Although various pharmacological activities of the shikonins have been documented, understanding the hierarchical regulation of these diverse bioactivities at the genome level is unsubstantiated. In this study, through cross examination between transcriptome and microRNA array analyses, we predicted that topical treatment of shikonin in vivo affects epithelial-mesenchymal transition (EMT) and the expression of related microRNAs, including 200a, 200b, 200c, 141, 205, and 429 microRNAs, in mouse skin tissues. In situ immunohistological analyses further demonstrated that specific EMT regulatory molecules are enhanced in shikonin-treated epidermal tissues. RT-PCR analyses subsequently confirmed that shikonin treatment downregulated expression of microRNA-205 and other members of the 200 family microRNAs. Further, expression of two RNA targets of the 200 family microRNAs in EMT regulation, Sip1 (Zeb2) and Tcf8 (Zeb1), was consistently upregulated by shikonin treatment. Enhancement of these EMT activities was also detected in shikonin-treated wounds, which repaired faster than controls. These results suggest that topical treatment with shikonin can confer a potent stimulatory effect on EMT and suppress the expression of the associated microRNAs in skin wound healing. Collectively, these cellular and molecular data provide further evidence in support of our previous findings on the specific pharmacological effects of shikonin in wound healing and immune modulation.

MicroRNA-154 (miR-154) has been identified as a tumor suppressor in several types of human cancers; however, its clinical significance and function in human hepatocellular carcinoma (HCC) remain unclear. The aim of the present study was to analyze the clinical significance and cellular function of miR-154 in HCC patients. The data showed that miR-154 expression was consistently lower in HCC tissues and cell lines compared to that in matched tumor-adjacent tissues and a normal hepatic cell line, and its expression was negatively correlated with tumor differentiation (P<0.01), TNM stage (P<0.01) and lymph node metastasis (P<0.01). Restoration of miR-154 expression in HepG2 cells inhibited cell proliferation, migration and invasion, and induced apoptosis and cell arrest at the G1 phase in vitro, as well as suppressed tumor growth in a nude mouse model. Using a luciferase assay, we identified that miR-154 was able to target the 3'-untranslated region (3'UTR) of ZEB2 mRNA. Then, we revealed that miR-154 was able to reduce ZEB2 expression at the levels of mRNA and protein using qRT-PCR and western blot analysis. Notably, restoration of expression of ZEB2 weakened miR-154-mediated suppression of tumor progression. In conclusion, these results indicate that miR-154 functions as a tumor suppressor in HCC by suppressing ZEB2, suggesting that miR-154 may serve as a potential target for HCC.

Nintedanib (BIBF1120) is a multi-targeted angiokinase inhibitor and has been evaluated in idiopathic pulmonary fibrosis and advanced non-small cell lung cancer (NSCLC) patients in clinical studies. In the present study, we evaluated the antitumor effects of nintedanib in 16 NSCLC cell lines and tried to identify microRNA (miRNA) associated with sensitivity to nintedanib. No correlations between FGFR, PDGFR and VEGFR family activation and sensitivity to nintedanib were found. The difference in miRNA expression profiles between 5 nintedanib-sensitive and 5 nintedanib-resistant cell lines was evaluated by miRNA array and quantitative RT-PCR analysis (qRT-PCR). Expression of miR-200b, miR-200a and miR-141 belonging to the miR-200 family which contributes to epithelial-mesenchymal transition (EMT), was significantly lower in 5 nintedanib-resistant than in 5 nintedanib-sensitive cell lines. We examined the protein expression of EMT markers in these 10 NSCLC cell lines. E-cadherin expression was lower, and vimentin and ZEB1 expression were higher in 5 nintedanib-resistant cell lines. PC-1 was the most sensitive of the NSCLC cell lines to nintedanib. We established nintedanib-resistant PC-1 cells (PC-1R) by the stepwise method. PC-1R cells also showed decreased expression of miR-200b, miR-141 and miR-429 and increased expression of ZEB1 and ZEB2. We confirmed that induction of miR-200b or miR-141 enhanced sensitivity to nintedanib in nintedanib-resistant A549 and PC1-R cells. In addition, we evaluated the response to gefitinib in combination with nintedanib after TGF-β1 exposure of A549 cells. Nintedanib was able to reverse TGF-β1-induced EMT and resistance to gefitinib caused by miR-200b and miR-141 upregulation and ZEB1 downregulation. These results suggested that the miR-200/ZEB axis might be predictive biomarkers for sensitivity to nintedanib in NSCLC cells. Furthermore, nintedanib combined with gefitinib might be a novel therapeutic strategy for NSCLC cells with EMT phenotype and resistance to gefitinib.

Although microRNA-200a (miR-200a) is frequently downregulated in cancer, its role in side population (SP) has not been investigated. In this study, 101 pairs of primary hepatocellular carcinoma (HCC) tissues and matched normal control tissues were analyzed for miR-200a expression and its clinicopathological value was determined. We found that miR-200a was downregulated in HCC/SP and this was associated metastasis. MiR-200a suppressed metastasis of SP cells. Overexpression of miR-200a in SP cells decreased metastasis-related markers and expression of ZEB2. The associations between miR-200a, SP cells and ZEB2 were validated in HCC. These findings reveal that miR-200a suppresses metastasis of SP cells by downregulating ZEB2.

Recently, lncRNA ZEB2-AS1 was identified as a lncRNA that promoted cancer progression. However, the biological function and the underlying mechanism of ZEB2-AS1 in pancreatic cancer had not been reported. In the current study, we revealed that the expression level of ZEB2-AS1 was elevated in pancreatic cancer cell lines and tissues. ZEB2-AS1 inhibition decreased cell growth and invasion in pancreatic cancer. Mechanismly, ZEB2-AS1 exerted as a ceRNA and negatively regulated miR-204 expression. In addition, HMGB1 was identified as a down-stream target of miR-204. The miR-204/HMGB1 axis mediated ZEB2-AS1's effect on pancreatic cancer. Our findings revealed that lncRNA ZEB2-AS1 may be a candidate prognostic biomarker and a target for new therapies in pancreatic cancer patients.

BACKGROUND

Carcinoma associated fibroblasts (CAFs), an important component of tumor microenvironment, are capable of enhancing tumor cells invasion and migration through initiation of epithelial-mesenchymal transition (EMT). MRC-5 fibroblasts are one of the CAFs expressing alpha-smooth muscle actin. It is ascertained that medium conditioned by MRC-5 fibroblasts stimulate motility and invasion of breast cancer cells. However, its role in hepatocellular carcinoma (HCC) is less clear. The aim of our study was to investigate the effect of MRC-5-CM on HCC and explore the underlying mechanisms.

RESULTS

Using a combination of techniques, the role of MRC-5-CM in HCC was evaluated. We determined that MRC-5-CM induced the non-classical EMT in Bel-7402 and MHCC-LM3 cell lines. Initiation of the non-classical EMT was mainly via quintessential redistribution of α-, β- and γ-catenin, P120 catenin, E-cadherin, and N-cadherin, rather than up-regulation of typical EMT-related transcription factors (i.e., Snail, Twist1, ZEB-1 and ZEB2). We also found that MRC-5-CM potentiated both the migration and invasion of Bel-7402 and MHCC-LM3 cells in mesenchymal movement mode through activation of the α6, β3, β4, β7 integrin/FAK pathway and upregulation of MMP2. The flow cytometric analysis showed that MRC-5-CM induced G1 phase arrest in Bel-7402 cells with a concomitant reduction of S phase cells. In contrast, MRC-5-CM induced S phase arrest in MHCC-LM3 cells with a concomitant reduction of cells in the G2/M phase. MRC-5-CM also inhibited apoptosis in Bel-7402 cells while inducing apoptosis in MHCC-LM3 cells.

CONCLUSIONS

Collectively, MRC-5-CM promoted HCC cell motility and invasiveness through initiation of the non-classical EMT, including redistribution of α-, β- and γ-catenin, P120 catenin, E-cadherin, and N-cadherin, activation of the integrin/FAK pathway, and upregulation of MMP2. Hence, MRC-5-CM exerted distinct roles in Bel-7402 and MHCC-LM3 cell viability by regulating cyclins, cyclin dependent kinases (CDKs), CDK inhibitors (CKIs), Bcl-2 family proteins and other unknown mechanosensors.

MiR-29b has been reported to be both a suppressor and a promoter in breast cancer (BC) cells proliferation and metastasis. Significant efforts have been made to explain the seemingly contradictory effects of miR-29b on BC, but no answer has yet been clearly verified. In this study, we overexpressed and knocked down miR-29b in BC cell lines, modulated expression of its downstream target gene TET1 and downregulated a downstream target gene of TET1, ZEB2, to explore the regulatory mechanism of miR-29b in BC cell proliferation, migration and epithelial-mesenchymal transition (EMT). Our results showed lower expression of miR-29b in BC samples and cell lines. Functional assays showed that miR-29b overexpression resulted in a higher cell proliferation, greater colony formation, higher migration rate and EMT. A dual luciferase assay identified TET1 as a direct target of miR-29b. As the promoting effects of miR-29b in the proliferation and metastasis of MDA-MB-231 and MCF-7, knockdown of TET1 also led to increased proliferation, colony formation, invasion and EMT. Further, we found that TET1 bound to the promoter of ZEB2, and siTET1 enhanced ZEB2 expression. Disruption of ZEB2 expression inhibited BC cells proliferation, colony formation and invasion. Our results establish the miR-29b/TET1/ZEB2 pathway in BC cell proliferation, migration and provide a theoretical basis for further research on the molecular mechanisms and new clinical treatments for BC.

The zinc-finger, E-box-binding homeobox-2 (Zeb2) gene encodes a SMAD-interacting transcription factor that has diverse roles in development and disease. Mutations at the hZeb2 locus cause Mowat-Wilson syndrome (MWS), a genetic disorder that is associated with mental retardation and other, case- and sex-dependent clinical features. Recent studies have detailed microRNA-mediated control of Zeb2, but little is known about the genomic context of this gene or of enhancer sequences that may direct its diverse functions. Here, we describe a novel transgenic rodent model in which Zeb2 regulatory sequence has been disrupted, resulting in a postnatal developmental phenotype that is autosomal dominant. The phenotype exhibits a genotype-by-sex interaction and manifests primarily as an acute attenuation of postnatal kidney development in males. Other aspects of embryonic and neonatal development, including neuronal, are unaffected. The transgene insertion site is associated with a 12 kb deletion, 1.2 Mb upstream of Zeb2, within a 4.1 Mb gene desert. A conserved sequence, derived from the deleted region, enhanced Zeb2 promoter activity in transcription assays. Tissue and temporal restriction of this enhancer activity may involve postnatal changes in proteins that bind this sequence. A control human/mouse VISTA enhancer (62 kb upstream of Zeb2) also up-regulated the Zeb2 promoter, providing evidence of a string of conserved distal enhancers. The phenotype arising from deletion of one copy of the extreme long-range enhancer indicates a critical role for this enhancer at one developmental stage. Haploinsufficiency of Zeb2 in this developmental context reflects inheritance of MWS and may underlie some sex-dependent, non-neural characteristics of this human inherited disorder.

Epidemiological and experimental studies have suggested that deregulated hepcidin-ferroportin (FPN) signaling is associated with the increased risk of cancers. However, the effects of deregulated hepcidin-FPN signaling on tumor behaviors such as metastasis and epithelial to mesenchymal transition (EMT) have not been closely investigated. In this study, LL/2 cancer cells were found to exhibit an impaired propensity to home into lungs, and a reduced ability to develop tumors was also demonstrated in lungs of Hamp1(-/-) mice. Moreover, hepatic hepcidin deficiency was found to considerably favor tumor-free survival in Hamp1(-/-) mice, compared with wild-type mice. These data thus underscored a contributive role of hepatic hepcidin in promoting lung cancer cell homing and fostering tumor progression. To explore the role of FPN in regulating tumor progression, we genetically engineered 4T1 cells with FPN over-expression upon induction by doxycycline. With this cell line, it was discovered that increased FPN expression reduced cell division and colony formation in vitro, without eliciting significant cell death. Analogously, FPN over-expression impeded tumor growth and metastasis to lung and liver in mice. At the molecular level, FPN over-expression was identified to undermine DNA synthesis and cell cycle progression. Importantly, FPN over-expression inhibited EMT, as reflected by the significant decrease of representative EMT markers, such as Snail1, Twist1, ZEB2, and vimentin. Additionally, there was also a reduction of lactate production in cells upon induction of FPN over-expression. Together, our results highlighted a crucial role of the hepcidin-FPN signaling in modulating tumor growth and metastasis, providing new evidence to understand the contribution of this signaling in cancers.

Paired-box 6 (PAX6) is an important transcription factor required for the function of human neuroectodermal epithelial tissues. Previous studies have suggested that it is also expressed in several types of tumors and has an oncogenic role. However, little is known about its role in non-small cell lung cancer (NSCLC). Here, we found that PAX6 expression levels were upregulated in human lung cancer tissues and correlated with poor clinical outcomes. PAX6 overexpression significantly promoted NSCLC epithelial-to-mesenchymal transition (EMT) and metastasis, whereas its knockdown inhibited these processes. PAX6 is commonly correlated with EMT-mediated stem cell transformation, thereby inducing cisplatin resistance. Using the RT² Profiler PCR Array, we found that WNT5A, EGFR, and ZEB2 were differentially regulated in response to PAX6 modulation. In addition, PAX6 directly bound to the promoter region of ZEB2. ZEB2 knockdown significantly reduced the expression and function of PAX6. ZEB2 was upregulated upon PAX6 overexpression and downregulated upon PAX6 knockdown, whereas E-cadherin expression negatively correlated with PAX6 levels. Moreover, p-PI3K and p-AKT were significantly enhanced by PAX6, which was reversed by the addition of the PI3K-AKT inhibitor, LY294002. These data suggest that PAX6 can mediate E-cadherin downregulation through the PI3K/AKT signaling pathway by directly binding the promoter region of ZEB2, thereby mediating cell migration, stem cell transformation, and cisplatin resistance; and ultimately, affecting survival in NSCLC patients.

Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the repression and activation of the pituitary transcription factor genes Hesx1 and Pou1f1, respectively. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome-wide analysis of PROP1 DNA binding and effects on gene expression in mutant mice, mouse isolated stem cells and engineered mouse cell lines. We determined that PROP1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to genes expressed in epithelial cells like Claudin 23, and to EMT inducer genes like Zeb2, Notch2 and Gli2. Zeb2 activation appears to be a key step in the EMT process. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.

miR-203 is a tumor suppressor that is disregulated in numerous malignancies including nasopharyngeal carcinoma (NPC). However, the role of miR-203 in suppressing tumor stemness, chemotherapy resistance as well as its molecular mechanisms are unclear. In this study, we observed that miR-203 suppressed cell migration, invasion, tumor stemness, and chemotherapy resistance to cisplatin (DDP) in vitro and in vivo. miR-203 exerted these effects by targeting ZEB2 and downstream epithelial-mesenchymal transition (EMT) and tumor stemness signals. Interestingly we observed that miR-203 expression was directly suppressed by ZEB2 via targeting its promoter, which significantly reduced cell migration, invasion, tumor stemness, and chemotherapy resistance in NPC cells. Finally, we found that miR-203 was negatively correlated with ZEB2 expression in NPC tissues and tumor spheres. Our data demonstrate a directly negative feedback loop between miR-203 and ZEB2 participating in tumor stemness and chemotherapy resistance, highlighting the therapeutic potential of targeting this signal for NPC chemotherapy.

Acute myeloid leukemia (AML) is a heterogeneous disease with complex molecular pathophysiology. To systematically characterize AML's genetic dependencies, we conducted genome-scale short hairpin RNA screens in 17 AML cell lines and analyzed dependencies relative to parallel screens in 199 cell lines of other cancer types. We identified 353 genes specifically required for AML cell proliferation. To validate the in vivo relevance of genetic dependencies observed in human cell lines, we performed a secondary screen in a syngeneic murine AML model driven by the MLL-AF9 oncogenic fusion protein. Integrating the results of these interference RNA screens and additional gene expression data, we identified the transcription factor ZEB2 as a novel AML dependency. ZEB2 depletion impaired the proliferation of both human and mouse AML cells and resulted in aberrant differentiation of human AML cells. Mechanistically, we showed that ZEB2 transcriptionally represses genes that regulate myeloid differentiation, including genes involved in cell adhesion and migration. In addition, we found that epigenetic silencing of the miR-200 family microRNAs affects ZEB2 expression. Our results extend the role of ZEB2 beyond regulating epithelial-mesenchymal transition (EMT) and establish ZEB2 as a novel regulator of AML proliferation and differentiation.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Increasing evidence suggests that microRNAs (miRNAs) are associated with HCC tumorigenesis. The present study was designed to define the role of miR-141 in HCC. The expression of miR-141 was significantly decreased in four HCC cell lines. Overexpression of miR-141 suppressed both the growth and the motility of HCC cells. Furthermore, we identified zinc finger E-box binding homeobox 2 (ZEB2) as a target of miR-141 and miR-141 functioned as a tumor suppressor via ZEB2 targeting in HCC. These data provide a novel potential therapeutic target for HCC treatment.

The microRNA-200 (miR-200) family plays a major role in specifying epithelial phenotype by preventing expression of the transcription repressors ZEB1 and ZEB2, which are well-known regulators of the epithelial-to-mesenchymal transition (EMT) in epithelial tumors including oral squamous cell carcinoma (OSCC). Here, we elucidated whether miR-200 family members control RNA-binding protein quaking (QKI), a newly identified tumor suppressor that is regulated during EMT. We predicted that miR-200a and miR-200b could recognize QKI 3'-UTR by analyzing TargetScan and The Cancer Genome Atlas head and neck squamous cell carcinoma (HNSCC) dataset. Forced expression of miR-200b/a/429 inhibited expression of ZEB1/2 and decreased cell migration in OSCC cell lines CAL27 and HSC3. QKI expression was also suppressed by miR-200 overexpression, and the 3'-UTR of QKI mRNA was directly targeted by miR-200 in luciferase reporter assays. Interestingly, shRNA-mediated knockdown of QKI led to pronounced EMT and protumor effects in both in vitro and in vivo studies of OSCC. Furthermore, high expression of QKI protein is associated with favorable prognosis in surgically resected HNSCC and lung adenocarcinoma. In conclusion, QKI increases during EMT and is targeted by miR-200; while, it suppresses EMT and tumorigenesis. We suggest that QKI and miR-200 form a negative feedback loop to maintain homeostatic responses to EMT-inducing signals.

Colorectal cancer remains the most common cause of cancer-related deaths worldwide and it continues to lack an effective treatment. Here, we found that zinc finger E-box binding homeobox 2 (ZEB2) was overexpressed in several colorectal cancer cell lines and colorectal cancer specimens relative to adjacent non-cancerous tissues. Although ZEB2 has been reported to be associated with several tumors, its involvement in colorectal cancer progression remains unclear. In this study, we investigated the biological functions and molecular mechanisms of ZEB2 underlying colorectal carcinoma metastasis and angiogenesis. HCT116 colorectal cancer cells were treated with ZEB2 shRNA or recombinant ZEB2, and the expression of ZEB2 was assessed using reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblotting, respectively. Ectopic expression of ZEB2 induced proliferation and epithelial-mesenchymal transition (EMT), and increased the metastatic capacity of HCT116 cells in vitro and in vivo. Furthermore, endothelial cell tube formation and angiogenesis in chick embryo chorioallantoic membrane (CAM) were accelerated by conditioned medium from ZEB2-overexpressing HCT116 cells. Further, overexpression of ZEB2 accelerated tumor growth and angiogenesis in xenotransplantation models. However, silencing endogenous ZEB2 caused an opposite outcome. Our results provide new evidence that ZEB2 promotes the progression of colon cancer, and thereby might represent a novel therapeutic target for colorectal carcinoma.

We describe a patient with an abnormal phenotype and a de novo CCR consisting of a reciprocal translocation between chromosomes 1 and 15 and an insertion of an interstitial segment from chromosome 2 within chromosome 1. The CCR was studied by QFQ banding and FISH. The apparently balanced rearrangement was further investigated with array-CGH, that uncovered three cryptic deletions on chromosome 2q. By means of BAC-FISH two deletions were located at the breakpoints of the insertion, at 2q14.3 and 2q31.2, and one at 2q22.2, in the region of 2q translocated on derivative 1. Consequently, in silico analysis of the deleted regions was performed. Among deleted genes, particularly interesting seems to be CNTNAP5, encoding a member of the neurexin superfamily. The three mouse orthologues are highly expressed in adult brain tissues. We speculate that loss of CNTNAP5 might contribute to the developmental language delay of this patient, similar to CNTNAP2, another member of the same protein family, whose alterations have been recently associated with delay in the age at first word in autistic patients. At clinical patient's evaluation, a Mowat-Wilson syndrome (MWS) like appearance was noted. The disease is caused by mutation or deletion of ZEB2 gene, located in our patient 794Kb distally to the 2q22.2 deletion, in the chromosome 2 segment inserted into the derivative 1. The loss of the gene has been excluded by the array-CGH results, but its proximity to the deleted segment and/or its insertion in a different genetic context might influence and consequently impair its expression. Our study confirms that array-CGH is a precious method to identify cryptic imbalances in CCR carriers and underlie the essential role of BAC-FISH as second step of analysis to assess the reciprocal position of the chromosomal segments involved in CCRs and the exact mapping of the imbalances.

E-cadherin expression is repressed by ZEB2/SIP1 while it is induced by KLF4. Independent data from the literature indicate that these two transcription factors could bind close to each other in the proximal region of the E-cadherin gene promoter. We have here explored a potential competition between ZEB2 and KLF4 for the binding to the E-cadherin promoter. We show an inverse correlation between ZEB2 expression levels and KLF4 recruitment on the E-cadherin promoter in three breast cancer cell lines and in A431/HA.ZEB2 cells in which ZEB2 expression is induced by doxycycline (DOX). We identified a region of the E-cadherin promoter bound by KLF4 which is necessary for the activation of the E-cadherin promoter activity after KLF4 overexpression. This region is localized between positions -28 and -10 and thus overlaps with one of the ZEB2 binding sites. Deleting the bipartite ZEB2 binding site results in increased KLF4 induced E-cadherin promoter activity. Taken together, our results suggest that E-cadherin expression in cancer cells is controlled by a balance between ZEB2 and KLF4 expression levels.

Osteosarcoma is the most common primary malignancy to arise from bone. The pathogenesis of osteosarcoma is unclear, and new therapy molecular target is needed. The miRNAs researched suggested that miRNAs are involved in the pathogenesis of osteosarcoma. MiR-141, which belong to miR-200 family, take a part in tumorigenesis. However, the role of miR-141 in the pathogenesis of osteosarcoma remained unclear. In this study, we focused on the miR-141 in osteosarcoma and found that the expression of miR-141 is lower in osteosarcoma. Overexpression of miR-141 not only inhibits osteosarcoma cell proliferation but also induces cell apoptosis. It is estimated that miR-141 played its role via ZEB1 and ZEB2. In all, miR-141 played a osteosarcoma-suppressing role via ZEB1 and ZEB2. Our finding may elucidate the miRNAs mechanism in osteosarcoma and provide a new molecule target for osteosarcoma therapy.

The epithelial-mesenchymal transition (EMT) process plays pivotal roles in regulatory mechanisms of embryogenesis and wound healing physiologically, and organ fibrosis, cancer progression, and metastasis pathologically. EMT is classified as primary, secondary, and tertiary during embryonic development. EMT contributes to repair of tissue injury and fibrogenesis by re-epithelialization and regeneration of fibroblasts, respectively. The hallmarks of EMT include loss of contact inhibition, remodeling of extracellular matrix, and reorganization of cytoskeleton, along with expression of mesenchymal markers and reduction of epithelial markers. Cancer cells acquire stemness, migration and invasive capability, evade apoptosis, and initiate metastasis to distant organs. Several EMT regulators including Snail, Zeb1, Zeb2, and Twist in solid tumor and Sox4, distal-less homeobox gene 4 (DLX4), Prdm14, Bmi1, and the forkhead box family in hematological malignancy are reviewed with regard to their signaling pathways, regulatory mechanisms, and clinical interactions.

A continuous cell line, ZEB2, was developed from zebrafish blastula-stage embryos expressing enhanced green fluorescent protein (GFP). Originally the rainbow trout spleen cell line, RTS34st, was used as feeders to initiate and maintain the cells through several passages. ZEB2 was then grown for 2 years without feeders in L-15 with 15% fetal bovine serum (FBS) for 120 population doublings. This new cell line, ZEB2J, was heteroploid, had detectable telomerase activity, and was adherent. After growing into monolayers, some cells continued to grow into mounds. Cultures expressed Pou-2 mRNA and contained many alkaline phosphatase and a few stage-specific embryonic antigen-1-positive cells. In dishes coated with a phospholipid polymer (2-methacryloxyloxyethyl phosphorylcholine, MPC), ZEB2J formed spherical aggregates. Aggregates attached to conventional culture plastic, and most cells that emerged from aggregates had typical epithelial-like shapes of ZEB2J, which suggests that ZEB2J had limited differentiation potential, despite expressing some stem cell properties. The fluorescence of ZEB2J allowed relationships with feeder cells to be studied. In MPC dishes, ZEB2J formed mixed spheroids with RTS34st. In adherent cocultures, RTS34st and other fish cell lines strongly stimulated the ZEB2J growth, which could be quantified specifically because ZEB2J expressed GFP. ZEB2J should be useful for optimizing culture conditions for zebrafish embryonic stem cells.

We explored molecular events associated with aging-induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle-aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age-phase-specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle-aged mice. These changes occurred with increment in TGFβ mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFβ-regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR-21 and miR-200c, but not miR-192, miR-200a, or miR-200b, was increased with aging. Increased miR-21 and miR-200c contents were associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Age-phase-specific regulation of matrix protein synthesis occurs and involves matrix protein-specific transcriptional and post-transcriptional mechanisms.

Long non-coding RNAs (lncRNAs) exert critical regulatory roles in the development and progression of several cancers. Plasmacytoma variant translocation 1 (PVT1), an lncRNA, was shown to be upregulated in clear cell renal cell carcinoma (ccRCC) in our study, while Kaplan-Meier curve and Cox regression analysis showed that high expression of PVT1 was associated with poor overall survival (OS) and disease free survival (DFS) in ccRCC patients. In vitro experiments revealed that PVT1 promoted renal cancer cell proliferation, migration, and invasion, while in vivo studies confirmed its oncogenic roles in ccRCC. Further bioinformatic analysis and RNA immunoprecipitation revealed that PVT1 could function as an oncogenic transcript partly through sponging miR-200s to regulate BMI1, ZEB1 and ZEB2 expression. Besides, a novel splicing variant of PVT1 lacking exon 4 (PVT1ΔE4) was found to have a higher expression in ccRCC and could also promote cell proliferation and invasion as the full-length transcript did. Besides, SRSF1 decreased the inclusion of exon 4 of full-length transcript and increased the relative expression of PVT1ΔE4 in ccRCC. Mechanistic investigations indicated that PVT1ΔE4 could also upregulate the expression of BMI1, ZEB1 and ZEB2 through interacting with miR-200s. Our study helps reveal new molecular events in ccRCC and provides promising diagnostic and therapeutic targets for this disease.