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MiR-4666-3p and miR-329 Synergistically Suppress the Stemness of Colorectal Cancer Cells via Targeting TGF-?/Smad Pathway.

ABSTRACT: Quiescent caner stem cells are identified as a subpopulation of colon cancer cells in dormant state and possess strong stem-cell like characteristics. Previously, we have identified this subpopulation in colorectal cancer (quiescent colon cancer stem cells, QCCSCs), and find QCCSCs are sensitive to the apoptotic effect of IFN-?, which is attributed to their high IFN-?R expression levels. Microarray and bioinformatic analysis indicate miR-4666-3p is low expressed in QCCSCs and target IFN-?R1/2, which is proved by luciferase assay and western-blot. Furthermore, we find miR-4666-3p could also target TGF-?R1 to block the activation of TGF-?1/Smad pathway, therefore function as a tumor suppressor gene to inhibit the stemness of colon cancer cells. Besides, compared with QCCSCs, we find the TGF-?1 expression also decreased with the weakening of stemness properties. In terms of mechanism, our result reveal TGF-?1 is the target gene of miR-329, which is also high expressed in non-QCCSCs. Thereafter, we perform gain- and loss- function experiments to confirm the synergistic effect between miR-4666-3p and miR-329 in blocking the activation of TGF-?/Smad pathway. Finally, we evaluate the expression of both miR-4666-3p and miR-329 in 73 tumor specimens and paired normal tissue, and find both two miRNAs are related to unfavorable prognosis and advanced tumor stage in colorectal cancer. Our study revealed a novel epigenetic regulation mechanism in colon cancer stem cells, which could be exploited as a novel therapeutic strategy for cancer treatment.

SUBMITTER: Ye J

PROVIDER: S-EPMC6880832 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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miR-4666-3p and miR-329 Synergistically Suppress the Stemness of Colorectal Cancer Cells via Targeting TGF-β/Smad Pathway.

Ye Jun J Lei Jiacai J Fang Qingqing Q Shen Yimin Y Xia Wenjie W Hu Xiaoge X Xu Qiuran Q Yuan Hongjun H Huang Jian J Ni Chao C

Frontiers in oncology 20191119

Quiescent caner stem cells are identified as a subpopulation of colon cancer cells in dormant state and possess strong stem-cell like characteristics. Previously, we have identified this subpopulation in colorectal cancer (quiescent colon cancer stem cells, QCCSCs), and find QCCSCs are sensitive to the apoptotic effect of IFN-γ, which is attributed to their high IFN-γR expression levels. Microarray and bioinformatic analysis indicate miR-4666-3p is low expressed in QCCSCs and target IFN-γR1/2, w ...[more]

PMID: 31824844

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Project description:BackgroundColorectal cancer is one of the most common malignancy in the world. It has been reported that cancer stem cells (CSCs) serve as the primary drivers of tumorigenesis and tumor progression. There is an urgent need to explore novel molecules that regulate CSCs or their signatures. Increasing evidence has shown that miRNAs are involved in tumorigenesis and progression. Here, we aim to explore the regulatory effect and mechanism of miR-3065-3p on the stemness of colorectal cancer.MethodsThe expression of miR-3065-3p in colorectal cancer and the association of miR-3065-3p expression with prognosis of patients with colorectal cancer were analyzed using TCGA dataset or clinical cases. Gain or loss of function in different models, including colorectal cancer cell lines and orthotopic xenograft or liver metastatic mouse model, were used to investigate the effects of miR-3065-3p on colorectal cancer stemness and metastasis in vitro and in vivo. Cancer stemness was analyzed by detecting the ability of migration and invasion, NANOG, OCT4, and SOX2 expression, ALDH activity and sphere formation. In addition, the interaction of miR-3065-3p and cytokine receptor-like factor 1 (CRLF1) was analyzed theoretically and identified by the luciferase reporter assay. Moreover, the correlation between CRLF1 expression and miR-3065-3p was analyzed in colorectal cancer tissues. Finally, the effect of CRLF1 on the stemness and metastasis of colorectal cancer in vitro and in vivo was assessed.ResultsIn this report, we found that miR-3065-3p was overexpressed in colorectal cancer and that its high expression was associated with poor prognosis of patients with colorectal cancer. miR-3065-3p promotes the stemness and metastasis of colorectal cancer. Furthermore, CRLF1 was the downstream target of miR-3065-3p and inhibited the stemness of colorectal cancer. In addition, CRLF1 expression was negatively correlated with miR-3065-3p in colorectal cancer tissues. And, CRLF1 mediated the effects of miR-3065-3p on promoting stemness of colorectal cancer cells.ConclusionOur data suggest that miR-3065-3p promoted the stemness and metastasis of colorectal cancer by targeting CRLF1. miR-3065-3p might serve as a promising prognostic marker as well as a therapeutic target for colorectal cancer.

Project description:BackgroundColorectal cancer (CRC) is the leading cause of cancer-related death worldwide. Exosome shave emerged as crucial regulators of intercellular communication and that abundant Circular RNAs (circRNAs) are enriched within exosomes. CircRNAs are novel members of noncoding RNAs regulating cancer proliferation and progression. However, the function and regulatory mechanism of cancer-derived exosomal circRNAs in CRC remains unclear.MethodsCRC cells-derived exosomes were characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blot. CCK-8, wound healing and transwell assays, and flow cytometry assays were conducted to assess whether exosomes would affect the proliferation, metastasis, and apoptosis of CRC cells, respectively. Moreover, we performed the RNA sequencing and RT-qPCR to identify circRNAs in exosome-stimulated CRC cells. Fluorescence in situ hybridization (FISH) assay was used to detect the cellular distribution of circPACRGL. Bioinformatic analyses (StarBase 2.0) were used to pool the miRNA targets of circPACRGL. Luciferase assays were performed to verify the direct interaction. Finally, flow cytometry was used to detect the differentiation of N1-N2 neutrophils.ResultsOur study identified a novel CRC-derived exosomal circRNA, circPACRGL. We found circPACRGL was significantly upregulated in CRC cells after tumor-derived exosomes addition. Moreover, circPACRGL serves as a sponge for miR-142-3p/miR-506-3p to facilitate the transforming growth factor-β1 (TGF-β1) expression. As a result, circPACRGL promoted CRC cell proliferation, migration and invasion, as well as differentiation of N1 to N2 neutrophils via miR-142-3p/miR-506-3p-TGF-β1 axis.ConclusionOur study, the first to reveal that cancer-derived exosomal circPACRGL plays an oncogenic role in CRC proliferation and metastasis, providing mechanistic insights into the roles of circRNAs in CRC progression and a valuable marker for CRC treatment.

Dataset Information

MiR-4666-3p and miR-329 Synergistically Suppress the Stemness of Colorectal Cancer Cells via Targeting TGF-?/Smad Pathway.

Publications

miR-4666-3p and miR-329 Synergistically Suppress the Stemness of Colorectal Cancer Cells via Targeting TGF-β/Smad Pathway.

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