and metastasis,such as cell proliferation,cell apoptosis,angiogenesis and cell adhesion.We selected ALX4 as a target gene of piR-19521 and confirmed that overexpression of piR-19521 promoted the expression of ALX4,whereas inhibition of piR-19521 suppressed the expression of ALX4.The expression of ALX4 was significantly up-regulated in CRCSCs compared with parental colorectal cancer cells.Overexpression of ALX4 in HCT116 cells induced the expression of CRCSC markers and SOX2 and increased the proportion of CRCSC populations.We further found that overexpression of ALX4 rescued the decline in stem cell marker expression and self-renewal capacity caused by pi R-19521 inhibition.Interestingly,piR-19521 was derived from an active enhancer element in colorectal cancer cells and enhanced the enhancer activity by regulating H3K27 ac levels in the vicinity of piR-19521 locus.Conclusions: Our study indicates that piR-19521 increases CRCSC-like properties by promoting ALX4 expression.We first reveal that piR-19521 regulates the activity of enhancers and functions as an enhancer RNA. |
