The role of miR-92 in regulating early development and metamorphosis of Japanese flounder Paralichthys olivaceus.

MicroRNAs are a class of short non-coding RNAs that contain approximately 22 nucleotides and play a regulatory role in RNA silencing and translational repression. miR-92 belongs to the miR-17-92 family and has a regulatory effect on cell proliferation, apoptosis, and expression of proto-oncogenes and tumor suppressor genes. However, its function in flatfish is unclear. In this study, we used farmed Japanese flounder, Paralichthys olivaceus, and showed that gata5 is a target gene of miR-92. Experiments on miR-92 overexpression indicated that gata5 and sox17 were downregulated, while the transcription level of ntl increased. By contrast, depletion of miR-92 resulted in increased gata5 and sox17 levels and reduced ntl level. Moreover, thiourea treatment indicated that miR-92 may inhibit the metamorphic development of Japanese flounder. Our study suggests that miR-92 regulates the fate of endoderm and mesoderm by controlling gata5.

Molecular determinants of nephron vascular specialization in the kidney.

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders.

GATA5 inhibits hepatocellular carcinoma cells malignant behaviours by blocking expression of reprogramming genes.

Evidence indicated that GATA5 may suppress hepatocellular carcinoma (HCC) cell malignant transformation, but the mechanism of how GATA5 affects cancer cell reprogramming to inhibit HCC malignant behaviour is still unclear. In this study, we report that the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM was significantly higher in HCC tissues compared to normal liver tissues. In contrast, the expression of GATA5 was significantly lower in HCC tissues compared to normal liver tissues. Transfection of CDH-GATA5 vectors into HCC cells (HLE, Bel 7402 and PLC/PRF/5 cells) increased the GATA5 expression and decreased the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM. Increased GATA5 expression by transfection with its expression vectors was also able to inhibit the cell growth, colony formation and capability of migration, invasion, while promoting apoptosis in HCC cells. Results revealed that GATA5 co-localization with ß-catenin in the cytoplasm, preventing ß-catenin from entering the nucleus. Treatment with the specific Wnt/ß-catenin pathway inhibitor salinomycin was able to reduce the expression of ß-catenin and reprogramming genes. Salinomycin exerted a similar influence as GATA5, and siRNA-GATA5 restored ß-catenin and reprogramming gene expression. This study demonstrates that an increase in the expression of GATA5 inhibits the expression of ß-catenin and reprogramming genes and suppresses tumour growth, colony formation, metastasis and invasion, while promoting apoptosis in HCC cells. The mechanism of GATA5 inhibiting the malignant behaviours of HCC cells may involve in the disruption of the Wnt/ß-catenin pathway and the reduction of reprogramming gene expression.

Prognostic DNA methylation markers for renal cell carcinoma: a systematic review.

AIM: Despite numerous published prognostic methylation markers for renal cell carcinoma (RCC), none of these have yet changed patient management. Our aim is to systematically review and evaluate the literature on prognostic DNA methylation markers for RCC. MATERIALS & METHODS: We conducted an exhaustive search of PubMed, EMBASE and MEDLINE up to April 2017 and identified 49 publications. Studies were reviewed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, assessed for their reporting quality using the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) criteria, and were graded to determine the level of evidence (LOE) for each biomarker. RESULTS: We identified promoter methylation of BNC1, SCUBE3, GATA5, SFRP1, GREM1, RASSF1A, PCDH8, LAD1 and NEFH as promising prognostic markers. Extensive methodological heterogeneity across the included studies was observed, which hampers comparability and reproducibility of results, providing a possible explanation why these biomarkers do not reach the clinic. CONCLUSION: Potential prognostic methylation markers for RCC have been identified, but they require further validation in prospective studies to determine their true clinical value.

WT1, MSH6, GATA5 and PAX5 as epigenetic oral squamous cell carcinoma biomarkers - a short report.

PURPOSE: Oral squamous cell carcinoma (OSCC) is a frequently occurring aggressive malignancy with a heterogeneous clinical behavior. Based on the paucity of specific early diagnostic and prognostic biomarkers, which hampers the appropriate treatment and, ultimately the development of novel targeted therapies, we aimed at identifying such biomarkers through a genetic and epigenetic analysis of these tumors. METHODS: 93 primary OSCCs were subjected to DNA copy number alteration (CNA) and methylation status analyses using methylation-specific multiplex ligation-dependent probe amplification (MS-MPLA). The genetic and epigenetic OSCC profiles obtained were associated with the patients' clinic-pathological features. RESULTS: We found that WT1 gene promoter methylation is a predictor of a better prognosis and that MSH6 and GATA5 gene promoter methylation serve as predictors of a worse prognosis. GATA5 gene promoter methylation was found to be significantly associated with a shorter survival rate. In addition, we found that PAX5 gene promoter methylation was significantly associated with tongue tumors. To the best of our knowledge, this is the first study that highlights this specific set of genes as epigenetic diagnostic and prognostic biomarkers in OSCC. CONCLUSIONS: Our data highlight the importance of epigenetically assessing OSCCs to identify key genes that may serve as diagnostic and prognostic biomarkers and, potentially, as candidate therapeutic targets.

Epigenetic and genetic variation in GATA5 is associated with gastric disease risk.

Gastric cancer incidence varies considerably among populations, even those with comparable rates of Helicobacter pylori infection. To test the hypothesis that genetic variation plays a role in gastric disease, we assessed the relationship between genotypes and gastric histopathology in a Colombian study population, using a genotyping array of immune-related single nucleotide polymorphisms (SNPs). Two synonymous SNPs (rs6061243 and rs6587239) were associated with progression of premalignant gastric lesions in a dominant-effects model after correction for multiple comparisons (p = 2.63E-07 and p = 7.97E-07, respectively); effect sizes were ß = -0.863 and ß = -0.815, respectively, where ß is an estimate of effect on histopathology scores, which ranged from 1 (normal) to 5 (dysplasia). In our replication cohort, a second Colombian population, both SNPs were associated with histopathology when additively modeled (ß = -0.256, 95 % CI = -0.47, -0.039; and ß = -0.239, 95 % CI = -0.45, -0.024), and rs6587239 was significantly associated in a dominant-effects model (ß = -0.330, 95 % CI = -0.66, 0.00). Because promoter methylation of GATA5 has previously been associated with gastric cancer, we also tested for the association of methylation status with more advanced histopathology scores in our samples and found a significant relationship (p = 0.001). A multivariate regression model revealed that the effects of both the promoter methylation and the exonic SNPs in GATA5 were independent. A SNP-by-methylation interaction term was also significant. This interaction between GATA5 variants and GATA5 promoter methylation indicates that the association of either factor with gastric disease progression is modified by the other.

Aberrant methylation of tumour suppressor genes WT1, GATA5 and PAX5 in hepatocellular carcinoma.

BACKGROUND: Aberrant hypermethylation of tumour suppressor genes (TSGs) occurring in hepatocellular carcinoma (HCC) could provide a mean of molecular characterisation of this cancer. The aim of this study was to investigate promoter methylation and gene expression of selected TSGs in HCC to identify candidate genes for further validation as potential biomarkers. METHODS: Methylation-specific multiplex ligation-dependent probe amplification method was used to measure the methylation status of 25 TSGs in 49 HCC samples and 36 corresponding non-cancerous liver tissue samples. Relative expression of the differentially methylated genes was assessed at the mRNA level using quantitative PCR. RESULTS: We observed a significantly higher methylation in genes WT1, PAX5, PAX6, PYCARD and GATA5 in HCC compared with control samples. The expression of PAX5 was significantly decreased by methylation; conversely methylation of WT1 was associated with higher mRNA levels. Methylation of GATA5 was significantly associated with overall survival and methylation of WT1 and PAX5 significantly varied between patients with ALBI score 1 vs. 2+3. Moreover, PAX5 was significantly more methylated in patients with tumour grade 2+3 vs. grade 1, and methylation of the PAX5 correlated with the patient's age at the time of diagnosis. CONCLUSIONS: HCC evince aberrant promoter methylation of WT1, PAX5, PAX6, PYCARD and GATA5 genes. Correlation between GATA5, WT1 and PAX5 methylation and clinical/histological parameters is suggestive of applicability of these markers in non-invasive (epi)genetic testing in HCC.

BMP4 depletion by miR-200 inhibits tumorigenesis and metastasis of lung adenocarcinoma cells.

BACKGROUND: MicroRNA-200 (miR-200) suppresses the epithelial-mesenchymal transition of various cancer cells, including lung adenocarcinoma cells. We found that bone morphogenetic protein 4 (BMP4) was decreased in miR-200-overexpressing cells and epithelial-like lung cancer cells. In this study, we investigated the mechanism and role of BMP4 depletion by miR-200 in murine lung adenocarcinoma cells. METHODS: BMP4 expression levels in murine lung cancer cells were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Promoter and 3'-untranslated region (UTR) luciferase reporter assays were performed to discover the mechanism of regulation of BMP4 by miR-200. Murine lung cancer cells were transfected with Bmp4 shRNAs, which were then injected into syngeneic mice to measure their tumorigenic and metastatic potential and cultured on Matrigel to study the influence of BMP4 on 3-D acinus formation. RESULTS: miR-200 down-regulated BMP4 via direct targeting of the GATA4 and GATA6 transcription factors that stimulate Bmp4 transcription. BMP4 up-regulated JAG2, an upstream factor of miR-200; therefore, JAG2, miR-200, and BMP4 form a regulatory loop. Bmp4 knockdown suppressed cancer cell growth, migration, and invasion and inhibited tumorigenesis and metastasis of lung cancer cells when injected into syngeneic mice. In addition, BMP4 was required for normal acinus formation in Matrigel 3-D culture of murine lung cancer cells, which may be mediated by MYH10, a downstream target of BMP4. CONCLUSION: BMP4 functions as a pro-tumorigenic factor in a murine lung cancer model, and its transcription is regulated by miR-200 and GATA4/6. Thus, we propose that BMP4 and its antagonists may be suitable therapeutic targets for the treatment of lung cancer.

Combined detection of plasma GATA5 and SFRP2 methylation is a valid noninvasive biomarker for colorectal cancer and adenomas.

AIM: To investigate GATA5, SFRP2, and ITGA4 methylation in plasma DNA as noninvasive biomarkers for colorectal cancer (CRC) or adenomas. METHODS: There were 57 CRC patients, 30 adenomas patients, and 47 control patients enrolled in this study. Methylation-specific polymerase chain reaction was used to determine the promoter methylation status of GATA5, SFRP2, and ITGA4 genes in plasma DNA, and their association with clinical outcome in CRC. The predictive ability of GATA5, SFRP2, and ITGA4 methylation, individually or in combination, to detect CRC or adenomas was further analyzed. RESULTS: Hypermethylated GATA5 was detected in plasma in 61.4% (35/57) of CRC cases, 43.33% (13/30) of adenoma cases, and 21.28% (10/47) of control cases. The hypermethylation of SFRP2 was detected in 54.39% (31/57), 40.00% (12/30), and 27.66% (13/47) in plasma samples from CRC, adenomas, and controls, respectively. ITGA4 methylation was detected in 36.84% (21/57) of plasma samples of CRC patients and in 30.00% (9/30) of plasma samples from patients with colorectal adenomas, and the specificity of this individual biomarker was 80.85% (9/47). Moreover, GATA5 methylation in the plasma was significantly correlated with larger tumor size (P = 0.019), differentiation status (P = 0.038), TNM stage (P = 0.008), and lymph node metastasis (P = 0.008). SFRP2 and ITGA4 methylation in plasma significantly correlated with differentiation status (SFRP2, P = 0.012; ITGA4, P = 0.007), TNM stage (SFRP2, P = 0.034; ITGA4, P = 0.021), and lymph node metastasis (SFRP2, P = 0.034; ITGA4, P = 0.021). From the perspective of predictive power and cost-performance, using GATA5 and SFRP2 together as methylation markers seemed the most favorable predictor for CRC (OR = 8.06; 95%CI: 2.54-25.5; P < 0.01) and adenomas (OR = 3.35; 95%CI: 1.29-8.71; P = 0.012). CONCLUSION: A combination of GATA5 and SFRP2 methylation could be promising as a marker for the detection and diagnosis of CRC and adenomas.

Comparative detection of aberrantly methylated DNA in preoperative and postoperative stool from patients with colorectal cancers.

BACKGROUND: Early detection of colorectal cancer (CRC) is crucial to reducing tumor-related mortality. Evaluating aberrantly methylated DNA in stool is promising for CRC screening. However, DNA methylation in the colonic epithelium of background mucosa may compromise stool DNA (sDNA) test results. Thus, we compared aberrant methylation of cancer-related genes in preoperative and postoperative sDNA, with the aim of demonstrating that a cancer-specific methylated allele in sDNA originates from CRCs. METHODS: Patients who were to undergo CRC resection in Kyushu University Hospital during 2003-2010 were prospectively enrolled. Preoperative (pre) stool samples from 54 patients, postoperative (post) samples from 52 of the patients and tumor samples were collected. Aberrant promoter methylation of CDH4 and GATA5 was assessed in the primary tumors by methylation-specific polymerase chain reaction (MSP) and in stool samples by real-time MSP. REULTS: Aberrant methylation of CDH4 and/or GATA5 was detected in 45 of CRC tissue samples (83.3%) and identified in 23 pre sDNA samples (42.3%) from CRC patients. Aberrant methylation was not found in pre sDNA obtained from CRC patients without aberrant methylation of these genes or in post sDNA in any patient. The detection rate of methylated alleles did not correlate with depth of invasion or tumor stage. CONCLUSION: Our findings demonstrate that aberrantly methylated alleles identified in sDNA originate from CRCs. Although tumor-specific aberrant methylation is found in sDNA from patients harboring early and advanced CRC throughout the colon and rectum, the sensitivity of this test needs to be improved for early detection of CRC.

Promoter hypermethylation may be an important mechanism of the transcriptional inactivation of ARRDC3, GATA5, and ELP3 in invasive ductal breast carcinoma.

Hypermethylation of promoter CpG islands represents an alternative mechanism to inactivate tumor suppressor genes. This study was to detect promoter methylation status and mRNA expression levels of ARRDC3, ELP3, GATA5, and PAX6, and to explore the association between methylation and expression in invasive ductal carcinomas (IDCs) and matched normal tissues (MNTs) from breast cancer patients. Aberrant gene methylation was observed as follows: ARRDC3 in 38.5 %, ELP3 in 73.1 %, GATA5 in 48.1 %, and PAX6 in 50.0 % of IDCs. mRNA expression of ARRDC3, ELP3, and GATA5 in IDCs showed a lower level than that in MNTs (P < 0.001, P = 0.001 and P < 0.001, respectively). For ARRDC3, both methylated and unmethylated IDCs showed significantly lower expression values compared to MNTs (P = 0.001 and P = 0.007, respectively). For ELP3 and GATA5, methylated tumors only showed significantly lower expression values compared to MNTs (P = 0.001 and P < 0.001, respectively). For ARRDC3 and GATA5, methylation was associated with their less fold change in IDCs (P = 0.049 and P = 0.020, respectively). Methylation of ARRDC3 was significantly associated with grades and lymph node status of IDCs (P = 0.036 and P = 0.002, respectively). Methylation frequency of ELP3 was higher in lymph node positive versus lymph node negative tumors (P = 0.020); whereas methylation frequency of PAX6 was lower in tumors with the ER negative samples (P = 0.025). Our data suggested that promoter hypermethylation may be an important mechanism of the transcriptional inactivation of ARRDC3, GATA5, and ELP3 in IDCs.

GATA5 and endothelial nitric oxide synthase expression in the ascending aorta is related to aortic size and valve morphology.

BACKGROUND: The pathogenesis of aortic dilatation in patients with congenital aortic valve anomalies is poorly understood. Recent studies suggest that alterations of gene expression may be related to ascending aortic aneurysm formation in these patients. Knockout of endothelial nitric oxide synthase (eNOS) and GATA5 is associated with bicuspid aortic valves in mice. To study the role of eNOS and GATA5 in human congenital aortic valve disease and aortic dilatation, we investigated their gene expression in aortic tissue from patients with unicuspid, bicuspid, and tricuspid aortic valves. METHODS: Samples from 84 patients (33 tricuspid, 32 bicuspid, and 19 unicuspid) were harvested intraoperatively from the ascending aorta. GATA5 and eNOS expression was determined by real-time polymerase chain reaction. RESULTS: GATA5 and eNOS expression in the aortic wall from patients with unicuspid aortic valves (GATA5: mean [M], 2.14; standard deviation [SD], 1.72; eNOS: M, 3.40; SD, 3.83) was significantly higher than in tricuspid aortic valves (GATA5: M, 1.12; SD, 0.80; eNOS: M, 1.00; SD, 0.74; each p < 0.05). Patients with bicuspid aortic valves (GATA5: M, 1.29, SD, 1.33; eNOS: M, 1.66; SD, 1.31) had a significantly higher eNOS expression than patients with tricuspid aortic valves (p < 0.05). The expression levels of eNOS and GATA5 correlated positively with each other and negatively with the ascending aortic diameter. CONCLUSIONS: Our data suggest that GATA5, possibly through upregulation of eNOS, plays a role in the development of aortic dilatation in patients with unicuspid and bicuspid aortic valves. The differential gene expression in patients with unicuspid compared with bicuspid aortic valves suggests that the pathogenesis of both aortic valve anomalies may be different.

GATA5 CpG island hypermethylation is an independent predictor for poor clinical outcome in renal cell carcinoma.

Transcriptional inactivation and CpG island (CGI) methylation of GATA transcription factor family members GATA3 and GATA5 have been reported for a few types of human cancer. Whether high-density CGI methylation of GATA3 or GATA5 is associated with the clinical course of patients with renal cell cancer (RCC) has not been clarified. Quantitative methylation-specific PCR assays were carried out to analyze 25 tumor cell lines including 6 RCC lines and 119 RCC and 87 adjacent normal tissues for the presence of densely methylated sequences. Methylation values were statistically compared with clinicopathological and recurrence-free survival (RFS) data for patients. Comparison of GATA3 and GATA5 methylation in different tumor cell lines revealed a marker-specific methylation characteristic with high and frequent signals for both methylation marks in RCC lines. GATA3 and GATA5 CGI relative methylation levels were found to be strongly associated with the state of metastasis (P=0.003 and P<0.001, respectively) and advanced disease (P=0.024 and P<0.001, respectively). Moreover, an independent decrease in RFS in Cox proportional hazard analysis was found for tumors exhibiting high GATA5 methylation (P<0.001, hazard ratio, 19.3; 95% confidence interval, 4.58-81.6). Epigenetic alterations in GATA family members may be associated with aggressive tumor phenotypes in RCC, and in the case of GATA5, may serve as a new independent molecular marker for aggressiveness and disease progression.

Decreased GATA5 mRNA expression associates with CpG island methylation and shortened recurrence-free survival in clear cell renal cell carcinoma.

BACKGROUND: GATA-5, a zinc-finger transcription factor and member of the GATA family proteins 1-6, is known to be involved in cellular differentiation. We recently found that tumor-specific hypermethylation of the GATA5 CpG island (CGI) occurs in renal cell carcinoma (RCC) and is associated with an adverse clinical outcome. In this study, we investigated whether epigenetic GATA5 alterations may result in changes in GATA5 mRNA expression levels and correlate with the observed prognostic impact of epigenetic changes in GATA5 in RCC. METHODS: Quantitative real-time reverse-transcribed polymerase chain reaction was applied to measure relative GATA5 mRNA expression levels in 135 kidney tissue samples, including 77 clear cell RCC (ccRCC) tissues and 58 paired adjacent normal renal tissue samples. Relative GATA5 expression levels were determined using the ΔΔCt method and detection of three endogenous control genes then compared to previously measured values of relative methylation. RESULTS: The mean relative GATA5 mRNA expression level exhibited an approximately 31-fold reduction in tumor specimens compared with corresponding normal tissues (p < 0.001, paired t-test). Decreased GATA5 mRNA expression was inversely correlated with increased GATA5 CGI methylation (p < 0.001) and was associated with shortened recurrence-free survival in ccRCC patients (p = 0.023, hazard ratio = 0.25). CONCLUSION: GATA5 mRNA expression is decreased in ccRCC, likely due to gene silencing by methylation of the GATA5 CGI. Moreover, reduced GATA5 mRNA levels were associated with a poor clinical outcome, indicating a possible role of GATA5 for the development of aggressive ccRCC phenotypes.

Expression and in situ localization of GATA4, 5 and 6†mRNAs in ovine conceptuses and uterine endometria during the peri-implantation period.

In vertebrates, six GATA transcription factors, GATA1 through GATA6, have been identified and GATA1-3 is known to be involved in hematopoietic developments, while GATA4-6 play roles in cardiac and endoderm developments. Recently, we and others have found that GATA2 and GATA3 found in the trophectoderm plays a role in gene expression specific to this cell type, but GATA4-6 have not been well characterized in early embryonic developments. Using quantitative polymerase chain reaction (qPCR) and in situ hybridization, we examined the expression of GATA4, 5 and 6 messenger RNAs (mRNAs) in ovine conceptuses and uteri during the peri-implantation period. In ovine conceptuses, GATA4, 5 and 6 transcripts were present on days 15, 17 and 21 (day 0 = day of mating), and high GATA5 and 6 mRNAs were found on day 21, most of which were localized in the trophectoderm and endoderm. Moreover, minute and substantial GATA4 and 5 mRNAs were found in days 15 and 21 uterine endometria, respectively. Increase in GATA4-6 transcripts in day 21 uteri indicates that in addition to GATA1-3, GATA4-6 may also play a potentially novel role in the development of ovine trophectoderm, endoderm and/or uterine endometria following conceptus attachment to the uterine epithelium.

Gata5 deficiency causes airway constrictor hyperresponsiveness in mice.

Gata5 is a transcription factor expressed in the lung, but its physiological role is unknown. To test whether and how Gata5 regulates airway constrictor responsiveness, we studied Gata5(-/-), Gata5(+/-), and wild-type mice on the C57BL/6J background. Cholinergic airway constrictor responsiveness was assessed invasively in mice without and with induction of allergic airway inflammation through ovalbumin sensitization and aerosol exposure. Gata5-deficient mice displayed native airway constrictor hyperresponsiveness (AHR) in the absence of allergen-induced inflammation. Gata5-deficient mice retained their relatively greater constrictor responsiveness even in ovalbumin-induced experimental asthma. Gata5 deficiency did not alter the distribution of cell types in bronchoalveolar lavage fluid, but bronchial epithelial mucus metaplasia was more prominent in Gata5(-/-) mice after allergen challenge. Gene expression profiles revealed that apolipoprotein E (apoE) was the fifth most down-regulated transcript in Gata5-deficient lungs, and quantitative RT-PCR and immunostaining confirmed reduced apoE expression in Gata5(-/-) mice. Quantitative RT-PCR also revealed increased IL-13 mRNA in the lungs of Gata5-deficient mice. These findings for the first time show that Gata5 regulates apoE and IL-13 expression in vivo and that its deletion causes AHR. Gata5-deficient mice exhibit an airway phenotype that closely resembles that previously reported for apoE(-/-) mice: both exhibit cholinergic AHR in native and experimental asthma states, and there is excessive goblet cell metaplasia after allergen sensitization and challenge. The Gata5-deficient phenotype also shares features that were previously reported for IL-13-treated mice. Together, these results indicate that Gata5 deficiency induces AHR, at least in part, by blunting apoE and increasing IL-13 expression.

Upstream stimulatory factor 1 activates GATA5 expression through an E-box motif.

Silencing of GATA5 gene expression as a result of promoter hypermethylation has been observed in lung, gastrointestinal and ovarian cancers. However, the regulation of GATA5 gene expression has been poorly understood. In the present study, we have demonstrated that an E (enhancer)-box in the GATA5 promoter (bp -118 to -113 in mice; bp -164 to -159 in humans) positively regulates GATA5 transcription by binding USF1 (upstream stimulatory factor 1). Using site-directed mutagenesis, EMSA (electrophoretic mobility-shift analysis) and affinity chromatography, we found that USF1 specifically binds to the E-box sequence (5'-CACGTG-3'), but not to a mutated E-box. CpG methylation of this E-box significantly diminished its binding of transcription factors. Mutation of the E-box within a GATA5 promoter fragment significantly decreased promoter activity in a luciferase reporter assay. Chromatin immunoprecipitation identified that USF1 physiologically interacts with the GATA5 promoter E-box in mouse intestinal mucosa, which has the highest GATA5 gene expression in mouse. Co-transfection with a USF1 expression plasmid significantly increased GATA5 promoter-driven luciferase transcription. Furthermore, real-time and RT (reverse transcription)-PCR analyses confirmed that overexpression of USF1 activates endogenous GATA5 gene expression in human bronchial epithelial cells. The present study provides the first evidence that USF1 activates GATA5 gene expression through the E-box motif and suggests a potential mechanism (disruption of the E-box) by which GATA5 promoter methylation reduces GATA5 expression in cancer.

ΔNp63α mediates proliferation and apoptosis in human gastric cancer cells by the regulation of GATA-6.

The oncogenic isoform of the p63 protein, ΔNp63α, has been found to be overexpressed in numerous human squamous cell carcinomas. However, the role of ΔNp63α in human gastric cancer remains unknown. To evaluate this role, we screened a panel of gastric cancer cell lines for ΔNp63α expression and found that they are correlated with the differentiation status of the cell lines. Using the MKN28 gastric cancer cell line for loss-of-function or gain-of-function of ΔNp63α in our experiments, we observed that forced expression of ΔNp63α promoted cell proliferation as assessed by the MTT and colony formation assays, and increased the GATA-6 expression. In contrast, down-regulation of ΔNp63α via small interfering RNA suppressed cell proliferation, induced cell apoptosis, and reduced the expression of GATA-6. In conclusion, our data suggest that ΔNp63α plays an important role in cell growth and proliferation of gastric cancer cells, which may be associated with the regulation of GATA-6 expression. This is the first study exploring the biological functions and the underlying mechanism of ΔNp63α during gastric cancer development. It also identifies potential targets for anti-tumor treatment.

GATA5 CpG island methylation in renal cell cancer: a potential biomarker for metastasis and disease progression.

UNLABELLED: GATA5 CpG island (CGI) methylation and transcriptional inactivation is involved in colorectal and gastric cancer. Whether DNA methylation of GATA5 affects clinical pathology is still unclear. In the present study, we analysed, for the first time, CGI methylation in RCC and its association with clinicopathological parameters and progression-free survival of patients. We show for the first time GATA5 CGI hypermethylation in RCC. Moreover, we found out that increased methylation is statistically associated with status of metastasis, progressive disease and shortened progression-free survival. The present study underline the necessity for further functional investigations as well as prospective survival analyses to clarify whether GATA5 promoter methylation can provide independent information for future clinical management of patients with RCC. OBJECTIVE: To investigate whether GATA5 CpG island (CGI) methylation occurs in renal cell carcinoma (RCC) and is associated with clinical, histopathological characteristics or progression-free survival of patients. PATIENTS AND METHODS: Methylation was quantified in 117 RCC samples and 89 paired adjacent normal tissues using quantitative combined bisulphite restriction analysis (COBRA). COBRA was evaluated in advance by pyrosequencing analyses of control RCC cell lines (coefficient of correlation, R = 0.95). Statistical analyses were carried out using the paired t-test for matched tumour tissue (TU) and adjacent normal tissue (adN) samples, logistic regression for comparisons of independent sample groups and Cox regression for analysis of progression-free survival. RESULTS: In the present study, we found a significant higher mean relative methylation in TU (20.4%) than in adN (7.9%, P < 0.001) in paired samples of all RCCs. Increased GATA5 methylation in tumours was associated with metastasis (P = 0.005) and decreased progression-free survival (P = 0.005, HR = 4.59) in the clear-cell RCC (ccRCC) group. CGI methylation in advanced ccRCCs (pT ≥3 and/or N1, M1 or G2-3/G3) exceeds those detected in localized tumours (pT ≤2, N0, M0, G1/G1-2) (27.8% vs 11.0%, P < 0.001). CONCLUSIONS: The association of GATA5 hypermethylation with metastasis and progression-free survival of patients indicates that epigenetic alterations of GATA5 participate in renal cell carcinogenesis. Moreover, GATA5 CGI methylation could serve as a biomarker for tumour progression, although prospective and functional investigations are necessary to clarify whether independent information for future clinical management of patients with RCC can be obtained.

MicroRNA-processing enzyme Dicer is required in epicardium for coronary vasculature development.

The epicardium is a sheet of epithelial cells covering the heart during early cardiac development. In recent years, the epicardium has been identified as an important contributor to cardiovascular development, and epicardium-derived cells have the potential to differentiate into multiple cardiac cell lineages. Some epicardium-derived cells that undergo epithelial-to-mesenchymal transition and delaminate from the surface of the developing heart subsequently invade the myocardium and differentiate into vascular smooth muscle of the developing coronary vasculature. MicroRNAs (miRNAs) have been implicated broadly in tissue patterning and development, including in the heart, but a role in epicardium is unknown. To examine the role of miRNAs during epicardial development, we conditionally deleted the miRNA-processing enzyme Dicer in the proepicardium using Gata5-Cre mice. Epicardial Dicer mutant mice are born in expected Mendelian ratios but die immediately after birth with profound cardiac defects, including impaired coronary vessel development. We found that loss of Dicer leads to impaired epicardial epithelial-to-mesenchymal transition and a reduction in epicardial cell proliferation and differentiation into coronary smooth muscle cells. These results demonstrate a critical role for Dicer, and by implication miRNAs, in murine epicardial development.