MicroRNA-520f-3p inhibits proliferation of gastric cancer cells via targeting SOX9 and thereby inactivating Wnt signaling.
ABSTRACT: MicroRNAs (miRNAs) are known to be important in a variety of cancer types. The specific expression and roles of miR-520f-3p in the context of gastric cancer (GC), however, remains unknown. Herein we determined miR-520f-3p expression to be significantly reduced in human GC cells compared to cells of the gastric epithelium, with comparable down-regulation also being evident in gastric cancer tissue samples and the low expression of this miRNA was positively correlated with features of more aggressive large tumor size (p?=?0.019), depth of invasion (p?=?0.008), and distant metastasis (p?=?0.037). We further found that lower levels of miR-520f-3p corresponded with poorer GC patient overall (p?=?0.003) and disease-free (p?=?0.036) survival. When over-expressed in GC cells, miR-520f-3p was able to impair their growth, proliferation, and survival, instead leading to the induction of apoptosis. We further found that miR-520f-3p was able to bind the SOX9 3'-UTR, thereby negatively regulating its expression in GC cells. Consistent with this model, SOX9 and miR-520f-3p expression were negatively correlated with one another in GC tissues. When SOX9 was upregulated, this was also able to abrogate miR-520f-3p-mediated inactivation of Wnt/?-catenin signaling. Together our findings thus suggest that miR-520f-3p can act to suppress GC progression, at least in part via suppressing SOX9 expression and thus disrupting Wnt/?-catenin signaling. Our results thus highlight potential novel therapeutic targets in GC worthy of future investigation.
Project description:<b>Rationale:</b> Inflammatory stimuli from the tumor microenvironment play important roles in cancer progression. However, the mechanism of promotion of cancer metastasis by inflammation in gastric cancer (GC) is poorly understood. <b>Methods:</b> The roles of NEK9 were validated <i>via</i> loss-of-function and gain-of-function experiments in vitro and in an animal model of metastasis. Cytoskeletal reorganization-associated molecules were detected by GST pull-down. The regulation of ARHGEF2 by NEK9 was investigated by phosphoproteomics analysis, immunoprecipitation (IP) and in vitro kinase assay. The transcriptional regulation of miR-520f-3p was studied using luciferase reporter and chromatin immunoprecipitation (ChIP). The expression of these proteins in GC tissues was examined by immunohistochemistry. <b>Results:</b> NEK9 directly regulates cell motility and RhoA activation in GC. The phosphorylation of ARHGEF2 by NEK9 is the key step of this process. NEK9 is a direct target of miR-520f-3p, which is transcriptionally suppressed by IL-6-mediated activation of STAT3. A decrease in miR-520f-3p leads to the amplification of IL-6/STAT3 by targeting GP130. A simultaneous elevation of the levels of NEK9, GP130 and p-STAT3 was confirmed in the lymph nodes and distant metastases. An increase in NEK9, GP130 and STAT3 is associated with reduced overall survival of GC patients. <b>Conclusion:</b> This study demonstrates that activation of STAT3 by IL-6 transcriptionally suppresses miR-520f-3p and diminishes the inhibitory effects of miR-520f-3p on NEK9 and GP130. An increase in GP130 enhances this signaling, and NEK9 directly influences cell motility and RhoA activation by targeting the phosphorylation of ARHGEF2. Targeting the IL-6-STAT3-NEK9 pathway may be a new strategy for GC treatment.
Project description:Hepatocellular carcinoma (HCC) is a prevalent solid tumor with a high global death rate. SRY box 9 (SOX9) has been reported as an oncogene in HCC by several studies, but the underlying mechanism remains largely unexplored. Here, we confirmed upregulation of SOX9 in HCC tissues and cell lines and validated that SOX9 facilitates proliferation, migration and invasion in HCC. We subsequently identified that the long non-coding RNA (lncRNA) SOX9 antisense RNA 1 (SOX9-AS1) is a neighbor gene to SOX9; SOX9-AS1 is also upregulated in HCC, and its expression is positively correlated with that of SOX9. In addition, SOX9-AS1 appears to have prognostic significance in HCC patients. We showed that SOX9-AS1 aggravates HCC progression and metastasis in vitro and in vivo. We demonstrated that SOX9-AS1 sponges miR-5590-3p to elevate SOX9 expression, and that SOX9 in turn transcriptionally activates SOX9-AS1. Moreover, we verified that SOX9-AS1 regulates SOX9 and its known downstream Wnt/β-catenin pathway so as to facilitate epithelial-to-mesenchymal transition. The results of our rescue assays suggest that SOX9-AS1 regulates HCC progression through SOX9 and the Wnt/β-catenin pathway. In conclusion, our study demonstrates that a SOX9-AS1/miR-5590-3p/SOX9 positive feedback loop drives tumor growth and metastasis in HCC through the Wnt/β-catenin pathway, suggesting SOX9-AS1 as a novel potential prognostic and treatment target for HCC.
Project description:BACKGROUND:Gastric cancer (GC) is a common malignancy and frequent cause of cancer-related death. Long non-coding RNAs (lncRNAs) have emerged as important regulators and tissue-specific biomarkers of multiple cancers, including GC. Recent evidence has indicated that the novel lncRNA LINC01133 plays an important role in cancer progression and metastasis. However, its function and molecular mechanism in GC remain largely unknown. METHODS:LINC01133 expression was detected in 200 GC and matched non-cancerous tissues by quantitative reverse transcription PCR. Gain- and loss-of-function experiments were conducted to investigate the biological functions of LINC01133 both in vitro and in vivo. Insights into the underlying mechanisms of competitive endogenous RNAs (ceRNAs) were determined by bioinformatics analysis, dual-luciferase reporter assays, quantitative PCR arrays, TOPFlash/FOPFlash reporter assay, luciferase assay, and rescue experiments. RESULTS:LINC01133 was downregulated in GC tissues and cell lines, and its low expression positively correlated with GC progression and metastasis. Functionally, LINC01133 depletion promoted cell proliferation, migration, and the epithelial-mesenchymal transition (EMT) in GC cells, whereas LINC01133 overexpression resulted in the opposite effects both in vitro and in vivo. Bioinformatics analysis and luciferase assays revealed that miR-106a-3p was a direct target of LINC01133, which functioned as a ceRNA in regulating GC metastasis. Mechanistic analysis demonstrated that miR-106a-3p specifically targeted the adenomatous polyposis coli (APC) gene, and LINC01133/miR-106a-3p suppressed the EMT and metastasis by inactivating the Wnt/?-catenin pathway in an APC-dependent manner. CONCLUSIONS:Our findings suggest that reduced expression of LINC01133 is associated with aggressive tumor phenotypes and poor patient outcomes in GC. LINC01133 inhibits GC progression and metastasis by acting as a ceRNA for miR-106a-3p to regulate APC expression and the Wnt/?-catenin pathway, suggesting that LINC01133 may serve as a potential prognostic biomarker and anti-metastatic therapeutic target for GC.
Project description:Upstream ORF (uORF) is a translational initiation element located in the 5'UTR of eukaryotic mRNAs. Studies have found that uORFs play an important regulatory role in many diseases. Based on The Cancer Genome Atlas database, the results of our experiments and previous research evidence, we investigated transcription factor AP-4 (TFAP4) and its uORF, LIM and SH3 protein 1 (LASP1), long noncoding RNA 00520 (LINC00520), and microRNA (miR)-520f-3p as candidates involved in glioma malignancy, which is a poorly understood process. Both TFAP4-66aa-uORF and miR-520f-3p were downregulated, and TFAP4, LASP1, and LINC00520 were highly expressed in glioma tissues and cells. TFAP4-66aa-uORF or miR-520f-3p overexpression or TFAP4, LASP1, or LINC00520 knockdown inhibited glioma cell proliferation, migration, and invasion, but promoted apoptosis. TFAP4-66aa-uORF inhibited the translation of TFAP4 by binding to the TFAP4 mRNA. MicroRNA-520f-3p inhibited TFAP4 expression by binding to its 3'UTR. However, LINC00520 could promote the expression of TFAP4 by competitively binding to miR-520f-3p. In addition, TFAP4 transcriptionally activated LASP1 and LINC00520 expression by binding to their promoter regions, forming a positive feedback loop of TFAP4/LINC00520/miR-520f-3p. Our findings together indicated that TFAP4-66aa-uORF inhibited the TFAP4/LINC00520/miR-520f-3p feedback loop by directly inhibiting TFAP4 expression, subsequently leading to inhibition of glioma malignancy. This provides a basis for developing new therapeutic approaches for glioma treatment.
Project description:Aberrant activation of Wnt/??catenin signaling is observed in >90% of colorectal cancer cases. microRNAs (miRNAs) regulate the expression of key genes in Wnt/??catenin signaling. As a result, abnormal expression of miRNAs regulates the activation of Wnt/??catenin signaling in several types of cancer. In the current study, it was demonstrated that miR?501?3p was overexpressed in colorectal tumor tissues compared to the adjacent normal tissues. Downregulation of miR?501?3p inhibited cell proliferation and sphere formation, while it induced cell cycle arrest at the G1 phase in colorectal cancer cells. Bioinformatics analysis results predicted that adenomatous polyposis coli (APC), a negative regulator of Wnt/??catenin signaling, was a potential target gene of miR?501?3p. Inhibition of miR?501?3p increased APC expression in colorectal cancer cells. Additionally, ??catenin was destabilized following miR?501?3p inhibition; immunofluorescence analysis revealed that ??catenin translocated from nucleus to cytoplasm. In addition, cyclin D1 and c?Myc, two well?characterized target genes of Wnt/??catenin signaling, were downregulated following miR?501?3p inhibition. Transfection of APC small interfering RNA re?activated ??catenin and stimulated the expression of cyclin D1 and c?Myc. Furthermore, silencing of APC reversed the miR?501?3p inhibitor?induced cell cycle disruption, and the inhibition of cell proliferation and sphere formation in colorectal cancer cells. In conclusion, the present study identified miR?501?3p as a novel regulator of Wnt/??catenin signaling in colorectal cancer cells via targeting APC, suggesting that miR?501?3p may act as a novel oncogenic miRNA in colorectal cancer.
Project description:<h4>Background</h4>Gastric cancer (GC) is one of the most common human cancers with the high rate of recurrence, metastasis and mortality. Aberrantly expressed microRNAs (miRNAs) are associated with invasion and metastasis in various human cancers. Recently, miR-188-5p has been indicated as an oncogene in GC since it promotes GC cell growth and metastasis. However, the underlying molecular mechanism remains to be fully defined.<h4>Methods</h4>Using Significance Analysis of Microarrays (SAM) screening, we identified that miR-188-5p is associated with overall survival and lymph node metastasis in patients with GC. The functional impact of miR-188-5p on GC metastasis was validated using in vitro and in vivo assays. The regulatory function of miR-188-5p on Wnt/?-catenin signaling activation through directly targeting PTEN was proven using quantitative real-time PCR, western blot analysis, a dual-luciferase assay, a Transwell assay, and immunofluorescence. Immunohistochemical analyses further confirmed the clinical significance of miR-188-5p in GC.<h4>Results</h4>MiR-188-5p diminishes tumor suppressor PTEN expression, and further increases phospho-Ser9 of GSK3? to activate Wnt/?-catenin signaling in GC. Consequently, miR-188-5p enhanced the migration and invasion of GC cells in vitro and tumor metastasis in vivo, whereas inhibition of miR-188-5p had the opposite effects. Moreover, miR-188-5p was negatively correlated with PTEN expression but positively correlated with nuclear ?-catenin staining in GC samples.<h4>Conclusions</h4>Our findings revealed a model of the miR-188-5p-PTEN-?-catenin axis in GC, which mediates the constitutive activation of Wnt/?-catenin signaling and promotes tumor metastasis, inferring that miR-188-5p is a potential therapeutic target to treat GC.
Project description:The Wnt/?-catenin pathway is constitutively active and promotes multiple tumor processes, including breast cancer metastasis. However, the underlying mechanism by which the Wnt/?-catenin pathway is constitutively activated in breast cancer metastasis remains unclear. Inhibition of Wnt antagonists is important for Wnt/?-catenin signaling activation, and post-transcriptional regulation of these antagonists by microRNAs (miRNAs) might be a possible mechanism underlying signaling activation. Regulation of nuclear pre-mRNA domain-containing 1A (RPRD1A) is a known inhibitor of cell growth and Wnt/?-catenin signaling activity, but the function and regulatory mechanism of RPRD1A in breast cancer have not been clarified. The aim of this study was to understand how regulators of the Wnt/?-catenin pathway may play a role in the metastasis of this cancer. <b>Methods:</b> RPRD1A expression and its association with multiple clinicopathological characteristics was analyzed immunohistochemically in human breast cancer specimens. miR-454-3p expression was analyzed using real-time PCR. RPRD1A or miR-454-3p knockdown and overexpression were used to determine the underlying mechanism of their functions in breast cancer cells. Xenografted tumor model, 3D invasive culture, cell migration and invasion assays and sphere formation assay were used to determine the biofunction of RPRD1A and miR-454-3p in breast cancer. Electrophoretic mobility shift assay (EMSA), luciferase reporter assay, and RNA immunoprecipitation (RIP) were performed to study the regulation and underlying mechanisms of RPRD1A and miR-454-3p expression and their correlation with the Wnt/?-catenin pathway in breast cancer. <b>Results:</b> The Wnt/?-catenin signaling antagonist RPRD1A was downregulated and its upstream regulator miR-454-3p was amplified and overexpressed in metastatic breast cancer, and both were correlated with overall and relapse-free survival in breast cancer patients. The suppression by miR-454-3p on RPRD1A was found to activate Wnt/?-catenin signaling, thereby promoting metastasis. Simultaneously, three other negative regulators of the Wnt/?-catenin pathway, namely, AXIN2, dickkopf WNT signaling pathway inhibitor (DKK) 3 and secreted frizzled related protein (SFRP) 1, were also found to be targets of miR-454-3p and were involved in the signaling activation. miR-454-3p was found to be involved in early metastatic processes and to promote the stemness of breast cancer cells and early relapse under both <i>in vitro</i> and <i>in vivo</i> conditions. <b>Conclusions:</b> The findings indicate that miR-454-3p-mediated suppression of Wnt/?-catenin antagonist RPRD1A, as well as AXIN2, DKK3 and SFRP1, sustains the constitutive activation of Wnt/?-catenin signaling; thus, miR-454-3p and RPRD1A might be potential diagnostic and therapeutic targets for breast cancer metastasis.
Project description:Cancer stem cells (CSCs) are involved in tumorigenesis, tumour recurrence and therapy resistance and Wnt signalling is essential for the development of the biological traits of CSCs. In non-small cell lung carcinoma (NSCLC), unlike in colon cancer, mutations in ?-catenin and APC genes are uncommon; thus, the mechanism underlying the constitutive activation of Wnt signalling in NSCLC remains unclear. Here we report that miR-582-3p expression correlates with the overall- and recurrence-free-survival of NSCLC patients, and miR-582-3p has an activating effect on Wnt/?-catenin signalling. miR-582-3p overexpression simultaneously targets multiple negative regulators of the Wnt/?-catenin pathway, namely, AXIN2, DKK3 and SFRP1. Consequently, miR-582-3p promotes CSC traits of NSCLC cells in vitro and tumorigenesis and tumour recurrence in vivo. Antagonizing miR-582-3p potently inhibits tumour initiation and progression in xenografted animal models. These findings suggest that miR-582-3p mediates the constitutive activation of Wnt/?-catenin signalling, likely serving as a potential therapeutic target for NSCLC.
Project description:BACKGROUND:EphA2 is a crucial oncogene in gastric cancer (GC) development and metastasis, this study aims to identify microRNAs that target it and serve as key regulators of gastric carcinogenesis. METHODS:We identified several potential microRNAs targeting EphA2 by bioinformatics websites and then analyzed the role of miR-302b in modulating EphA2 in vitro and in vivo of GC, and it's mechanism. RESULTS:Our analysis identified miR-302b, a novel regulator of EphA2, as one of the most significantly downregulated microRNA (miRNA) in GC tissues. Overexpression of miR-302b impaired GC cell migratory and invasive properties robustly and suppressed cell proliferation by arresting cells at G0-G1 phase in vitro. miR-302b exhibited anti-tumor activity by reversing EphA2 regulation, which relayed a signaling transduction cascade that attenuated the functions of N-cadherin, ?-catenin, and Snail (markers of Wnt/?-catenin and epithelial-mesenchymal transition, EMT). This modulation of EphA2 also had distinct effects on cell proliferation and migration in GC in vivo. CONCLUSIONS:miR-302b serves as a critical suppressor of GC cell tumorigenesis and metastasis by targeting the EphA2/Wnt/?-catenin/EMT pathway.
Project description:BACKGROUND AIMS: Gastric cancer is the most frequent gastrointestinal tumor in adults and is the most lethal form of human cancer. Despite of the improvements in treatments, the underlying mechanism of gastric carcinogenesis is not well known. To define novel modulators that regulate susceptibility to tumorgenesis, we focused on miR-219-2-3p. METHODS: Quantitative RT-PCR was employed to investigate the level of miR-219-2-3p in gastric cancer (GC) tissues (n = 113) and their matched adjacent normal tissues (n = 113). In vitro cell proliferation, apoptosis assays, cell migration, and invasion assays were performed to elucidate biological effects of miR-219-2-3p. Since silencing of miRNA by promoter CpG island methylation may be an important mechanism in tumorgenesis, GC cells were treated with 5-aza-2'-deoxycytidine and trichostatin A, and expression changes of miR-219-2-3p were subsequently examined by quantitative RT-PCR. Finally, the methylation status of CpG island upstream of miR-219-2-3p was analyzed by methylation-specific PCR in GC tissues (n = 22). RESULTS: miR-219-2-3p was down-regulated in GC and cell lines. In addition, the experiments documented the lower expression of miR-219-2-3p in GC specimens with higher grade and later stage tumors. Meanwhile, miR-219-2-3p exerted antiproliferative, proapoptotic, and antimetastatic roles and reduced levels of p-ERK1/2 in GC cells. Furthermore, 5-aza-2'-deoxycytidine and trichostatin A increased the expression (~2 fold) of miR-219-2-3p in GC cells. By methylation-specific PCR, DNA methylation in the upstream region of miR-219-2-3p was detected in both adjacent normal tissues and cancer tissues. As expected, the methylation level was considerably higher in the miR-219-2-3p down-regulated group than up-regulated group. CONCLUSIONS: miR-219-2-3p is potentially involved in gastric cancer progression and metastasis by regulating ERK1/2-related signal pathways, which may provide a novel therapeutic strategy for treatment of gastric cancer. Methylation mechanism may be involved in modulating the expression level of miR-219-2-3p in gastric cancer.