MicroRNAs (miRNAs) play critical roles in the growth, metastasis and therapeutic resistance of liver cancer. Accumulating evidence suggests that miR‑498 is aberrantly expressed in several human malignancies. However, the role and underlying mechanism of miR‑498 in liver cancer remain unclear. In the present study, we investigated the potential roles and clinical value of miR‑498 in liver cancer. We found that the miR‑498 expression level was significantly lower in liver cancer patient tissues than that in healthy control tissues. The expression of miR‑498 was also decreased in liver cancer cell lines compared to that noted in a normal human normal liver cell line. miR‑498 overexpression markedly inhibited liver cancer cell proliferation, migration and invasion. miR‑498 overexpression induced cell cycle arrest and apoptosis while it suppressed epithelial‑mesenchymal transition (EMT) in liver cancer cells. Bioinformatic analysis and luciferase reporter assay further identified zinc finger E‑box binding homeobox 2 (ZEB2) as a novel target of miR‑498. Furthermore, ZEB2 knockdown recapitulated the inhibitory effects of miR‑498 overexpression in liver cancer cells. ZEB2 overexpression rescued the inhibition of liver cancer cell proliferation, migration, and invasion by miR‑498, indicating that ZEB2 acts as a downstream effector of miR‑498 in liver cancer cells. Thus, we demonstrated that miR‑498 suppresses the growth and metastasis of liver cancer cells, partly at least, by directly targeting ZEB2, suggesting that miR‑498 may serve as a potential biomarker for the diagnosis and therapy of liver cancer.

BACKGROUND

Endometrial cancer (EC) is the most common gynecologic malignancy, but the molecular events involved in the development and progression of EC remain unclear. This study aimed to explore epigenetic modification of genes and miRNAs involved in EC development.

METHODS

Ishikawa and AN3CA cells were treated with 5'-Aza-2-deoxycytidine or histone deacetylase inhibitor. The expression of miRNAs and related genes were detected by PCR and Western blot. Promoter methylation was detected by bisulfite specific PCR sequencing. The proliferation, colony formation, cell cycle progression, migration and invasion of EC cells were evaluated by MTT, soft agar assay, flow cytometry, wound healing and invasion assay, respectively.

RESULTS

Aberrant expression of miRNAs including miR-200b, miR-130a/b, miR-625 and miR-222 was associated with tumorigenesis and metastasis in endometrial cancer. Silencing of miR-130b induced E-cadherin expression, while ectopic expression of miR-130b and knockdown of DICER1 increased the expression of Vimentin, zeb2, N-cadherin, Twist and Snail in EC cells. Furthermore, 5'-Aza-2-deoxycytidine and Histone deacetylase (HDAC) inhibitor inhibited the proliferation, colony formation, migration and invasion of EC cells, accompanied by reduced MMP secretion.

CONCLUSIONS

Our study provides the first description of epigenetic modification of epithelial mesenchymal transition associated genes and miRNAs in EC cells, which are extensively involved in the regulation of gene expression and subsequent accumulation of malignant features of EC cells.

MicroRNA (miR)-145-5p has been reported to function as a suppressor of cancer and plays an important role in cancer invasiveness. Epithelial-mesenchymal transition (EMT) is an important process in cancer invasion and migration. However, the involvement of miR-145-5p in EMT in human gastric cancer (GC) remains unclear. In this study, we aimed to investigate the molecular mechanisms by which miR-145-5p regulates EMT in GC invasiveness. We used quantitative real-time polymerase chain reaction to investigate the miR-145-5p expression level in GC and matched normal tissues. The effects of miR-145-5p on GC cell invasion and migration abilities were evaluated using Transwell models. The relationships among miR-145-5p and zinc-finger E-box binding homeobox 2 (ZEB2), E-cadherin, and N-cadherin were analyzed by quantitative real-time polymerase chain reaction and Western blot analyses. miR-145-5p levels in primary GC tissues obtained from 60 patients were significantly downregulated, compared to those in paired normal tissues. Lauren classification, depth of tumor invasion, lymph node metastasis, lymphatic invasion, and tumor-node-metastasis stage were associated with miR-145-5p expression. miR-145-5p inhibits the expression of the candidate target gene ZEB2 to delay the invasion and migration of GC cells. ZEB2 acts as transcriptional repressor of E-cadherin, while miR-145-5p is known to suppress N-cadherin directly to regulate EMT. Therefore, we concluded that miR-145-5p may target N-cadherin and ZEB2 directly to influence EMT.

OBJECTIVE

Down-regulation of miR-138 is observed in a variety of cancers, which suggests that miR-138 may be involved in cancer pathogenesis. Our current work aimed to evaluate the effects of miR-138 in adriamycin (ADM)-resistant human NSCLC cells.

METHODS

Cell proliferation was determined by MTT assay. Real-time PCR and western blot were performed to detect the mRNA and protein expression levels. The target of miR-138 was validated by luciferase activity assay.

RESULTS

Compared with the chemosensitive parental cells, miR-138 was remarkably decreased in A549/ADM and NCI-H23/ADM cells. Ectopic expression of miR-138 sensitized chemoresistant tumor cells to ADM administration. In addition, the epithelial-mesenchymal transition (EMT) related markers E-cadherin or vimentin was up-regulated or down-regulated upon the overexpression of miR-138 in NSCLC cells. Further studies identified zinc finger E-box-binding homeobox 2 (ZEB2) as the target of miR-138 and up-regulation of miR-138 suppressed the mRNA and protein expression of ZEB2. Notably, luciferase reporter assay confirmed that ZEB2 was a direct target of miR-138.

CONCLUSIONS

Our study demonstrates that miR-138 sensitizes NSCLC cells to ADM via EMT, suggesting that miR-138 might be a potential therapeutic target for drug-resistant NSCLC patients.

Long noncoding RNA activated by transforming growth factor-β (lncRNA-ATB) is a novel lncRNA, which is recently reported to have critical roles in carcinogenesis and progression of several cancers. However, the expression, clinical values, biological roles, and underlying molecular mechanisms of lncRNA-ATB in osteosarcoma are still known. In this study, we measured lncRNA-ATB expression in serum and osteosarcoma tissues of osteosarcoma patients, analyzed its diagnostic and prognostic values. Serum lncRNA-ATB is increased in osteosarcoma patients and could accurately discriminate osteosarcoma patients from healthy controls. LncRNA-ATB is also upregulated in osteosarcoma tissues and cell lines, and positively associated with Enneking stage, metastasis and recurrence. Increased lncRNA-ATB level indicates poor recurrence-free survival and overall survival. Functional experiments demonstrated that overexpression of lncRNA-ATB enhances osteosarcoma cells proliferation, migration, and invasion, and while depletion of lncRNA-ATB inhibits osteosarcoma cells proliferation, migration, and invasion. Mechanistically, we found that lncRNA-ATB inhibits miR-200s, and upregulates miR-200s target genes ZEB1 and ZEB2. Additionally, the roles of lncRNA-ATB on osteosarcoma cells proliferation, migration, and invasion in vitro, and osteosarcoma tumor growth in vivo are dependent on the regulation of miR-200s. Taken together, this study suggests that lncRNA-ATB may be a potential diagnostic and prognostic biomarker and a therapeutic target for osteosarcoma.

Epithelial-to-mesenchymal transition (EMT) is associated with poor prognosis and metastasis in hepatocellular carcinoma. We have previously demonstrated an in vivo model of liver cancer in which mesenchymal cells post-EMT demonstrate a high rate of invasive growth and metastasis. Here, we investigate the role of microRNA 200 (miR-200) family members and epigenetic modifications on the maintenance of mesenchymal/metastatic phenotype after EMT. Mesenchymal cells post-EMT demonstrates high levels of E-box repressors Zeb1 and Zeb2 and downregulation of four miR-200 family members (miR-200a, miR-200b, miR-200c and miR-429). In addition, DNA sequencing after bisulfite modification demonstrates that several CpG sites within the E-cadherin promoter are methylated in mesenchymal cells. In mesenchymal cells, forced expression of miR-200b results in a significant increase in E-cadherin and a reduction in cell migration/invasion. Despite these mesenchymal-to-epithelial transition (MET) changes in vitro, there is no significant change in metastatic potential after miR-200b upregulation in vivo. After the mesenchymal cells were treated with combination of DNA methyltransferase (DNMT) inhibitor and upregulation of miR-200b, invasive phenotype was significantly reduced and metastatic potential was eliminated. Direct targeting of E-cadherin with short hairpin RNA does not restore metastatic potential after DNMT inhibition and miR-200b re-expression. In addition, restoration of E-cadherin alone was unable to block metastatic potential in primary mesenchymal cells. In conclusion, targeting mesenchymal liver cancer cells with miR-200b and DNMT inhibitor reduces metastatic potential irrespective of E-cadherin expression. Thus, the broader differentiation and MET effects of DNMT inhibition and miR-200b must be considered in terms of rescuing metastatic potential.

The transforming growth factor-β1 (TGF-β1)/Smad3 signaling pathway has a central role in pathogenesis of lung fibrosis. In the present study, we investigated if all-trans retinoic acid (ATRA) could attenuate fibrosis in bleomycin (BLM)-induced lung fibrosis in rats through regulating TGF-β1/Smad3 signaling. Beginning on day 14 after BLM administration, the ATRA I and II groups of rats received daily oral administration of ATRA for 14 days. All rats were killed on day 28. Lung tissue sections were prepared and subject to histological assessment, and expression levels of proteins involved in the TGF-β1 signaling cascade and epithelial-mesenchymal transition (EMT) were evaluated by transmission electron microscopy (TEM), quantitative real-time polymerase chain reaction (qRT-PCR), western blot procedure, and immunohistochemical or immunofluorescence staining. BLM significantly increased the alveolar septum infiltrates, inflammatory cell infiltrates, and collagen fibers. These BLM-induced changes were significantly ameliorated by ATRA treatment. In addition, BLM significantly increased levels of lung fibrosis markers α-SMA, hydroxyproline (Hyp), collagen I, Snail, and Twist, whereas significantly decreased E-cadherin expression. ATRA treatment largely reversed BLM-induced changes in these lung fibrosis markers. ATRA also blocked BLM-induced activation of the TGF-β1/Smad3 signaling pathway in lung tissues, including expression of TGF-β1, Smad3, p-Smad3, zinc-finger E-box-binding homeobox 1 and 2 (ZEB1 and ZEB2), and the high-mobility group AT-hook 2 (HMGA2). Our results suggest that ATRA may have potential therapeutic value for lung fibrosis treatment.

There is an urgent need to identify targeting molecules to control invasion and metastasis in cancer patients. We first isolated cancer stem cells (CSCs) from SKOV3 ovarian cancer cells and then investigated the role of melatonin in invasiveness and migration of CSCs compared to SKOV3 cells. The proportion of CSCs in SKOV3 cells was as low as 1.28% with overexpression of both CD133 and CD44. The ability of spheroid formation along with SOX2 overexpression revealed a high self-renewal potential in isolated cells. Melatonin (3.4 mM) inhibited proliferation of CSCs by 23% which was confirmed by a marked decrease in protein expression of Ki67, as a proliferation marker. Applying luzindole, a melatonin receptor 1, 2 inhibitor, partially abolished anti-proliferative effect of melatonin. Melatonin also decreased Epithelial mesenchymal transition (EMT) related gene expressions including ZEB1, ZEB2, snail and vimentin with increase in E-cadherin as a negative EMT regulator. Incubation of CSCs with melatonin showed a marked decrease in matrix metalloproteinase 9 (MMP9) expression and activity. Melatonin also inhibited CSCs migration in a partially receptor dependent and PI3k and MAPK independent manner. Melatonin can be considered as an important adjuvant to control invasion and metastasis especially in patients with high melatonin receptor expression.

Many epithelial-mesenchymal transition (EMT)-promoting transcription factors have been implicated in tumorigenesis and metastasis as well as chemoresistance of cancer. However, the underlying mechanisms mediating these processes are unclear. Here, we report that Foxq1, a forkhead box-containing transcription factor and EMT-inducing gene, promotes stemness traits and chemoresistance in mammary epithelial cells. Using an expression profiling assay, we identified Twist1, Zeb2, and PDGFRα and β as Foxq1 downstream targets. We further show that PDGFRα and β can be directly regulated by Foxq1 or indirectly regulated through the Foxq1/Twist1 axis. Knockdown of both PDGFRα and β results in more significant effects on reversing Foxq1-promoted oncogenesis in vitro and in vivo than knockdown of either PDGFRα or β alone. In addition, PDGFRβ is a more potent mediator of Foxq1-promoted stemness traits than PDGFRα. Finally, pharmacologic inhibition or gene silencing of PDGFRs sensitizes mammary epithelial cells to chemotherapeutic agents in vitro and in vivo. These findings collectively implicate PDGFRs as critical mediators of breast cancer oncogenesis and chemoresistance driven by Foxq1, with potential implications for developing novel therapeutic combinations to treat breast cancer.

The epithelial-mesenchymal transition (EMT) correlates with disruption of cell-cell adhesion, loss of cell polarity and development of epithelial cell malignancy. Identifying novel molecules that inhibit EMT has profound potential for developing mechanism-based therapeutics. We previously demonstrated that the endoplasmic reticulum protein 29 (ERp29) is a novel factor that can drive mesenchymal-epithelial transition (MET) and induce cell growth arrest in MDA-MB-231 cells. Here, we show that ERp29 is an important molecule in establishing epithelial cell integrity during the MET. We demonstrate that ERp29 regulates MET in a cell context-dependent manner. ERp29 overexpression induced a complete MET in mesenchymal MDA-MB-231 cells through downregulating the expression of transcriptional repressors (for example, Slug, Snai1, ZEB2 and Twist) of E-cadherin. In contrast, overexpression of ERp29 induces incomplete MET in basal-like BT549 cells in which the expression of EMT-related markers (for example, vimentin; cytokeratin 19 (CK19) and E-cadherin) and the transcriptional repressors of E-cadherin were not altered. However, ERp29 overexpression in both cell-types resulted in loss of filamentous stress fibers, formation of cortical actin and restoration of an epithelial phenotype. Mechanistic studies revealed that overexpression of ERp29 in both cell-types upregulated the expression of TJ proteins (zonula-occludens-1 (ZO-1) and occludin) and the core apical-basal polarity proteins (Par3 and Scribble) at the membrane to enhance cell-cell contact and cell polarization. Knockdown of ERp29 in the epithelial MCF-7 cells decreased the expression of these proteins, leading to the disruption of cell-cell adhesion. Taken together, ERp29 is a novel molecule that regulates MET and epithelial cell integrity in breast cancer cells.

Accumulating evidence demonstrated that abnormal expression of long non-coding RNAs (lncRNAs) was closely associated with cancer development including retinoblastoma (RB). LncRNA X inactive specific transcript (XIST) has been found to function as an oncogene or a tumor suppressor in several cancers. However, the role and underlying mechanism of XIST in RB have not been clarified. The expression of XIST, microRNA (miR)- 101, zinc finger E-box binding homeobox (ZEB) 1, and ZEB2 was detected in human RB tissues and cell lines. The effects of XIST on the proliferation, migration, invasion, epithelial to mesenchymal transition (EMT), and apoptosis of RB cells were evaluated after downregulation of XIST. Furthermore, the mechanism of XIST was mainly focused on miR-101/ZEB1 or ZEB2 signaling. We found the expression of XIST, ZEB1 and ZEB2 was increased, whereas miR-101 was reduced in RB tissues and cells. Knockdown of XIST significantly suppressed the proliferation, migration, invasion and EMT, but promoted the apoptosis and caspase-3 activity. Moreover, we found that XIST functioned as a competing endogenous RNA (ceRNA) for miR-101 to regulate the de-repression of its endogenous targets ZEB1 and ZEB2. In conclusion, these findings suggest that XIST may facilitate the progression of RB through acting as a ceRNA for miR-101 to mediate the expression of ZEB1 and ZEB2. This may provide novel therapeutic options for RB.

BACKGROUND

Epithelial mesenchymal transition (EMT) is thought to be an essential feature of malignant tumor cells when they spread into the stroma. Despite the extracellular acidity of tumor tissues, the effect of acidic extracellular pH (pH e ) on EMT in carcinoma models, including the Lewis lung carcinoma (LLC) model, remains unclear.

METHODS

High and low metastatic LLC variants were generated by repeated tail vein injection of metastatic cells. DMEM/F12 medium, which has been supplemented with 15 mM HEPES, 4 mM phosphoric acid, and 1 g/L NaHCO3 and adjusted to the desire pH with HCl or NaOH, was used for cell culture. EMT marker gene expression was determined by quantitative reverse transcription-polymerase chain reaction. Migration and invasion activities were analyzed by wound healing assay and the Boyden chamber assay through Matrigel®, respectively.

RESULTS

Low metastatic variant LLCm1 cells showed a cobble-stone like morphology at pH e 7.4. At pH e 6.8, however, their morphology became fibroblastic, similar in shape to high metastatic variant LLCm4 cells. Steady state levels of matrix metalloproteinase-9 (Mmp9) mRNA were induced by acidic pH e , maximizing at pH 6.8, with the levels of Mmp9 mRNA higher in LLCm4 than in LLCm1 cells. Both variants showed decreased levels of E-cadherin and increased levels of vimentin at pH e 6.8. Acidic pH e also induced expression of mRNAs encoding the E-cadherin repressors, Zeb2, Twist1 and Twist2, as well as enhancing cell motility and in vitro invasion through Matrigel®.

CONCLUSIONS

Acidic pH e can induce EMT in some types of carcinoma.

Reversing epithelial-to-mesenchymal transition (EMT) in cancer cells has been widely considered as an approach to combat cancer progression and therapeutic resistance, but a limited number of broadly comprehensive investigations of miRNAs involved in this process have been conducted. In this study, we screened a library of 1120 miRNA for their ability to transcriptionally activate the E-cadherin gene CDH1 in a promoter reporter assay as a measure of EMT reversal. By this approach, we defined miR-520f as a novel EMT-reversing miRNA. miR-520f expression was sufficient to restore endogenous levels of E-cadherin in cancer cell lines exhibiting strong or intermediate mesenchymal phenotypes. In parallel, miR-520f inhibited invasive behavior in multiple cancer cell systems and reduced metastasis in an experimental mouse model of lung metastasis. Mechanistically, miR-520f inhibited tumor cell invasion by directly targeting ADAM9, the TGFβ receptor TGFBR2 and the EMT inducers ZEB1, ZEB2, and the snail transcriptional repressor SNAI2, each crucial factors in mediating EMT. Collectively, our results show that miR-520f exerts anti-invasive and antimetastatic effects in vitro and in vivo, warranting further study in clinical settings. Cancer Res; 77(8); 2008-17. ©2017 AACR.

Tumor stem cell marker Doublecortin-like kinase1 (DCLK1) is upregulated in several solid tumors. The role of DCLK1 in hepatocellular carcinoma (HCC) is unclear. We immunostained tissues from human livers with HCC, cirrhosis controls (CC), and non-cirrhosis controls (NCC) for DCLK1. Western blot and ELISA analyses for DCLK1 were performed with stored plasma samples. We observed increased immunoreactive DCLK1 in epithelia and stroma in HCC and CCs compared with NCCs, and observed a marked increase in plasma DCLK1 from patients with HCC compared with CC and NCC. Analysis of the Cancer Genome Atlas' HCC dataset revealed that DCLK1 is overexpressed in HCC tumors relative to adjacent normal tissues. High DCLK1-expressing cells had more epithelial-mesenchymal transition (EMT). Various tumor suppressor miRNAs were also downregulated in HCC tumors. We evaluated the effects of DCLK1 knockdown on Huh7.5-derived tumor xenograft growth. This was associated with growth arrest and a marked downregulation of cMYC, and EMT transcription factors ZEB1, ZEB2, SNAIL, and SLUG via let-7a and miR-200 miRNA-dependent mechanisms. Furthermore, upregulation of miR-143/145, a corresponding decrease in pluripotency factors OCT4, NANOG, KLF4, and LIN28, and a reduction of let-7a, miR-143/145, and miR-200-specific luciferase activity was observed. These findings suggest that the detection of elevated plasma DCLK1 may provide a cost-effective, less invasive tool for confirmation of clinical signs of cirrhosis, and a potential companion diagnostic marker for patients with cirrhosis and HCC. Our results support evaluating DCLK1 as a biomarker for detection and as a therapeutic target for eradicating HCC.

The differentiation of human pluripotent stem cells (hPSCs) to insulin-expressing beta islet-like cells is a promising in vitro model system for studying the molecular signaling pathways underlying beta cell differentiation, as well as a potential source of cells for the treatment of type 1 diabetes. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate many biological processes, including cellular differentiation. We studied the miRNA and mRNA expression profiles of hPSCs at five stages of in vitro differentiation along the pancreatic beta cell lineage (definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitor and hormone-expressing endocrine cells) in the context of samples of primary human fetal pancreas and purified adult islet cells using microarray analysis. Bioinformatic analysis of the resulting data identified a unique miRNA signature in differentiated beta islet cells, and predicted the effects of key miRNAs on mRNA expression. Many of the predicted miRNA-mRNA interactions involved mRNAs known to play key roles in the epithelial-mesenchymal transition process and pancreatic differentiation. We validated a subset of the predictions using qRT-PCR, luciferase reporter assays and western blotting, including the known interaction between miR-200 and ZEB2 (involved in epithelial-mesenchymal transition) and the novel interaction between miR-200 and SOX17 (a key transcription factor in specification of definitive endoderm). In addition, we found that miR-30d and let-7e, two miRNAs induced during differentiation, regulated the expression of RFX6, a transcription factor that directs pancreatic islet formation. These findings suggest that precise control of target mRNA expression by miRNAs ensures proper lineage specification during pancreatic development.

MicroRNAs (miRs) have been demonstrated to regulate various biological processes in human cancer, including non-small cell lung cancer (NSCLC). However, little evidence has been provided regarding the exact role of miR-200c in mediating the malignant progression of NSCLC, as well as the underlying mechanism. The present study aimed to investigate the putative role of miR‑200c in the progression of NSCLC. The expression levels of miR‑200c were significantly reduced in NSCLC cell lines compared with in normal lung epithelial cells, as determined by reverse transcription‑quantitative polymerase chain reaction. Overexpression of miR‑200c significantly suppressed cell migration and invasion of A549 NSCLC cells. Results of a luciferase reporter assay further identified zinc finger E‑box‑binding homeobox 2 (ZEB2) as a direct target gene of miR‑200c, and the expression of ZEB2 was shown to be suppressed in A549 cells overexpressing miR‑200c. Furthermore, small interfering RNA‑mediated inhibition of ZEB2 suppressed the migration and invasion of A549 cells. In addition, since ZEB2 is an epithelial‑mesenchymal transition (EMT) regulator, the role of miR‑200c in the regulation of EMT in NSCLC cells was further examined. Results of a western blot analysis indicated that overexpression of miR‑200c upregulated E‑cadherin, and downregulated N‑cadherin and vimentin expression in A549 cells, thus suggesting that EMT was suppressed. Based on these results, the present study suggested that miR‑200c was able to inhibit the metastasis of NSCLC cells by targeting ZEB2. Therefore, miR-200c may be considered as a potential candidate for the treatment of NSCLC.

Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8⁺ T cell memory compartment. Following viral infection, CD8⁺ T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8⁺ T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8⁺ T cell stemness and highlight its therapeutic potential.

Hepatocellular carcinoma (HCC) remains one of the most common types of cancer worldwide and prognosis remains poor. Previous studies have suggested that long non‑coding RNAs (lncRNAs) may be key regulators of tumor development and progression in HCC. It has been determined that 61‑72% of transcribed regions contain lncRNAs in the antisense orientation (aslncRNAs). However, the function of aslncRNAs in HCC remains to be elucidated. The present study investigated the function of the aslncRNA zinc finger E‑box binding homeobox 2 antisense RNA 1 (ZEB2‑AS1) in 40 HCC tissues and 5 different human HCC cell lines using reverse transcription‑quantitative polymerase chain reaction. Additionally, the expression levels of ZEB2‑AS1 were downregulated by transfection of small interfering RNAs (siRNAs) to determine whether ZEB2‑AS1 is capable of affecting cell proliferation, invasion and metastasis by regulating ZEB2, vimentin, fibronectin, E‑cadherin and N‑cadherin expression levels. The results of the present study demonstrated that the expression levels of ZEB2‑AS1 were greater in HCC tissues when compared with the adjacent normal tissues. Furthermore, ZEB2‑AS1 expression was significantly associated with the size of the primary tumor, intrahepatic metastasis and tumor-node-metastasis stage. The Kaplan‑Meier survival curves suggested that patients with high ZEB2‑AS1 expression levels experienced the lowest overall and recurrence‑free survival rates compared with those that had low expression levels. In addition, the current study demonstrated that the downregulation of ZEB2‑AS1 was associated with decreased tumor growth and metastasis in HCC by the regulation of the expression levels of epithelial mesenchymal transition-induced markers. In conclusion, lncRNA ZEB2‑AS1 may be used as a valuable biomarker in patients with HCC.

Gastric cancer is the fifth most common malignancy in the world, with Eastern Asia as one of areas with the highest incidence rates. Trastuzumab, a HER2-targeting antibody, combined with chemotherapy has been successfully employed for the gastric cancer patients with HER2 overexpression/amplification. However, trastuzumab resistance is a major problem in clinical practice. Here we observed that the trastuzumab-resistant gastric cancer cell line NCI-N87/TR expressed high levels of epithelial-mesenchymal transition factors and demonstrated increased migration and invasion capability compared with NCI-N87 cells. Downregulated E-cadherin and increased N-cadherin, TGF-β, ZEB1, ZEB2, TWIST1, and Snail were detected in NCI-N87/TR cells. We also found that miR-200c was downregulated in NCI-N87/TR cells compared with parental cells NCI-87 by qRT-PCR. Treatment with TGF-β downregulated the expression of miR-200c and upregulated ZEB2, and significantly decreased the trastuzumab sensitivity of NCI-N87 cells. miR-200c restored trastuzumab sensitivity and inhibited migration and invasion through suppressing ZEB1 and ZEB2. In summary, TGF-β/ZEB2 axis plays an encouraging role in trastuzumab resistance of gastric cancer, while miR-200c overexpression downregulates ZEB1/ZEB2 and resensitizes drugs resistance. Our findings might provide a potential therapeutic strategy for trastuzumab resistance of gastric cancer.

Invasion and migration of glioblastoma multiforme (GBM) is a multistep process and an important phenotype that causes this disease to invade surrounding tissues in the brain. Recent studies have highlighted that miRNAs play a pivotal role in controlling GBM cell plasticity. In this report, we used wound healing and transwell assays to identify a novel role of miR-139-5p in inhibition of GBM cell migration and invasion. Bioinformatics coupled with luciferase and Western blot assays also revealed that miR-139-5p inhibited expression of ZEB1 and ZEB2, which are master regulators of tumor metastasis. MiR-139-5p specifically interacts with the 3'-UTR regions of ZEB1 and ZEB2, attenuating their expression in GBM cells. To corroborate this finding, we rescued ZEB1 and ZEB2 expression and found partial but significant increases in miR-139-5p-suppressed invasion of GBM cells. The biological relevance of our study was validated by analyzing levels of miR-139-5p in GBM tissue. We found that its expression significantly downregulated compared to normal tissue and shorter overall survival rates in patients with lower miR-139-5p expression. These results confirm that miR-139-5p suppresses GBM migration and invasion and highlight its potential as a biomarker and therapeutic target for treating GBM.

MicroRNA-215 (miR-215) has previously been demonstrated to be dysregulated in a number of human malignancies and to be correlated with tumor progression. However, the expression and function of miR-215 in non-small cell lung cancer (NSCLC) has remained to be elucidated. Therefore, the present study aimed to investigate the effects of miR-215 in NSCLC tumorigenesis and development. Reverse transcription-quantitative polymerase chain reaction was used to evaluate miR-215 expression in NSCLC cell lines and primary tumor tissues. The association between miR-215 expression and certain clinicopathological factors was also determined, and the effects of miR-215 on the biological behavior of NSCLC cells were investigated. In addition, the potential regulatory function of miR-215 on zinc finger E-box-binding homeobox 2 (ZEB2) expression was examined. miR-215 expression was significantly downregulated in NSCLC cell lines and clinical specimens. Reduced miR-215 expression was significantly associated with lymph node metastasis and advanced TNM stage. Overexpression of miR-215 inhibited NSCLC cell proliferation, invasion and migration, and promoted cell apoptosis in vitro, and suppressed tumorigenicity in vivo. Furthermore, luciferase reporter assay analysis identified ZEB2 as a direct target of miR-215. These findings indicated that miR-215 may act as a tumor suppressor in NSCLC and may serve as a novel therapeutic agent for miR-based therapy.

Mowat-Wilson syndrome (MWS) is caused by a heterozygous mutation or deletion of the ZEB2 gene. It is characterized by a distinctive facial appearance in association with intellectual disability (ID) and variable other features including agenesis of the corpus callosum, seizures, congenital heart defects, microcephaly, short stature, hypotonia, and Hirschsprung disease. The current study investigated the behavioral phenotype of MWS. Parents and carers of 61 individuals with MWS completed the Developmental Behavior Checklist. Data were compared with those for individuals selected from an epidemiological sample of people with ID from other causes. The behaviors associated with MWS included a high rate of oral behaviors, an increased rate of repetitive behaviors, and an under-reaction to pain. Other aspects of the MWS behavioral phenotype are suggestive of a happy affect and sociable demeanor. Despite this, those with MWS displayed similarly high levels of behavioral problems as those with intellectual disabilities from other causes, with over 30% showing clinically significant levels of behavioral or emotional disturbance. These findings have the potential to expand our knowledge of the role of the ZEB2 gene during neurodevelopment. Furthermore, they are a foundation for informing interventions and management options to enhance the independence and quality of life for persons with MWS.

Renal cell carcinoma (RCC) is associated with a high frequency of metastasis and only few therapies substantially prolong survival. Honokiol, isolated from Magnolia spp. bark, has been shown to exhibit pleiotropic anticancer effects in many cancer types. However, whether honokiol could suppress RCC metastasis has not been fully elucidated. In the present study, we found that honokiol suppressed renal cancer cells' metastasis via dual-blocking epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. In addition, honokiol inhibited tumor growth in vivo. It was found that honokiol could upregulate miR-141, which targeted ZEB2 and modulated ZEB2 expression. Honokiol reversed EMT and suppressed CSC properties partly through the miR-141/ZEB2 axis. Our study suggested that honokiol may be a suitable therapeutic strategy for RCC treatment.

Recently, epithelial to mesenchymal transition (EMT) has been shown to represent a feature of dedifferentiating hepatocytes in vitro. Three-dimensional soft collagen gels can antagonize but not completely abolish this effect. Hormonal additives to culture media are known to maintain differentiated hepatocyte functions. Therefore, we studied whether insulin and dexamethasone antagonize EMT in cultured hepatocytes. Both hormones antagonized but not completely abolished certain morphological features of EMT. Dexamethasone antagonized acquisition of fibroblastoid shape, whereas insulin favored bile canaliculi formation. In a subsequent step, we analyzed expression of a battery of EMT-related genes. Of all markers tested, vimentin and snail-1 correlated best with morphological features of EMT. Interestingly, dexamethasone reduced expression levels of both vimentin and snail-1, whereas the influence of insulin was less pronounced. An important result of this study is that 12 out of 17 analyzed EMT markers were transcriptionally influenced by dexamethasone (vimentin, snail-1, snail-2, HNF4 alpha, Twist-1, ZEB2, fibronectin, occludin, MMP14, claudin-1, cytokeratin-8, and cytokeratin-18), whereas the remaining factors seemed to be less dependent on dexamethasone. In conclusion, EMT markers in hepatocytes can be classified as dexamethasone-dependent versus -independent.