miR-26a/HOXC9 Dysregulation Promotes Metastasis and Stem Cell-Like Phenotype of Gastric Cancer

Xudong Peng, Qingjie Kang, Rui Wan, Ziwei Wang
2018 Cellular Physiology and Biochemistry  
Background/Aims: Previous studies demonstrated that HOXC9 acts as an oncogene in several tumors. The aim of this study was to explore whether HOXC9 promotes gastric cancer (GC) progression and elucidate the underlying molecular mechanisms. Methods: HOXC9 expression in GC tissues and adjacent non-cancer tissues was detected by quantitative RT-PCR (qRT-PCR) and immunohistochemistry. The functional effects of HOXC9 on proliferation, metastasis and stem cell-like phenotype were evaluated by
more » ... experiments in GC cells. The effect of miR-26a on HOXC9 was investigated by gain-and loss-of-function assays and luciferase reporter assay. Nude mouse models were established to test the effect of miR-26a and HOXC9 on tumorigenesis and metastasis of GC cells in vivo. Results: Herein, we showed that HOXC9 was upregulated in GC tissues and associated with a poor prognosis. HOXC9 knockdown inhibited the metastasis and stem cell-like phenotype of GC cells without significant effects on cell proliferation. In addition, we identifed HOXC9 as a direct target of miR-26a. Restoration of miR-26a in GC cells downregulated HOXC9 and reversed its promoting effect on metastasis and self-renewal, whereas miR-26a silencing upregulated HOXC9. In vivo experiments showed that HOXC9 knockdown suppressed tumorigenesis and lung metastasis of GC cells in nude mice, and these effects were mimicked by restoration of miR-26a. Conclusion: The present study demonstrates that HOXC9 promotes the metastasis and stem cell-like phenotype of GC cells, and this phenomenon can be reversed by restoration of miR-26a. Peng et al.: miR-26a/HOXC9: a Novel Axis Regulating the Progression of Gastric Cancer the poor prognosis of GC patients [1] . In the past few decades, molecular targeted therapy has become an alternative therapeutic approach with the in-depth study of molecular mechanism of tumor progression. For example, a monoclonal antibody against VEGFR2 called ramucirumab and a monoclonal antibody against HER2 called trastuzumab have been used in the clinical treatment of GC [2, 3] . However, we are still far from identifying a radical cure for GC. To develop new therapies for GC, it is necessary to further explore the molecular mechanisms that regulate GC progression. HOXC9 is a member of the homeobox (HOX) gene family, which contains a homologous domain involved in embryonic development [4] . Recent studies suggest the crucial role of HOXC9 in cancer progression. HOXC9 is highly expressed in glioblastoma and associated with a poor prognosis. HOXC9 silencing activates DAPK1-Beclin1 pathway-induced autophagy in glioblastoma cells, thereby suppressing the proliferation, metastasis, and tumorigenesis of cancer cells [5] . CD133 + glioblastoma cells are tumorigenic stem-like cells. Okamoto et al. showed that HOXC9 is overexpressed in CD133 + glioblastoma cells, suggesting that HOXC9 is involved in the acquisition of stem cell-like phenotype [6] . HOXC9 is highly expressed in breast cancer cells and promotes the switch from a proliferative to an invasive phenotype [7] . However, the role of HOXC9 in GC has not been reported. We analyzed the differential expression profiles of GC tissues and paracancerous tissues using three microarray datasets obtained from the GEO database and found that the HOXC9 gene was significantly upregulated in GC tissues compared with adjacent tissues. The logFC value was 3.92, 3.15, and 4.81, respectively [8] [9] [10] . Members of the microRNA (miR)-26 family, including miR-26a and miR-26b, act as tumor suppressor genes and are downregulated in esophageal squamous cancer and hepatocellular carcinoma [11, 12] . A recent study reported that downregulation of miR-26a in GC is indicative of poor prognosis, whereas restoration of miR-26a suppresses the proliferation and metastasis of GC cells [13] . In the present study, the results of bioinformatics analysis identified a conserved binding site for miR-26 (miR-26a/b) in the 3ʹ-untranslated region (3ʹ-UTR) of HOXC9 mRNA, indicating that miR-26 may have an inhibitory effect on HOXC9. However, the interaction remains to be verified. In the present study, we showed that HOXC9 was overexpressed in GC tissues and promoted the metastasis and stem cell-like phenotype of GC cells. We demonstrated that loss of miR-26a expression led to the upregulation of HOXC9. The present study is the first to demonstrate the role of the miR-26a /HOXC9 axis in the malignant behaviors of GC cells. Materials and Methods Gene expression datasets and online survival analysis Three human GC microarray datasets, GSE103236 (10 patients), GSE79973 (10 patients), and GSE65801 (32 patients) were obtained from the Gene Expression Omnibus (GEO) database (http:// www.ncbi.nlm.nih.gov/geo) [14] . Paired GC and adjacent tissues were collected from 52 patients who had undergone radical gastrectomy for GC and did not receive radiotherapy and chemotherapy before surgery. The expression of HOXC9 in GC and matched adjacent tissues was analyzed by GEO2R. The prognostic effect of HOXC9 mRNA in GC was assessed using an online database, Kaplan-Meier Plotter (http://kmplot.com/), which contains gene expression data and survival information of 631 GC patients. Patient samples were divided into a high expression group and a low expression group by auto select best cutoff, and the overall survival (OS) and progression-free survival (PFS) were assessed by a Kaplan-Meier survival plot and the log rank test [15] . Cells and GC specimens. The human gastric cancer BGC823, MKN45, MKN28, and SGC7901 cell lines were obtained from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China). HEK293 cell line was obtained from the central laboratory of Chongqing Medical University. Cells were cultured in DMEM (Hyclone, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, PAN biotech, Germany) under a humidified
doi:10.1159/000493502 pmid:30205370 fatcat:l5ynkibgt5ctvlorca42jwbllu