DNA-methylation-mediated silencing of miR-486-5p promotes colorectal cancer proliferation and migration through activation of PLAGL2/IGF2/?-catenin signal pathways.
ABSTRACT: As one of the most common cancers worldwide, colorectal cancer (CRC) causes a large number of mortality annually. Aberrant expression of microRNAs (miRNAs) is significantly associated with the initiation and development of CRC. Further investigations regarding the regulatory mechanism of miRNAs is warranted. In this study, we discovered that miR-486-5p was remarkably downregulated in CRC, which partially results from higher DNA methylation in the promoter region detected by using methylation-specific PCR, bisulfite sequencing PCR, and DNA demethylation treatment. Besides, decreased miR-486-5p was obviously associated with advanced TNM stage, larger tumor size, lymphatic metastasis, and poor prognosis in CRC. Upregulated miR-486-5p inhibited the proliferation and migration of CRC through targeting PLAGL2 expression and subsequent repressing IGF/?-catenin signal pathways both in vitro and in vivo. Notably, plasma miR-486-5p expression was significantly upregulated in CRC patients and we identified plasma miR-486-5p as a novel diagnostic biomarker of CRC using receiver operating characteristic (ROC) curve analysis. Moreover, exploration in GEO dataset revealed that circulating miR-486-5p is tumor derived through being packaged into secretory exosomes. Taken together, our data demonstrated that miR-486-5p promotes colorectal cancer proliferation and migration through activation of PLAGL2/IGF2/?-catenin signal pathway, which is a promising therapeutic target of CRC treatment.
Project description:Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cancer deaths. Increasing evidences supports the idea that the poor prognosis of patients is related to the presence of cancer stem cells (CSCs), a cell population able to drive cancer recurrence and metastasis. The deregulation of microRNAs (miRNAs) plays a role in the formation of CSC. We investigated the role of hsa-miR-486-5p (miR-486-5p) in CRC, CSCs, and metastasis, in order to reach a better understanding of the biomolecular and epigenetic mechanisms mir-486-5p-related. The expression of miR-486-5p was investigated in three different matrices from CRC patients and controls and in CSCs obtained from the CRC cell lines HCT-116, HT-29, and T-84. In the human study, miR-486-5p was up-regulated in serum and stool of CRC patients in comparison with healthy controls but down-regulated in tumor tissue when compared with normal mucosa. miR-486-5p was also down-regulated in the sera of metastatic patients. In vitro, miR-486-5p was down-regulated in CSC models and it induced an inhibitory effect on stem factors and oncogenes in the main pathways of CSCs. Our results provide a step forward in understanding the role of mir-486-5p in CRC and CSC, and suggest that further studies are needed to investigate its diagnostic and prognostic power, possibly in combination with other biomarkers.
Project description:MicroRNAs (miRNAs/miRs) regulate the levels of transcripts and serve a critical function in the regulation of tumor microenvironments. Therefore, miRNA levels in cancer tissues are thought to be potential biomarkers for immunotherapy. From a phase I trial of a vaccine treatment using 5 human leukocyte antigen (HLA)-A*2402-restricted peptides (registration no. UMIN000004948), colorectal cancer (CRC) tissues were obtained from 8 patients and normal colorectal tissues from 5 patients via surgery. From a phase II trial using the same peptides (registration no. UMIN000001791), CRC tissues were obtained from 16 patients from the HLA-A*2402-matched group and 10 patients from the HLA-A*2402-unmatched group. These tissues were used for miRNA microarray analysis. As the first step, cancer tissues from the phase I study were used and 10 candidate miRNAs were selected by comparing the miRNA expression between two groups; one with improved prognosis and the other with poor prognosis. The miRNAs were subsequently validated using the cases enrolled in the phase II study. Significantly improved prognoses were identified in 16 patients in the HLA-A*2402-matched group with high expression of miR-196b-5p and low expression of miR-378a-3p and miR-486-5p. There was no difference in prognosis in the 10 patients in the HLA-A*2402-unmatched group. Therefore, high miR-196b expression and low miR-378a-3p and miR-486-5p expression were indicated as useful biomarkers for prediction of the efficacy of vaccine treatment for patients with metastatic CRC. In a planned phase III study, expression levels of these 3 miRNAs (miR-196b-5p, miR-378a-3p and miR-486-5p) may be useful biomarkers for assessing patients who are likely to have an improved outcome following vaccination.
Project description:BACKGROUND:We previously demonstrated that the pleomorphic adenoma gene like-2 (PLAGL2) is involved in the pathogenesis of Hirschsprung disease. Enhanced PLAGL2 expression was observed in several malignant tumours. However, the exact function of PLAGL2 and its underlying mechanism in colorectal cancer (CRC) remain largely unknown. METHODS:Immunohistochemical analysis of PLAGL2 was performed. A series of in vitro and in vivo experiments were conducted to reveal the role of PLAGL2 in the progression of CRC. RESULTS:Enhanced PLAGL2 expression was significantly associated with EMT-related proteins in CRC. The data revealed that PLAGL2 promotes CRC cell proliferation, migration, invasion and EMT both in vitro and in vivo. Mechanistically, PLAGL2 promoted the expression of ZEB1. PLAGL2 enhanced the expression and nuclear translocation of ?-catenin by decreasing its phosphorylation. The depletion of ?-catenin neutralised the regulation of ZEB1 that was caused by enhanced PLAGL2 expression. The small-molecule inhibitor PNU-74654, also impaired the enhancement of ZEB1 that resulted from the modified PLAGL2 expression. The depletion of ZEB1 could block the biological function of PLAGL2 in CRC cells. CONCLUSIONS:Collectively, our findings suggest that PLAGL2 mediates EMT to promote colorectal cancer metastasis via ?-catenin-dependent regulation of ZEB1.
Project description:Evidence has shown that microRNAs (miRNAs) participate in the progression of CRC. Previous studies have indicated that miR-214-3p is abnormally expressed in various malignant tumors. However, the biological function it plays in CRC and the potential mechanism are unclear. Here, we demonstrated that miR-214-3p was obviously downregulated in CRC. Moreover, we found a strong correlation between the miR-214-3p level and tumor size and lymphatic metastasis. Furthermore, when miR-214-3p was decreased by an Lv-miR-214-3p inhibitor, the proliferation and migration of SW480 and HCT116 cells were significantly increased. As expected, the ability of proliferation and migration was significantly suppressed when miR-214-3p was overexpressed in DLD1 cells. According to the dual-luciferase reporter results, PLAGL2 was found to be a direct downstream molecule of miR-214-3p. Chromatin immunoprecipitation (CHIP) confirmed that MYH9, a well-known cytoskeleton molecule in CRC, was a direct targeting gene of PLAGL2. Silencing PLAGL2 or MYH9 could reverse the effect of a miR-214-3p inhibitor on CRC cells. In summary, our studies proved that low expression of miR-214-3p and overexpression of downstream PLAGL2 in CRC indicated a poor prognosis. MiR-214-3p suppressed the malignant behaviors of colorectal cancer by regulating the PLAGL2/MYH9 axis. MiR-214-3p might be a novel therapeutic target or prognostic marker for CRC.
Project description:Metastasis remains a big barrier for the clinical treatment of colorectal cancer (CRC). Our previous proteomics analysis identified DJ-1 as a potential metastasis biomarker of CRC. In this study, we found that DJ-1 was upregulated in CRC. The levels of DJ-1 were closely correlated with the depths of invasion and predicted patient outcome. Enforced expression of DJ-1 could enhance CRC proliferation and metastasis in vitro and in vivo by stimulating Wnt-?-catenin signaling. Specifically, DJ-1-induced ?-catenin nuclear translocation stimulated TCF transcription activity, which promoted BMP4 expression for CRC cell migration and invasion, and elevated CCND1 expression for CRC cell proliferation, respectively. Furthermore, DJ-1-induced Wnt signaling activation was dependent on PLAGL2 expression. In conclusion, our study demonstrates that DJ-1 can promote CRC metastasis by activating PLAGL2-Wnt-BMP4 axis, suggesting novel therapeutic opportunities for postoperative adjuvant therapy in CRC patients.
Project description:To identify microRNAs (miRNAs, miRs) with potential roles in lung fibrogenesis, we performed genome-wide profiling of miRNA expression in lung tissues from a silica-induced mouse model of pulmonary fibrosis using microarrays. Seventeen miRNAs were selected for validation via qRT-PCR based on the fold changes between the silica and the control group. The dysregulation of five miRNAs, including miR-21, miR-455, miR-151-3p, miR-486-5p and miR-3107, were confirmed by qRT-PCRs in silica-induced mouse model of pulmonary fibrosis and were also confirmed in a bleomycin (BLM)-induced mouse lung fibrosis. Notably, miR-486-5p levels were decreased in the serum samples of patients with silicosis, as well as in the lung tissues of patients with silicosis and idiopathic pulmonary fibrosis (IPF). In addition, as determined by luciferase assays and Western blotting, SMAD2, a crucial mediator of pulmonary fibrosis, was identified to be one of target genes of miR-486-5p. To test the potential therapeutic significance of this miRNA, we overexpressed miR-486-5p in animal models. At day 28, miR-486-5p expression significantly decreased both the distribution and severity of lung lesions compared with the silica group (P?<?0.01). In addition, miR-486-5p had a similar effect in the BLM group (P?<?0.001). These results indicate that miR-486-5p may inhibit fibrosis.
Project description:Glioblastoma multiforme (GBM) and other solid malignancies are heterogeneous and contain subpopulations of tumor cells that exhibit stem-like features. Our recent findings point to a dedifferentiation mechanism by which reprogramming transcription factors Oct4 and Sox2 drive the stem-like phenotype in glioblastoma, in part, by differentially regulating subsets of miRNAs. Currently, the molecular mechanisms by which reprogramming transcription factors and miRNAs coordinate cancer stem cell tumor-propagating capacity are unclear. In this study, we identified miR-486-5p as a Sox2-induced miRNA that targets the tumor suppressor genes PTEN and FoxO1 and regulates the GBM stem-like cells. miR-486-5p associated with the GBM stem cell phenotype and Sox2 expression and was directly induced by Sox2 in glioma cell lines and patient-derived neurospheres. Forced expression of miR-486-5p enhanced the self-renewal capacity of GBM neurospheres, and inhibition of endogenous miR-486-5p activated PTEN and FoxO1 and induced cell death by upregulating proapoptotic protein BIM via a PTEN-dependent mechanism. Furthermore, delivery of miR-486-5p antagomirs to preestablished orthotopic GBM neurosphere-derived xenografts using advanced nanoparticle formulations reduced tumor sizes in vivo and enhanced the cytotoxic response to ionizing radiation. These results define a previously unrecognized and therapeutically targetable Sox2:miR-486-5p axis that enhances the survival of GBM stem cells by repressing tumor suppressor pathways. SIGNIFICANCE: This study identifies a novel axis that links core transcriptional drivers of cancer cell stemness to miR-486-5p-dependent modulation of tumor suppressor genes that feeds back to regulate glioma stem cell survival.
Project description:Tumour hypoxia contributes to poor treatment outcome in locally advanced rectal cancer (LARC) and circulating extracellular vesicles (EVs) as potential biomarkers of tumour hypoxia and adverse prognosis have not been fully explored. We examined EV miRNAs from hypoxic colorectal cancer cell lines as template for relevant miRNAs in LARC patients participating in a prospective biomarker study (NCT01816607). Five cell lines were cultured under normoxia (21% O2) or hypoxia (0.2% O2) for 24 h, and exosomes were isolated by differential ultracentrifugation. Using a commercial kit, exosomes were precipitated from 24 patient plasma samples collected at the time of diagnosis. Exosome size distribution and protein cargo were determined by cryo-electron microscopy, nanoparticle tracking analysis, immunoblotting and flow cytometry. The vesicles harboured strong cell line-specific miRNA profiles with 35 unique miRNAs differentially expressed between hypoxic and normoxic cells. Six of these miRNAs were considered candidate-circulating markers of tumour hypoxia in the patients based on the frequency or magnitude of variance in hypoxic versus normoxic cell line experiments and prevalence in patient plasma. Of these, low plasma levels of exosomal miR-486-5p and miR-181a-5p were associated with organ-invasive primary tumour (p = 0.029) and lymph node metastases (p = 0.024), respectively, both attributes of adverse LARC prognosis. In line with this, the plasma level of exosomal miR-30d-5p was elevated in patients who experienced metastatic progression (p = 0.036). Our strategy confirmed that EVs from colorectal cancer cell lines were exosomes containing the oxygen-sensitive miRNAs 486-5p, 181a-5p and 30d-5p, which were retrieved as circulating markers of high-risk LARC.
Project description:BACKGROUND:Our previous study revealed that PLAGL2 or POFUT1 can promote tumorigenesis and maintain significant positive correlations in colorectal cancer (CRC). However, the mechanism leading to the co-expression and the underlying functional and biological implications remain unclear. METHODS:Clinical tumor tissues and TCGA dataset were utilized to analyze the co-expression of PLAGL2 and POFUT1. Luciferase reporter assays, specially made bidirectional promoter vectors and ectopic expression of 3'UTR were employed to study the mechanisms of co-expression. In vitro and in vivo assays were performed to further confirm the oncogenic function of both. The sphere formation assay, immunofluorescence, Western blot and qRT-PCR were performed to investigate the effect of both genes in colorectal cancer stem cells (CSCs). FINDINGS:PLAGL2 and POFUT1 maintained co-expression in CRC (r?=?0.91, p?<?.0001). An evolutionarily conserved bidirectional promoter, rather than post-transcriptional regulation by competing endogenous RNAs, caused the co-expression of PLAGL2 and POFUT1 in CRC. The bidirectional gene pair PLAGL2/POFUT1 was subverted in CRC and acted synergistically to promote colorectal tumorigenesis by maintaining stemness of colorectal cancer stem cells through the Wnt and Notch pathways. Finally, PLAGL2 and POFUT1 share transcription factor binding sites, and introducing mutations into promoter regions with shared transcription regulatory elements led to a decrease in the PLAGL2/POFUT1 promoter activity in both directions. INTERPRETATION:Our team identified for the first time a bidirectional promoter pair oncogene, PLAGL2-POFUT1, in CRC. The two genes synergistically promote the progression of CRC and affect the characteristics of CSCs, which can offer promising intervention targets for clinicians and researchers. FUND: National Nature Science Foundation of China, the Hunan province projects of Postgraduate Independent Exploration and Innovation of Central South University.
Project description:Cancer cells undergo epithelial-to-mesenchymal transition (EMT) in response to hypoxia. Exosomes produced in tumor microenvironments carry microRNAs (miRNAs) that affect proliferation, metastasis, and EMT. Hypoxic regulation of EMT is associated with telomerase content and stability, but the underlying mechanisms remain unclear. We identified a targeting relationship between tumor-suppressing miR-1255b-5p and human telomerase reverse transcriptase (hTERT) via clinical screening of serum samples in colorectal cancer (CRC) patients. EMT suppression via exosomal miR-1255b-5p delivery was investigated by assessing hTERT expression, Wnt/?-catenin signaling, and telomerase activity. We revealed that hypoxia directly affected exosomal miR-1255b-5p content, the delivery of which between CRC cells significantly impacted cell invasion, EMT-related protein expression, and telomerase stability. Specifically, miR-1255b-5p suppressed EMT by inhibiting Wnt/?-catenin activation via hTERT inhibition. Hypoxia reduced exosomal miR-1255b-5p secretion by CRC cells, thereby increasing hTERT expression to enhance EMT and telomerase activity. In a mouse CRC model, hypoxic exosomes containing overexpressed miR-1255b-5p attenuated EMT, tumor progression, and liver metastasis. Our results suggest the antitumor role of miR-1255b-5p and its involvement in the regulation of hTERT-mediated EMT. We propose that miRNA-targeted regulation of telomerase is a promising therapeutic strategy for future CRC treatment.