ZEB1 Levels Research Articles

169P - M-phase phosphoprotein 8 promotes gastric cancer growth and metastasis via p53/Bcl-2 and EMT-related signalling pathways

BackgroundThe main issue of this study is to demonstrate whether M-phase phosphoprotein 8 (MPP8) affect gastric tumour growth and metastasis. MethodsRetrospective study was proceeded in 280 patients’ surgical specimens with different disease stages. Lentivirus delivery of short hairpin RNA targeting MPP8 was also used to explore the function of MPP8 in gastric cancer cells. Quantitative real-time PCR and western blot were applied to measure apoptosis and metastasis relative biomarker. ResultsThe present study revealed that MPP8 expression was higher in surgical specimens from patients with gastric cancer (vs. normal adjacent tissues), and that elevated expression was associated with male sex (vs. female sex), intermediate differentiation (vs. poorly differentiated cancer), and later stage (vs. earlier stage). Furthermore, MPP8 overexpression in tumour tissues was marginally associated with a poor prognosis, with a significant relationship between MPP8 overexpression and prognosis among patients with poorly differentiated gastric cancer. Inhibition of MPP8 in these cells significantly suppressed proliferation and colony formation, promoted apoptosis, and repressed invasion. Furthermore, silencing of MPP8 remarkably increased the expression of apoptosis-related proteins (p53, Bax, and PARP), and down-regulated Bcl-2 expression. Silencing of MPP8 also decreased the expression of metastasis pathway-related proteins (N-cadherin and vimentin), and as well as the levels of anti-oncogene ZEB1, MET, and KRAS mRNA. ConclusionsThese results suggest that MPP8 plays a critical role in regulating gastric cancer apoptosis and metastasis via the p53/Bcl-2 and EMT-related signalling pathways, respectively, which may indicate that MPP8 is a promising target for treating gastric cancer. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Effect of miR-145 on Migration and Invasion of Ovarian Cancer Cells

Objective To evaluate the effect of miR-145 on migration and invasion of ovarian cancer cells.Methods The effect of miR-145 overexpression on the expression levels of miR-145 and zeb-2 were detected with qRT-PCR and Western blotting.The changes of in vitro migration and invasion were examined using Transwell assay.Target genes of miR-145 were predicted by bioinformatics software.Dual-luciferase reporter assay were used to verify zeb-2 as a direct target of miR-145.zeb-2 siRNA was transiently transfected in SKOV3 and 3AO cells,Transwell was used to examine in vitro migration and invasion abilities.Results The migration and proliferation of SKOV3(t=10.752,P=0.000;t=5.617,P=0.005)and 3AO cells(t=10.111,P=0.001;t=21.746,P=0.000)decreased significantly after overexpression of miR-145.The results of dual-luciferase reporter assay showed that the relative luciferase activity of co-transfected miR-145 mimic and WT 3'UTR expression vectors was significantly lower than that of co-transfected mimic control and WT 3'UTR expression vectors(SKOV3:t=4.572,P=0.010;3AO:t=3.528,P=0.024).There was no significant difference in relative luciferase activity between co-transfected miR-145 mimic/MUT 3'UTR expression vector cells and co-transfected mimic control/MUT 3'UTR expression vector cells(SKOV3:t=0.227,P=0.831;3AO:t=0.040,P=0.970).Real-time quantitative PCR showed that the zeb-2 expressions in SKOV3(t=1.490,P=0.211)and 3AO cells(t=0.114,P=0.914)were not significantly different from negative control after 48 h of miR-145 overexpression.Western blot analysis showed that the expression of zeb-2 protein in SKOV3(t=3.769,P=0.020)and 3AO cells(t=4.452,P=0.011)decreased significantly compared with negative control after 72 h of miR-145 overexpression.Seventy-two hours after transfection of zeb-2 siRNA,Western blotting showed that the expression of zeb-2 protein in SKOV3(t=4.660,P=0.010)and 3AO cells(t=4.594,P=0.010)was significantly down-regulated.Transwell assay showed that the migration and invasion abilities of SKOV3(t=18.655,P=0.000;t=18.026,P=0.000)and 3AO cells(t=5.500,P=0.005;t=8.780,P=0.001)were significantly decreased.Conclusion miR-145 may inhibit the migration and invasion of ovarian cancer cells by targeting zeb-2.

LncRNA ZEB1-AS1 down-regulation suppresses the proliferation and invasion by inhibiting ZEB1 expression in oesophageal squamous cell carcinoma.

Multiple studies have unveiled that long non‐coding RNAs (lncRNAs) play a pivotal role in tumour progression and metastasis. However, the biological role of lncRNA ZEB1‐AS1 in oesophageal squamous cell carcinoma (ESCC) remains under investigation, and thus, the current study was to investigate the functions of ZEB1‐AS1 in proliferation and invasion of ESCC. Here, we discovered that ZEB1‐AS1 and ZEB1 were markedly up‐regulated in ESCC tissues and cells relative to their corresponding normal control. ZEB1‐AS1 and ZEB1 overexpressions were both related to TNM staging and lymph node metastasis as well as poor prognosis in ESCC. The hypomethylation of ZEB1‐AS1 promoter triggered ZEB1‐AS1 overexpression in ESCC tissues and cells. In addition, ZEB1‐AS1 knockdown mediated by siRNA markedly suppressed the proliferation and invasion in vitro in EC9706 and TE1 cells, which was similar with ZEB1 siRNA treatment, coupled with EMT alterations including the up‐regulation of E‐cadherin level as well as the down‐regulation of N‐cadherin and vimentin levels. Notably, ZEB1‐AS1 depletion dramatically down‐regulated ZEB1 expression in EC9706 and TE1 cells, and ZEB1 overexpression obviously reversed the inhibitory effects of proliferation and invasion triggered by ZEB1‐AS1 siRNA. ZEB1‐AS1 shRNA evidently inhibited tumour growth and weight, whereas ZEB1 elevation partly recovered the tumour growth in ESCC EC9706 and TE1 xenografted nude mice. In conclusion, ZEB1‐AS1 overexpression is tightly involved in the development and progression of ESCC, and it exerts the antitumour efficacy by regulating ZEB1 level in ESCC.

Circular RNA circ-TSPAN4 promotes lung adenocarcinoma metastasis by upregulating ZEB1 via sponging miR-665.

BackgroundCancer metastasis is responsible for 90% of cancer‐related deaths. Recently, circular RNA (circRNA) is deemed to be an important regulator of cancer progression. However, little is known about the role of circRNA in the metastasis of lung adenocarcinoma (LUAD). Herein, we investigated the clinical implication and regulatory effect of circ‐TSPAN4 (hsa_circ_0020732) in LUAD.MethodsGene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE104854) was used to identify the aberrantly expressed circRNAs in LUAD. The expression levels of circ‐TSPAN4, miR‐4731‐5p, miR‐665, and ZEB1 were determined by quantitative reverse transcription PCR (qRT‐PCR). The functional experiments were carried out with wound healing and transwell assays. And, the luciferase reporter and RNA pull‐down assays were employed to examine the crosstalk between circ‐TSPAN4, miR‐665, and ZEB1. In vivo metastasis experiment was tested by the lung metastasis model.ResultsCirc‐TSPAN4 was significantly upregulated in LUAD tissues and cell lines. The increased circ‐TSPAN4 was linked to advanced tumor‐node‐metastasis stage, lymph node and distant metastasis, and poor outcome. Lentivirus‐mediated stably circ‐TSPAN4 knockdown dramatically attenuated the metastatic ability of LUAD cells both in vitro and in vivo. Mechanistically, circ‐TSPAN4 directly interacted with miR‐665, but not miR‐4731‐5p, to increase the expression of ZEB1, which is a well‐known metastasis trigger. Importantly, the reduced metastatic capacity caused by circ‐TSPAN4 depletion was partially rescued by miR‐665 silencing or ZEB1 overexpression.ConclusionsCirc‐TSPAN4 plays a pivotal metastasis‐promoting role in LUAD through acting as a sponge for miR‐665 and upregulating ZEB1.

Interleukin-8/β-catenin mediates epithelial-mesenchymal transition in ameloblastoma.

Epithelial-mesenchymal transition (EMT) is important in the tooth development and tumor invasion. We investigated the effect of interleukin-8 (IL-8) on the EMT process in primary-cultured ameloblastoma tumor cells (AM-P) and ameloblastoma immortalized tumor cells (AM-L) and its underlying mechanism. IL-8 levels in ameloblastomas were detected by immunofluorescence staining and ELISA. AM-P cells and AM-L cells were stimulated with IL-8, and EMT transcription factors, total β-catenin and phosphorylated-β-catenin (p-β-catenin) levels were determined by Western blot analysis and immunofluorescence staining. β-catenin siRNA was used to knockdown β-catenin expression in AM-P cells and AM-L cells stimulated with IL-8. IL-8 was highly expressed in the solid ameloblastomas. IL-8 promoted the EMT process in ameloblastoma tumor cells in vitro, as evidenced by decreased E-cadherin and increased vimentin, twist and zeb1 levels. IL-8 also increased total β-catenin and p-β-catenin expression in ameloblastoma tumor cells, and β-catenin knockdown partially inhibited the EMT process in tumor cells, as evidenced by increased E-cadherin, and decreased vimentin and zeb1 levels. IL-8 could promote EMT in ameloblastoma tumor cells by activating β-catenin and its downstream transcription factor zeb1.

Two Novel Tri-Aryl Derivatives Attenuate the Invasion-Promoting Effects of Stromal Mesenchymal Stem Cells on Breast Cancer.

The concept of Epithelial-Mesenchymal Transition (EMT) to promote carcinoma progression has been recognized as a venue for research on novel anticancer drugs. Triaryl template-based structures are one of the pivotal structural features found in a number of compounds with a wide variety of biological properties including anti-breast cancer. Among the various factors triggering EMT program, cyclooxygenase-2 (COX-2), NF-κB as well as the transforming growth factor-beta (TGF-β) have been widely investigated. Here, we aim to investigate the effect of two novel compounds A and B possessing triaryl structures, which interact with both COX-2 and TGF-β active sites and suppress NF-κB activation, on EMT in a co-culture system with breast cancer and stromal cells. MDA-MB-231 and bone-marrow mesenchymal stem (BM-MS) cells were co-cultured in a trans-well plate. Migration, matrigel-based invasion and colony formation in soft agar assays along with Real- time PCR and Western blot analysis were performed to examine the effect of compounds A and B on the invasive properties of MDA-MB-231 cells after 72 hours of co-culturing with BM-MSCs. In addition, TGF-beta interaction was investigated by Localized Surface Plasmon Resonance (LSPR). BM-MSCs enhanced migration, invasion and anchorage-independent growth of the co-cultured MDAMB- 231 cells. A reduction in E-cadherin level concomitant with an increase in vimentin and N-cadherin levels following the co-culture implied EMT as the underlying process. Compounds A and B inhibited invasion and anchorage-independent growth of breast cancer cells co-cultured with BM-MSCs at 10µM. The observed inhibitory effects along with an increase in E-cadherin and a reduction in vimentin and ZEB2 levels suggest that the anti-invasive properties of compounds A and B might proceed through the blockade of stromal cell-induced EMT, mediated by their interaction with TGF-beta. These findings introduce compounds A and B as novel promising agents, which prevent EMT in invasive breast cancer cells.

Silencing LINC00511 inhibits cell proliferation, migration, and epithelial-mesenchymal transition via the PTEN-AKT-FOXO1 signaling pathway in lung cancer.

Lung cancer is the most common cause of cancer-related death worldwide. Long noncoding RNAs (lncRNAs) are longer than 200 nt transcripts and are not translated into proteins. Increasing evidence has shown that lncRNAs are associated with several biological processes in cancer. However, the roles of LINC00511 in lung cancer progression remain unknown. In the present study, we confirmed that LINC00511 knockdown significantly inhibited cell proliferation and migration in A549, SPCA1, and H460 cells. Western blot results showed that silencing LINC00511 inhibited epithelial-mesenchymal transition (EMT), which resulted in decreased expression levels of ZEB2, N-cadherin, and vimentin and increased expression levels of E-cadherin. Additionally, silencing LINC00511 significantly upregulated PTEN mRNA and protein expression, increased FOXO1, and inactivated AKT. Furthermore, we found that PTEN knockdown reversed the inhibition of cell migration and proliferation induced by LINC00511 siRNA, markedly reduced p-FOXO1 expression, and promoted p-AKT expression and EMT in A549 and H460 cells. Therefore, these findings revealed that LINC00511 functions as an oncogene through the PTEN-AKT-FOXO1 signaling pathway in lung cancer, providing a potential target of metastasis in lung cancer.

Luteolin exhibits anti-breast cancer property through up-regulating miR-203

Luteolin is a representative of natural flavonoid that has anti-tumour properties. This study designed to check its impact on breast cancer and the underlying mechanisms. MDA-MB-453 and MCF-7 cells were administrated with luteolin and the following techniques were carried out: CCK-8 assay, FITC-PI double-staining and Western blot. qRT-PCR analysis was utilized to see the effects of luteolin on miR-203 expression. Besides, miR-203 expression was silenced by transfection with specific inhibitor. Luteolin remarkably declined MDA-MB-453 and MCF-7 cells viability and accelerated apoptosis which accompanied by Bax up-regulation, Bcl-2 down-regulation and Caspase-3 cleavage. Also, luteolin impeded TGFβ1-induced EMT, as evidenced by the decreased levels of Vimentin, Zeb1 and N-cadherin, as well as the increased level of E-cadherin. miR-203 was highly expressed in 22 pair of breast cancer tissues than the matched paracancerous tissues. Luteolin could elevate miR-203 level. Besides, luteolin’s anti-tumour effects were partially eliminated by miR-203 silence. Further, luteolin inhibited Ras/Raf/MEK/ERK signalling, while the inhibitory effects were flattened by miR-203 silence. Luteolin significantly reduced breast cancer cells growth and EMT. Luteolin exerted its anti-tumour effects possibly involved the elevated expression of miR-203 and the inhibited Ras/Raf/MEK/ERK signalling.

Loss of miR-873 contributes to gemcitabine resistance in triple-negative breast cancer via targeting ZEB1.

Gemcitabine-based chemotherapy is commonly applied for the treatment of breast cancer in a clinical setting. However, acquired resistance to chemotherapy primarily results in treatment failure and eventually culminates in patient mortality. Aberrant expression of microRNAs (miRNAs) has been demonstrated to be implicated in the development of chemoresistance; however, the role of miR-873 in the chemoresistance of breast cancer and its underlying mechanism have not been completely elucidated. Herein, using cell viability assays, the present study demonstrated that overexpression of miR-873 sensitized triple-negative breast cancer (TNBC) cells (MDA-MB-231 and BT549) towards gemcitabine treatment, while inhibition of miR-873 promoted resistance of TNBC cells to gemcitabine exposure. The 3' untranslated region of zinc finger E-box binding homeobox 1 (ZEB1) was predicted as a candidate target of miR-873, and the regulatory association between ZEB1 and miR-873 was validated with a dual luciferase assay. Reverse transcription-quantitative polymerase chain reaction and western blot analysis confirmed that miR-873 mimics reduced ZEB1 at mRNA and protein levels in MDA-MB-231 and BT549 cells. As ZEB1 was previously reported to interact with Yes associated protein (YAP) to promote cancer progression. The present study observed that miR-873 overexpression decreased the expression of YAP target genes AXL receptor tyrosine kinase, connective tissue growth factor and cysteine rich angiogenic inducer 61 at mRNA and protein levels. Additionally, elevation of the ZEB1 level and reduction of the miR-873 level were detected in gemcitabine-resistant MDA-MB-231 (MDA-MB-231GEMr) cells, which were accompanied with stronger proliferative ability, compared with parental cells. Overexpression of miR-873 or ZEB1 knockdown reversed chemoresistance of MDA-MB-231GEMr cells by inducing a notable cell growth arrest upon gemcitabine exposure. In conclusion, the data obtained by the present study demonstrated that the decrease of miR-873 promoted the development of gemcitabine resistance in TNBC via elevation of ZEB1 expression, which indicated that miR-873 may be a promising predictor for gemcitabine sensitivity in patients with TNBC.

A ZEB1/p53 signaling axis in stromal fibroblasts promotes mammary epithelial tumours

Accumulating evidence indicates that the zinc-finger transcription factor ZEB1 is predominantly expressed in the stroma of several tumours. However, the role of stromal ZEB1 in tumour progression remains unexplored. In this study, while interrogating human databases, we uncover a remarkable decrease in relapse-free survival of breast cancer patients expressing high ZEB1 levels in the stroma. Using a mouse model of breast cancer, we show that ZEB1 inactivation in stromal fibroblasts suppresses tumour initiation, progression and metastasis. We associate this with reduced extracellular matrix remodeling, immune cell infiltration and decreased angiogenesis. ZEB1 deletion in stromal fibroblasts increases acetylation, expression and recruitment of p53 to FGF2/7, VEGF and IL6 promoters, thereby reducing their production and secretion into the surrounding stroma. Importantly, p53 ablation in ZEB1 stroma-deleted mammary tumours sufficiently recovers the impaired cancer growth and progression. Our findings identify the ZEB1/p53 axis as a stroma-specific signaling pathway that promotes mammary epithelial tumours.

The role of Zeb1 in the pathogenesis of morbidly adherent placenta.

Zinc finger E-box-binding homeobox 1 (Zeb1) is a promoter of epithelial-mesenchymal transformation, which may serve an important role in morbidly adherent placenta (MAP). In the present study, the protein expression levels of Zeb1 were examined in the placenta tissues of 60 patients, including 20 patients with placenta accreta (PA) and 20 patients with placenta previa without PA (UPA) and 20 patients in late pregnancy that delivered by cesarean section (normal). The expression levels of Zeb1, N-cadherin, vascular endothelial growth factor (VEGF), Tumor necrosis factor-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2), and tumor necrosis factor-related apoptosis-inducing ligand-receptor 3 (TRAIL-R3) were higher in PA tissues compared with in normal control tissues. The expression levels of E-cadherin and TRAIL-R2 were decreased in PA tissues compared with in normal control tissues. These findings indicated that Zeb1 may serve an important role in placental attachment, thus promoting the development of dangerous PA. Overexpression of Zeb1 may upregulate the expression levels of N-cadherin, VEGF, TRAIL-R3, cyclin D1 and Bcl-2, and downregulate the expression levels of E-cadherin and TRAIL-R2. In addition, Zeb1 regulated the viability, apoptosis and migration of HTR-8/SV neo cells and human umbilical vein endothelial cells by regulating the Akt pathway. In conclusion, these findings indicated that Zeb1 may promote placental implantation by activating the Akt signaling pathway, thus providing a theoretical basis for investigating the causes of MAP.

Schisandrin B inhibits TGF-β1-induced epithelial-mesenchymal transition in human A549 cells through epigenetic silencing of ZEB1

Purpose/Aim: More and more evidences suggest that airway remodeling of fibrotic lung diseases may be associated with epithelial-mesenchymal transition (EMT) of human A549 cells induced by transforming growth factor (TGF)-β1. Schisandrin B (Sch B) is the highest content of dibenzocyclooctadiene lignans in Schisandra chinensis. In this study, we assessed the inhibitory influences of Sch B on TGF-β1-stimulated EMT in human A549 cells. Materials and Methods: The influences of Sch B on cell viability, invasion and metastasis in TGF-β1-induced human A549 cells were detected by MTT, wound healing and transwell invasion assays. The expression levels of α-SMA, E-cadherin, ZEB1 and Twist1 were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. The enrichment of H3K4me3 and H3K9me3 at the ZEB1 promoter was determined by ChIP analysis. Results: Experimental results showed that Sch B increased the expression of the epithelial phenotype marker E-cadherin and inhibited the expression of the mesenchymal phenotype marker α-SMA during EMT induced by TGF-β1. The enhancement in invasion and migration of TGF-β1-induced A549 cells was inhibited by Sch B. Sch B also repressed the expression of ZEB1 transcription factor in EMT, by increasing the enrichment of H3K9me3 at the ZEB1 promoter to repress its transcription while the expression of the Twist1 transcription factor was unaffected. Conclusions: Our data suggest that Sch B can prevent TGF-β1-stimulated EMT in A549 cells through epigenetic silencing of ZEB1, which may be clinically related to the efficient treatment of EMT-associated fibrotic diseases.

MicroRNA-155 and FOXP3 jointly inhibit the migration and invasion of colorectal cancer cells by regulating ZEB2 expression.

The study aimed to explore whether microRNA-155 and FOXP3 could regulate invasive and migratory capacities of colorectal cancer (CRC) cells by mediating Zinc finger E-box binding homeobox 2 (ZEB2) expression. Dual-luciferase reporter gene assay was performed to detect the binding condition between microRNA-155, FOXP3, and ZEB2. Protein and mRNA levels of ZEB2 in CRC cells were detected after overexpression of microRNA-155 and FOXP3 by Western blot and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), respectively. In vitro experiments were conducted using HCT116 and SW620 cell lines. We first detected expression levels of microRNA-155, FOXP3, and ZEB2 in the normal colorectal epithelial cell line (NCM460) and CRC cell lines (HCT116 and SW620) by qRT-PCR. Protein expressions of ZEB2, E-cadherin, and vimentin in WT, LV-GFP, and LV-FOXP3 groups were detected. Wound healing assay and transwell assay were conducted to determine the regulatory effects of microRNA-155 and FOXP3 on invasive and migratory capacities of CRC cells, respectively. Dual-luciferase reporter gene assay found that FOXP3 binds to the promoter and intron regions of ZEB2, and microRNA-155 binds to the 3'UTR region of wild-type ZEB2. Overexpression of FOXP3 downregulated mRNA and protein levels of ZEB2. ZEB2 was highly expressed, whereas microRNA-155 and FOXP3 were lowly expressed in HCT116 and SW620 cells than NCM460 cells. MicroRNA-155 overexpression upregulated E-cadherin and downregulated vimentin in CRC cells. Overexpression of FOXP3 and microRNA-155 inhibited invasive and migratory capacities of CRC cells. MicroRNA-155 and FOXP3 can jointly regulate ZEB2 expression, thereby inhibiting the migration and invasion of colorectal cancer cells.

Abstract 166: MMP12 promotes EMT-mediated tumor aggressiveness in lung cancer cells

Abstract Epithelial mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis. Matrix metalloproteinases (MMPs) have a major role in remodeling the extracellular matrix (ECM) and are implicated in the invasion and metastasis of various malignancies. MMP12 is involved in many pathological processes including cancer, but has not been fully understood yet. To investigate the role of MMP12 in lung cancer cells, we examined the effect of overexpression or knockdown of MMP12 in A549 cells and measured the protein expression level of EMT markers. MMP12 overexpression induced considerable EMT phenotypic changes compared to the control group, both in vitro and in vivo. The mRNA levels of EMT markers, snail2/slug, snail1, zeb1, TGFβ-1, and fibronectin, increased significantly upon MMP12 overexpression. We observed that changes in the expression level of ECM components such as, collagen, lamin, and proteoglycans led to MMP12 overexpression-induced EMT. These alterations promoted the aggressiveness of the A549 cells, evident from changes in functional properties of the cell, such as proliferation, migration, invasion, and adhesion capacity. On the contrary, MMP12 suppression abrogated malignancy of A549 cells. MMP12 knockdown in A549 cells led to decreased mRNA expression of EMT marker proteins associated with adherence, invasion, and migration. Our results suggest that MMP12 is a key regulator of EMT in non-small cell lung cancer (NSCLC). MMP12 overexpression in A549 cells results in changes in the expression of EMT markers and ECM composition. Interestingly, the dynamic changes in the ECM components by MMP12 overexpression cause EMT reprogramming and induce malignant functional properties. Thus, we suggest that the control of MMP12 expression can be a potential target for new therapeutic strategies of NSCLC. Citation Format: Na-Kyung Han, Yoon-Jin Lee, Hae-June Lee. MMP12 promotes EMT-mediated tumor aggressiveness in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 166.

Expression and Role of MicroRNAs from the miR-200 Family in the Tumor Formation and Metastatic Propensity of Pancreatic Cancer

MicroRNAs from the miR-200 family are commonly associated with the inhibition of the metastatic potential of cancer cells, following inhibition of ZEB transcription factors expression and epithelial-to-mesenchymal transition. However, previous studies performed in pancreatic adenocarcinoma revealed a more complex picture challenging this canonical model. To gain better insights into the role of miR-200 family members in this disease, we analyzed the expression of miR-200a, miR-200b, miR-200c, miR-141, miR-429, and miR-205, and ZEB1, ZEB2, and CDH1 in pancreatic tumors and matching normal adjacent parenchyma and patient-derived xenografts. We found that miR-200a, miR-429, and miR-205 are frequently overexpressed in pancreatic tumors, whereas CDH1 is downregulated, and ZEB1 and ZEB2 levels remain unchanged. Furthermore, we measured a positive correlation between miR-200 family members and CDH1 expression, and a negative correlation between ZEB1 and miR-200c, miR-141, and miR-205 expression, respectively. Interestingly, we identified significant changes in expression of epithelial-to-mesenchymal transition regulators and miR-200 members in patient-derived xenografts. Lastly, functional studies revealed that miR-141 and miR-429 inhibit the tumorigenic potential of pancreatic cancer cells. Taken together, this comprehensive analysis strongly suggests that miRNAs from the miR-200 family, and in particular miR-429, may act as a tumor suppressor gene in pancreatic cancer.

Upregulated TTYH2 expression is critical for the invasion and migration of U2OS human osteosarcoma cell lines

Ion channels have recently emerged as stable biomarkers and anticancer targets particularly when the applications of the currently available therapeutic regimens are limited, as in case of osteosarcoma, a malignant bone tumor. Here, we evaluated the expression of TTYH2, a presumably calcium-activated chloride channel, in a human osteosarcoma cell line U2OS. We used small-interfering RNA (siRNA)-mediated gene silencing to demonstrate the downregulation in the expression of TTYH2 that resulted in the decrease in the invasion and migration, but not proliferation, of U2OS cells. The expression levels of Slug and ZEB1, the transcription factors involved in epithelial-mesenchymal transition, significantly reduced after TTYH2 silencing. Based on these results, we suggest that TTYH2 may serve as a novel target for the treatment of osteosarcoma.

MicroRNA‑30a regulates cell proliferation, migration, invasion and apoptosis in human nasopharyngeal carcinoma via targeted regulation of ZEB2.

MicroRNA-30a (miR-30a) was previously reported to serve as a tumor suppressor able to inhibit the development and progression of certain types of cancer. A number of previous studies demonstrated that zinc finger E-box binding homeobox 2 (ZEB2) may be regulated by miR-30a in clear cell renal cell carcinoma and breast cancer. However, the function of miR-30a in human nasopharyngeal carcinoma (NPC) remains unclear. The present study aimed to investigate the association between miR-30a and ZEB2 in NPC. Therefore, the expression levels of miR-30a and ZEB2 were measured in human NPC cells and tissues from patients with NPC, and the present results suggested that the expression level of miR-30a was significantly decreased in NPC tissues compared with paracancerous tissues. The direct interaction between miR-30a and the untranslated region of ZEB2 was examined using the dual-luciferase reporter assay, and ZEB2 was identified as a direct target of miR-30a. Additionally, the effects of miR-30a and ZEB2 overexpression on cell proliferation, migration, invasion and apoptosis were additionally investigated. Functional experiments identified that overexpression of miR-30a increased apoptosis and suppressed cell proliferation, cell migration and cell invasion by directly targeting ZEB2. Collectively, the present study suggested that miR-30a may serve an important role in the progression of NPC and may represent a novel target for the treatment of patients with NPC.

Gene regulation and prognostic indicators of lung squamous cell carcinoma: TCGA‐derived miRNA/mRNA sequencing and DNA methylation data

Lung squamous cell carcinoma (LSCC) is a common cancer worldwide, and this study aimedto investigate the key regulatory networks and prognostic indicators of LSCC. MicroRNA (miRNA)/messenger RNA (mRNA) sequencing and DNA methylation data were obtained from the Cancer Genome Atlas. Differentially expressed miRNAs (DEmiRNAs) and genes (DEGs) were identified by the limma package. Then, the transcription factors (TFs) of DEmiRNAs/DEGs, as well as the targets of miRNAs, were predicted by the TFmiR online tool. Using the t test, aberrant methylation was detected in TF binding sites (TFBSs) in promoters. Finally, integrated network and survival analyses were conducted using SPSS software. We obtained 104 DEmiRNAs and 4,491 DEGs, and validated 2,113 DEGs (VDEGs). Then, 103 TFs, 295 TFs, and 14 DEmiRNAs were predicted to target 95 DEmiRNAs, 821 DEGs and 283 DEGs, respectively. After TF-DEmiRNA/DEG and TF-DEmiRNA-DEG networks were constructed (e.g., E2F1-CDC25A, miR29a-RAN, miR326-TBL1XR1), five feedforward loops between ZEB1 and miR-141/200a/200b/200c/429 were found. Furthermore, VDEGs CDC25A, RAN, TBL1XR1as well as miR-130b and miR-590 were negatively correlated with survival rates. E2F1-CDC25A, miR29a-RAN, miR326-TBL1XR1, and the feedforward loops between ZEB1/ZEB2 and miR-141/200a/200b/200c/429 might participate in LSCC development. Compared with BEAS-2B cells, the SK-MES-1 cells presented a higher expression level of miR-141, miR-200a, miR-200b, miR-200c but a lower expression level of ZEB1. Overexpressed miR-200c significantly attenuated the expression of ZEB1 and ZEB2and inhibited the proliferation and migration of SK-MES-1 cells (all p < 0.05). In addition, CDC25A, miR-200a, miR-200b, miR-200c, miR-130b, and miR-590 are potential prognostic indicators of LSCC.

SNHG1 contributes to proliferation and invasion by regulating miR-382 in breast cancer.

Introduction: Long non-coding RNAs (lncRNAs) are key regulators in multiple cancers. lncRNA, SNHG1, was shown to be associated with tumorigenesis. However, little is known about the role SNHG1 plays in breast cancer. The aim of the study was to study the role and underlying mechanism of SNHG1 regulation in breast cancer.Methods: Quantitative real-time PCR was used to measure the levels of SNHG1, miR-382 and ZEB1 levels in breast cancer tissue or cells. The proliferation, colony formation, migration and invasion of breast cancer cells, under SNHG1 knockdown achieved by transfection of SNHG1-specific siRNAs, were assessed by Cell Counting Kit-8, colony forming, scratch wound and transwell assays. Bioinformatical analysis and luciferase assay were used to explore the interaction between SNHG1 and its potential miRNA target. Western blot was used to evaluate the expression of epithelial-to-mesenchymal transition (EMT) markers. MDA-MB-231 cells with or without SNHG1 knockdown were used to initiate tumor xenografts in vivo. Tumor growth and expression of SNHG1, miR-382-5p and EMT markers were evaluated.Results: SNHG1 upregulation was observed in breast cancer tissues and cells. Knockdown of SNHG1 attenuated breast cancer proliferation, colony formation, migration and invasion. A miRNA, miR-382-5p, was identified as the target of SNHG1. A reciprocal negative regulation was found between SNHG1 and miR-382-5p. SNHG1 knockdown attenuated EMT both in vitro and in vivo. miR-382-5p transfection reversed the tumor-promoting role by SNHG1. In vivo, SNHG1 knockdown decreased breast tumor growth.Conclusion: SNHG1 promotes breast cancer through the regulation of miR-382-5p and EMT markers. Our results report SNHG1 as a novel miRNA that govern the progression of breast cancer, providing a potential new therapeutic target in breast cancer.

MicroRNA-3662 targets ZEB1 and attenuates the invasion of the highly aggressive melanoma cell line A375.

BackgroundCutaneous melanoma is the most aggressive form of skin cancer. It accounts for approximately 5% of all cutaneous malignancies and is currently responsible for the majority of skin cancer-related deaths. However, the exact mechanisms responsible for the occurrence of melanoma, in particular the invasive growth in normal skin or muscle tissue, remain unknown.Materials and methodsmiR-3662, a microRNA is a potential tumor suppressor targeting zinc finger E-box binding homeobox 1 (ZEB1), which functions as a key regulator of the epithelial-mesenchymal transition (EMT) process. This microRNA was identified using an online database (miRDB) and expression was confirmed by Western blot analysis. Quantitative polymerase chain reaction (qPCR) was used to examine whether miR-3662 inhibits the EMT process in the aggressive melanoma cell line, A375, through the modification of the expression of invasion-related genes in A375 cells. The effects of miR-3662 on the in vivo growth of A375 cells were examined in a nude mouse model.ResultsUsing virtual screening of the miRDB database, miR-3662 was shown to target the 3ʹ untranslated region (UTR) of the ZEB1 gene. Expression of miR-3662 via a lentivirus vector significantly decreased protein levels of ZEB1 and inhibited the growth of A375 cells in vitro and in vivo. The reduction in ZEB1 expression induced by miR-3662 resulted in EMT inhibition in A375 cells and decreased the relative expression of metastasis genes.ConclusionDown-regulation of ZEB1’s expression via miR-3662 lentivirus vectors significantly decreased the in vitro and in vivo growth of the highly aggressive melanoma cell line A375.