Background: HOXD9, a Hox family member, is involved in cancer growth and metastasis. But, its regulation mechanism at the molecular level particularly in colorectal cancer (CRC), is mostly unknown. Methods: The HOXD9 protein expression levels were analyzed using immunofluorescence, immunohistochemistry (IHC) assays, and western blot. The in vivo and in vitro roles of HOXD9 in CRC were determined using colony formation and EdU incorporation, CCK-8, wound scratch and transwell invasion assay, and animal models. Results: Expression of HOXD9 was higher in CRC than in matched healthy tissues.High expression of HOXD9 has significantly associated with the American Joint Committee on Cancer (AJCC) stages, tumor differentiation, lymph node metastasis, and other serious invasions, and it had a poor prognosis. In vitro, HOXD9 encouraged proliferation, movement and EMT processes in cells of CRC. Also, TGF-β1 promoted the expression of HOXD9 and this effect was dependent on the dose and downregulation of HOXD9 repressed TGF-β1 -induced EMT. In vivo, HOXD9 promoted the invasive and metastasis of CRC cells via orthotopic implantation. Conclusions: The ectopic expression of HOXD9 promoted the invasion metastasis in cells of the colorectal tumor by induction of EMT in vitro and vivo. | 3933 LIU et aL How to cite this article: Liu M, Xiao Y, Tang W, et al. HOXD9 promote epithelial-mesenchymal transition and metastasis in colorectal carcinoma.
Background The deubiquitinating enzyme ubiquitin-specific protease 3 (USP3) plays a crucial role in numerous biological processes. The aberrant expression of USP3 may have an important role in tumor development. However, the mechanism by which USP3 promotes gastric cancer (GC) metastasis remains largely unknown. Methods Effects of USP3 on the progression of GC in vivo and in vitro and the potential underlying mechanisms have been investigated utilizing proteomics, RT-PCR, western blotting, immunohistochemistry, immunofluorescence, cell invasion and migration assays and xenograft tumor models. Results USP3 expression was upregulated in GC compared with matched normal tissues and was predictive of poor survival. USP3 also promoted migration and epithelial-to-mesenchymal transition (EMT) in GC cells. Moreover, TGF-β1 induced USP3 expression, and USP3 knockdown inhibited TGF-β1-induced EMT. Furthermore, we utilized Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to identify differentially expressed proteins in USP3-overexpressing cells compared with control cells. Importantly, we found that SUZ12 is indispensable for USP3-mediated oncogenic activity in GC. We observed that USP3 interacted with and stabilized SUZ12 via deubiquitination. SUZ12 knockdown inhibited USP3-induced migration and invasion, as well as EMT in GC cells. Examination of clinical samples confirmed that USP3 expression was positively correlated with SUZ12 protein expression and that the levels of USP3 or SUZ12 protein were negatively correlated with the levels of E-cadherin protein. Conclusions These findings identify USP3 as a critical regulator. The USP3-SUZ12 axis might promote tumor progression and could be a potential therapeutic candidate for human GC. Electronic supplementary material The online version of this article (10.1186/s13046-019-1270-4) contains supplementary material, which is available to authorized users.
Background: The transcription factor HOXD9 is one of the members of the HOX family, which plays an important role in neoplastic processes. However, the role of HOXD9 in the growth and metastasis of gastric cancer (GC) remains to be elucidated. Methods:In vitro functional role of HOXD9 and RURY3 in GC cells was determined using the TMA-based immunohistochemistry, western blot, EdU incorporation, gelatin zymography, luciferase, chromatin Immunoprecipitation (ChIP) and cell invasion assays. In vivo tumor growth and metastasis were conducted in nude mice.Results: HOXD9 is overexpressed in GC cells and tissues. The high expression of HOXD9 was correlated with poor survival in GC patients. Functionally, HOXD9 expression significantly promoted the proliferation, invasion and migration of GC cells. Mechanically, HOXD9 directly associated with the RUFY3 promoter to increase the transcriptional activity of RUFY3. Inhibition of RUFY3 attenuated the proliferation, migration and invasiveness of HOXD9-overexpressing GC cells in vitro and in vivo. Moreover, both HOXD9 and RUFY3 were highly expressed in cancer cells but not in normal gastric tissues, with their expressions being positively correlated. Conclusions:The evidence presented here suggests that the HOXD9-RUFY3 axis promotes the development and progression of human GC.
Background/Aims: Metastasis is the primary cause of colorectal cancer (CRC)-related death. However, the molecular mechanisms underlying metastasis in CRC remain unclear. Methods: We evaluated mRNA and protein expression levels by quantitative real-time reverse transcription PCR, western blotting, immunofluorescence, tissue microarrays, and immunohistochemistry assays. We also assessed the migration and invasion abilities of CRC cells in vitro by wound healing assays, invasion and migration assays, western blot analysis, and immunofluorescence. Tumor metastasis was evaluated in nude mice in vivo. Results: A positive correlation was observed between the expression patterns of Forkhead box k1 (FOXK1) and Snail in CRC. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that Snail directly bound to and activated the human FOXK1 gene promoter. Moreover, the Snail-FOXK1 axis promote epithelial mesenchymal transition (EMT)-mediated CRC cell invasion and metastasis. FOXK1 and Snail expression levels were correlated with tumor progression and served as significant predictors of overall survival in patients with CRC. Furthermore, overexpression of FOXK1 induced the EMT by upregulating the expression of cysteine-rich angiogenic inducer 61 (Cyr61). Luciferase assays showed that Cyr61 was a direct transcriptional target of FOXK1. Down regulation of Cyr61 decreased FOXK1-enhanced “CRC cell” migration, invasion, and metastasis. Additionally, FOXK1 expression was positively correlated with Cyr61 expression and was associated with poor prognosis. Conclusions: The Snail/FOXK1/Cyr61 signaling axis regulates the EMT and metastasis of CRC.
BackgroundIt has been widely reported that epithelial-mesenchymal transition (EMT) is associated with malignant progression in gastric cancer (GC). Integration of the molecules related to EMT for predicting overall survival (OS) is meaningful for understanding the role of EMT in GC. Here, we aimed to establish an EMT-related gene signature in GC.MethodsTranscriptional profiles and clinical data of GC were downloaded from The Cancer Genome Atlas (TCGA). We constructed EMT-related gene signature for predicting OS by using univariate Cox regression and least absolute shrinkage and selection operator (LASSO) regression analyses. Time-dependent receiver operating characteristic (ROC), Kaplan-Meier analysis were performed to assess its predictive value. A nomogram combining the prognostic signature with clinical characteristics for OS prediction was established. And its predictive power was estimated by concordance index (C-index), time-dependent ROC curve, calibration curve and decision curve analysis (DCA). GSE62254 dataset from Gene Expression Omnibus (GEO) was used for external validation. Quantitative real-time PCR (qRT-PCR) was used to detected the mRNA expression of the five EMT-related genes in human normal gastric mucosal and GC cell lines. To further understand the potential mechanisms of the signature, Gene Set Enrichment Analysis (GSEA), pathway enrichment analysis, predictions of transcription factors (TFs)/miRNAs were performed.ResultsA novel EMT-related gene signature (including ITGAV, DAB2, SERPINE1, MATN3, PLOD2) was constructed for OS prediction of GC. With external validation, ROC curves indicated the signature’s good performance. Patients stratified into high- and low-risk groups based on the signature yielded significantly different prognosis. Univariate and multivariate Cox regression suggested that the signature was an independent prognostic variable. Nomogram for prognostication including the signature presented better predictive accuracy and clinical usefulness than the similar model without risk score to some extent with external validation. The qRT-PCR assays suggested that high expression of the five EMT-related genes could be found in human GC cell lines compared with normal gastric mucosal cell line. GSEA and pathway enrichment analysis revealed that focal adhesion and ECM-receptor interaction might be the two important pathways to the signature.ConclusionOur EMT-related gene signature may have practical application as an independent prognostic factor in GC.
Purpose Growing evidence indicates that aberrant expression of microRNAs contributes to tumor development. However, the biological role of microRNA-4490 (miR-4490) in gastric cancer (GC) remains to be clarified. Methods To explore the function of miR-4490 in GC, we performed colony formation, EdU incorporation, qRT-PCR, Western blotting, in situ hybridization (ISH), immunohistochemistry (IHC), flow cytometry, ChIP and dual-luciferase reporter assays. In addition, the growth, migration and invasion capacities of GC cells were evaluated. Results We found that miR-4490 was significantly downregulated in primary GC samples and in GC-derived cell lines compared with normal controls, and that this expression level was negatively correlated with GC malignancy. Exogenous miR-4490 expression not only reduced cell cycle progression and proliferation, but also significantly inhibited GC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, we found that miR-4490 directly targets USP22, which mediates inhibition of GC cell proliferation and EMT-induced metastasis in vitro and in vivo. Moreover, we found through luciferase and ChIP assays that transcription factor POU2F1 can directly bind to POU2F1 binding sites within the miR-4490 and USP22 promoters and, by doing so, modulate their transcription. Spearman's correlation analysis revealed a positive correlation between USP22 and POU2F1 expression and negative correlations between miR-4490 and USP22 as well as miR-4490 and POU2F1 expression in primary GC tissues. Conclusion Based on our results we conclude that miR-4490 acts as a tumor suppressor, and that the POU2F1/miR-4490/USP22 axis plays an important role in the regulation of growth, invasion and EMT of GC cells.
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