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The association of microRNAs (miRs) with cancer progression has been established in many cancers including esophageal squamous cell carcinoma (ESCC). A public microarray database showed that the expression of miR-150 was lower in ESCC than in normal esophageal mucosa. Here, we focused on ZEB1, epithelial-mesenchymal-transition (EMT)-inducer, as a target gene of miR-150 based on in silico predictions. The purpose of this study was to clarify the clinicopathological significance of miR-150 in ESCC, and to investigate miR-150′s EMT-regulatory ability. Quantitative RT-PCR was used to evaluate miR-150 expression in 108 curative resected ESCC samples to determine the clinicopathological significance. Moreover, we examined the in vitro and in vivo function of miR-150 via degradation of ZEB1. MiR-150 expression was significantly lower in cancer tissues compared to adjacent non-cancerous tissues (P < 0.001). Low expression of miR-150 in ESCC contributed to malignant potential, such as tumor depth, lymph node metastasis, lymphatic invasion, venous invasion, clinical staging, and poor prognosis (P < 0.05). In vitro assays showed that EMT-inducer-ZEB1 is a new direct target of miR-150. Moreover, miR-150 induced MET-like changes in TE-8 cells through ZEB1 degradation (e.g., E-cadherin expression, vimentin repression, epithelial morphology, and suppression of migration ability), and significantly inhibited tumorigenicity and tumor growth in a mouse xenograft model. Analysis of the regulation of ZEB1 by miR-150 could provide new insights into preventing metastasis and also suggests novel targeted therapeutic strategies in ESCC. (Cancer Sci 2013; 104: 48–54)
Progress in perioperative management and definitive or adjuvant therapy has led to improved survival of esophageal squamous cell carcinoma (ESCC) patients. However, for patients with advanced disease, prognosis remains poor.[1-3] Local ESCCs directly invade other organs, presenting serious obstacles to radical resection, a characteristic which enhances local recurrence. Moreover, ESCCs cause early lymphatic and hematogenous disseminations more frequently compared to other solid gastrointestinal cancers.[4, 5] Therefore, clinical indicators that accurately predict ESCC progression and prognosis are essential for improving patient survival.
Recently, microRNAs (miRs) have attracted attention for their involvement in the regulation of gene expression. miRs are small non-coding RNAs, approximately 18–25 nucleotides in length, which partially bind to the 3′-untranslated region (3′-UTR) of target mRNAs, leading to mRNA degradation and/or translational repression. Many miRs play an essential role in cellular processes, such as proliferation, differentiation, apoptosis, and cancer progression, depending on their specific gene targets. To find cancer-associated miRs in ESCC, we re-analyzed GSE6188 in the Gene Expression Omnibus public microarray database. In this way, we detected five downregulated miRs in ESCC compared to normal esophageal mucosa (Fig. S1). First, we examined the clinical significance of miR-133 in ESCC samples and validated the high expression of miR-133 in ESCC relative to normal mucosa. However, we could not show the prognostic value or a correlation with clinicopathological factors in miR-133 analysis. MiR-375 had previously been reported to function as a tumor suppressing miR via IGF1R in ESCC. Therefore, we focused on miR-150, known to be downregulated in malignant lymphoma, chronic myeloid leukemia, mantle cell lymphoma, and pituitary tumor, and upregulated in osteosarcoma. On the other hand, miR-150 represses MYB which is associated with cancer progression in many malignancies.[14, 15] Therefore, we suggested that miR-150 may function as a tumor suppressing miR in ESCC.
The epithelial-mesenchymal transition (EMT) and the mesenchymal-epithelial transition (MET) have attracted attention as regulatory mechanisms of invasion and metastasis in many cancers including ESCC. EMT-induced cancer cells are more efficient at forming cancer stem cells with invasive and tumorigenic phenotypes. Therefore, EMT-regulatory miRs in cancers have been considered as new diagnostic and therapeutic tool for human malignancies.[18, 19] In this study, we focused on EMT-inducers ZEB1 as target genes of miR-150 based on in silico miR target prediction tools. ZEB1 were previously reported to be associated with EMT induction.
The purpose of this study was to clarify the clinicopathological significance of miR-150 in ESCC, and to investigate miR-150-mediated regulation of EMT. Therefore, we examined the expression levels of miR-150 in clinical ESCC samples, and demonstrated the direct binding of miR-150 to ZEB1′s 3′-UTR using a luciferase reporter assay. Furthermore, miR-150 functional analysis was performed in TE-8 cells which express ZEB1, and repress E-cadherin, similar to EMT-induced cancer cells.
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In this study, we found that the expression level of miR-150 in T was lower than in N, consistent with data from the previous microarray expression analysis. We also showed that miR-150 may function as an inducer of MET-like changes in vitro and as an inhibitor of tumorigenicity in vivo by targeting the EMT-inducers ZEB1.
EMT has important roles in cancer invasion, metastasis and cancer stem cell properties.[16, 17] EMT-inducer ZEB1 was identified as a new miR-150 target in this study. ZEB1 were previously reported to be associated with cancer progression, and are likely necessary for EMT. Note that TGF-beta-induced EMT is inhibited in squamous cells by suppression of ZEB1 via blocking of cellular senescence programs. On the other hand, it was reported that cancer stem cell properties (including tumorigenicity in colon and pancreatic cancers) are promoted by suppression of the stemness-inhibiting miR-200 family by a ZEB/miR-200 feedback loop. In this study, we demonstrated that ZEB1 -targeting by miR-150 could suppress E-cadherin expression, migration ability, and tumorigenicity in ESCC cells. In ESCC, ZEB1 may control not only EMT via TGF-beta signals but also cancer stem cells via the miR-200 family.
In this study, miR-150 was able to induce MET-like changes and to suppress tumorigenicity in TE-8 cells. While the EMT has been shown to promote cancer migration and intravasation from primary cancer in metastatic cascade in many cancers, the meaning of the MET in metastasis is still controversial. Recently, induction of the MET at the metastatic site was reported to facilitate metastatic colonization in mouse models. The MET induced by miR-200 was shown to promote the development of metastasis by controlling E-cadherin and Sec23a expression, thereby mediating the secretion of metastasis-suppressive proteins.[25, 26] MiR-150 controls the EMT-MET by controlling ZEB1/miR-200 loop; however, miR-150 itself has many targets that are known to be involved in cancer progression, including Myb, Notch, and CXCR4, and therefore is unlikely to target Sec23a, as suggested by in silico analyses.[14, 27, 28] Indeed, premiR-150 transfection in ESCC cell lines suppressed tumorigenic progression in vivo (Fig. 4), and use of an miR-150 inhibitor suppressed E-cadherin expression and promoted proliferation and migration in a separate ESCC cell line, TE-15 (Fig. S2). Therefore, these data suggest that induction of miR-150 in ESCC cells acts via regulation of miR-200 as well as other targets. Future studies are needed in order to clarify the connection between miR-150 and the metastatic cascade.
Previous studies have demonstrated that siRNA administered systemically to humans could inhibit specific genes via an RNA interference mechanism. Small RNAs, including miRs, have attracted attention as potential new tools for cancer therapeutic strategies.[30, 31] Some groups have reported the potential of targeting specific miRs in cancer therapy.[32, 33] Currently, the regulation of ZEB1 by miR-150 in the human circulatory system is not well understood. In the future, miR150 administration to patients with ESCC may provide a promising new therapeutic strategy for suppressing cancer metastasis and reducing cancer recurrence via ZEB1-mediated MET induction in ESCC cells.
In conclusion, our data indicate that downregulated-miR-150 is associated with poor prognosis and cancer progression in ESCC. EMT-inducer ZEB1 is regulated by miR-150, which may function as a regulator of EMT and MET in ESCC. The regulation by miR-150 could provide new insights into preventing metastasis and also provide a promising novel candidate for targeted therapeutic strategies in ESCC.