MiR-92b inhibited cells EMT by targeting Gabra3 and predicted prognosis of triple negative breast cancer patients.

OBJECTIVE: Triple negative breast cancer (TNBC) is a subtype of breast cancer without the three markers, which has a poor prognosis than other types. Recently, studies have identified that microRNA-92b (miR-92b) acted as potential oncogene in tumor progression, however, the biological roles of miR-92b in TNBC remain unknown. The purpose of this study was to investigate the functions of miR-92b and verify its effect on the regulation of Gabra3 in TNBC. MATERIALS AND METHODS: Dual-Luciferase reporter assay was recruited to confirm whether miR-92b directly binds to the 3'-untranslated region (3'-UTR) of Gabra3 mRNA in TNBC. Transwell assay was employed to analyze the capacities of migration and invasion. Western blot was applied to evaluate the expression of the special proteins that including Gabra3, epithelial-mesenchymal transition (EMT) markers and GAPDH. RESULTS: We demonstrated that miR-92b was remarkably low expressed in TNBC tissues and cell lines, and particularly in inhibiting the migration, invasion and EMT of TNBC cells. On the contrary, Gabra3 was significantly overexpressed in TNBC tissues and cell lines in comparison with the corresponding paracancerous tissues and the normal breast epithelial cell line. The expression of miR-92b had a negative correlation with the expression of Gabra3 in TNBC tissues. Downregulation of Gabra3 could inhibit the migration, invasion and EMT of TNBC cells. MiR-92b mediated the expression of Gabra3 through directly binding to the 3'-UTR of Gabra3 mRNA. In addition, low expression of miR-92b or overexpression of Gabra3 predicted poor prognosis of TNBC patients. CONCLUSIONS: MiR-92b inhibited the migration and invasion-mediated EMT through directly targeting the 3'-UTR of Gabra3 mRNA in triple negative breast cancer. The newly identified miR-92b/Gabra3 axis may make it to be a new target for clinical diagnosis and treatment of TNBC.

MiR-1178-3p promotes the proliferation, migration and invasion of nasopharyngeal carcinoma Sune-1 cells by targeting STK4.

Nasopharyngeal carcinoma (NPC) is a malignant tumor with high invasive and metastatic properties. Dysregulation of miRNAs may contribute to disease progression by targeting disease-related genes. In this study we aimed to elucidate the role and function of aberrantly expressed miRNA in NPC tumorigenesis. We found that miR-1178-3p was highly expressed in NPC tissues. Overexpression of miR-1178-3p promoted the proliferation, migration and invasion of NPC cells in vitro. In contrast, knocking down endogenous miR-1178-3p by miRNA-specific inhibitor significantly suppressed the above phenotypes. Moreover, miR- 1178-3p was shown to negatively regulate serine/threonine-protein kinase 4 (STK4), an NPC-related tumor suppressor gene, in the post-transcriptional level. Furthermore, STK4 overexpression abolished miR-1178- 3p-induced cell proliferation, migration and invasion through STK4-mediated dephosphorylation of AKT. Our results indicate that miR-1178-3p acts as an oncomiRNA in NPC by suppressing STK4 expression, and inhibition of miR-1178-3p may become a therapeutic potential for NPC.

[Empirical therapy of bloodstream infections caused by extended-spectrum ß-lactamase-producing Enterobacteriaceae].

OBJECTIVE: To compare the clinical outcomes and costs associated with carbapenems and ß-lactam/ß-lactamase inhibitor combinations (BLBLIs) for the empirical treatment of patients with extended-spectrum ß-lactamase (ESBL)-positive Enterobacteriaceae bloodstream infections (BSIs). METHODS: The medical records of individuals diagnosed with ESBL-producing Escherichia coli and Klebsiella pneumoniae BSIs between January 2014 and June 2015 at Changhai Hospital were reviewed. Patients were divided into two groups based on the empirical therapy (carbapenems group and BLBLIs group). Propensity score matching in a 1∶1 ratio was used to match the patients from two groups. Clinical outcomes and costs were compared before and after matching. RESULTS: One hundred and fifty-eight patients were analyzed, 93 in the carbapenems group and 65 in the BLBLIs group. Before matching, the two groups were significantly different in department distribution, tumor rate, deep vein catheter rate, urinary catheter rate, nasogastric tube rate, and mechanical ventilation rate (all P<0.05), and the carbapenems group had longer total length of stay (LOS) and post-BSI LOS (26.0 vs 18.0 d, P=0.029 and 12.0 vs 10.0 d, P=0.044) , higher hospital cost and daily hospital cost (84 120 vs 39 000 ï¿¥, P<0.001 and 3 451 vs 2 574 ï¿¥, P=0.002). After matching, the two groups had no significant differences in covariates such as sex, age, department distribution, pathogens, comorbidities, invasive interventions, LOS before BSI, multiple admissions, surgical rate during hospitalization and delayed antimicrobial therapy (all P>0.05). Finally, there were no differences between two groups in mortality, post-BSI LOS, total LOS, hospital cost and antimicrobial cost (all P>0.05). CONCLUSION: BLBLIs may provide a reasonable carbapenem-sparing option for the empirical treatment of ESBL producers.

DNA methylation reactivates GAD1 expression in cancer by preventing CTCF-mediated polycomb repressive complex 2 recruitment.

Levels of γ-aminobutyric acid (GABA) and glutamic acid decarboxylase 1 (GAD1), the enzyme that synthesizes GABA, are significantly increased in neoplastic tissues. However, the mechanism underlying this increase remains elusive. Instead of silencing gene transcription, we showed that the GAD1 promoter was hypermethylated in both colon and liver cancer cells, leading to the production of high levels of GAD1. GAD1 is a target gene that is silenced by H3K27me3. The key locus responsible for GAD1 reactivation was mapped to a DNA methylation-sensitive CTCF-binding site (CTCF-BS3) within the third intron of GAD1. Chromosome configuration capture (3C) analysis indicated that an intrachromosomal loop was formed by CTCF self-dimerisation in normal cells (CTCF binds to both unmethylated CTCF-BS3 and CTCF-BS2). The CTCF dimer then interacted with suppressor of zeste 12 homologue (SUZ12), which is a domain of Polycomb repressive complex 2 (PRC2), promoting the methylation of H3K27 and the silencing of GAD1 expression. This silencing was shown to be inhibited by DNA methylation in cancer cells. These findings strongly suggest that GAD1 is reactivated by DNA methylation, which provided a model for DNA methylation and the active orchestration of oncogenic gene expression by CTCF in cancer cells.

A 39-gene signature is associated with early occurrence of distant metastasis in primary lymph-node negative breast cancers.

Risk factors of the development of distant metastasis in primary node-negative breast cancer patients are heterogeneous. Identification of patients at high risk of early distant metastasis is of important clinical significance. In the current study, using the already published datasets, we develop a gene signature that can robustly predict early distant metastasis for patients with primary node-negative breast cancer. We identified a 39-gene signature, which were associated with distant metastasis and shorter distant metastasis free survival (DMFS) in node-negative breast cancers. Using the survival prediction analysis method in BRB-Array tools, this signature can stratify patients into early- and late- distant metastasis subgroups with different DMFS in VDX training dataset (AUC=0.734, P < 0.01). And we further validated the reliability of the prognostic value of this 39-gene signature in another two independent breast cancer cohorts (NKI dataset, AUC=0.642, P<0.0167; TRANSBIG dataset, AUC=0.711, P<0.0167). Furthermore, the early distant metastasis subgroups defined by the 39-gene signature exhibited a significant association with ER negative status and more aggressive molecular subtypes in all three datasets, and with poor differentiation status in two datasets. In summary, we developed a novel distant metastasis-related gene signature for predicting early occurrence of distant metastasis in node-negative breast cancers, what might be useful in making treatment decisions for these early metastasis patients.

Molecular events are associated with resistance to vinblastine in bladder cancer.

Bladder cancer occurs in the majority of cases in males, which represents the fourth highest incident cancer in men and tenth in women. It is associated with a high rate of recurrence, and prognosis is poor once the cancer metastasizes to distant sites. Transitional cell cancer (TCC) is the most predominant histological type. Bladder cancer is highly chemosensitive. However, the presence of acquired drug resistance is one of the primary impediments to the success of chemotherapy. To differentiate and delineate the molecular events, we developed drug resistant human transitional bladder cancer T24 cells (DRC) by treating cells with the increasing concentration of vinblastine. We found that DRC was resistant to vinblastine in comparison to parental T24 cells. We analyzed the contributory factors that may be involved in the development of resistance. As expected, expression of permeability glycoprotein (P—gp) was up—regulated in DRC. In addition, levels of Caveolin—1 (Cav—1), Fatty acid synthase (FASN) and Cytochrome P450 (CYP450) were elevated in DRC. Downregulation of these proteins by respective specific pharmacological inhibitors and/or by siRNAs resensitized cells to vinblastine. These results suggested that differential levels of P—gp, Cav—1 and FASN except CYP450 play a major role in acquired resistant phenotype in bladder cancer.

AML1/ETO cooperates with HIF1α to promote leukemogenesis through DNMT3a transactivation.

The mechanisms by which AML1/ETO (A/E) fusion protein induces leukemogenesis in acute myeloid leukemia (AML) without mutagenic events remain elusive. Here we show that interactions between A/E and hypoxia-inducible factor 1α (HIF1α) are sufficient to prime leukemia cells for subsequent aggressive growth. In agreement with this, HIF1α is highly expressed in A/E-positive AML patients and strongly predicts inferior outcomes, regardless of gene mutations. Co-expression of A/E and HIF1α in leukemia cells causes a higher cell proliferation rate in vitro and more serious leukemic status in mice. Mechanistically, A/E and HIF1α form a positive regulatory circuit and cooperate to transactivate DNMT3a gene leading to DNA hypermethylation. Pharmacological or genetic interventions in the A/E-HIF1α loop results in DNA hypomethylation, a re-expression of hypermethylated tumor-suppressor p15(INK4b) and the blockage of leukemia growth. Thus high HIF1α expression serves as a reliable marker, which identifies patients with a poor prognosis in an otherwise prognostically favorable AML group and represents an innovative therapeutic target in high-risk A/E-driven leukemia.

COUP-TFII regulates metastasis of colorectal adenocarcinoma cells by modulating Snail1.

BACKGROUND: Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII, also known as NR2F2) promotes metastasis by functioning in the tumour microenvironment; however, the role of COUP-TFII in colorectal cancer remains unknown. METHODS: Human colon adenocarcinoma tissues were collected to test COUP-TFII expression. Wound-healing and cell invasion assay were used to evaluate migration and invasion of cells. Chicken ovalbumin upstream promoter-transcription factor II and related protein expression was assessed by immunostaining, immunoblotting and real-time PCR assay. Tamoxifen-inducible COUP-TFII knockout mice were employed to test COUP-TFII functions on colon cancer metastasis in vivo. RESULTS: Elevated expression of COUP-TFII in colorectal adenocarcinoma tissue correlated with overexpression of the Snail1 transcription factor. High COUP-TFII expression correlated with metastasis and shorter patient survival. Chicken ovalbumin upstream promoter-transcription factor II regulated the migration and invasion of cancer cells. With Snail1, COUP-TFII inhibited expression of adherence molecules such as ZO-1, E-cadherin and ß-catenin in colorectal cancer cells. Overexpression of COUP-TFII was required for cancer cells to metastasise in vivo. Chicken ovalbumin upstream promoter-transcription factor II regulated the transcription and expression of Snail1 by directly targeting the Snail1 promoter and regulated associated genes. CONCLUSIONS: Chicken ovalbumin upstream promoter-transcription factor II was crucial for colorectal cancer metastasis and regulated cell migration and metastasis in conjunction with Snail1. Chicken ovalbumin upstream promoter-transcription factor II was found to be a biomarker associated with patient survival and colorectal cancer metastasis.

Effects of anthopleurin-Q on the intracellular free Ca2+ concentration in cultured rat cortical neurons.

The present study was designed to investigate the mechanism underlying the intracellular free Ca(2+) concentration ([Ca(2+)]i) modulated by Anthopleurin-Q (AP-Q), a sea anemone toxin, using whole-cell patch clamp and fluorescence digital imaging techniques. Results indicated that the overall Ca(2+) concentration could be augmented in presence of AP-Q. The increase of [Ca(2+)]i induced by AP-Q was eliminated in Na(+)-free solution, Ca(2+)-free solution or in presence of TTX. However, the Ca(2+) increase induced by AP-Q could not be influenced by cyclopiazonic acid (CPA), a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase pump. We furthermore demonstrated that voltage-gated calcium channels (VGCCs) blocker verapamil, or inhibitor of the reverse operation Na(+)-Ca(2+) exchanger NiCl2 attenuated AP-Q-induced [Ca(2+)]i elevation. Furthermore, the inactivation process of Na(+) current (I Na ) was significantly delayed with slightly change of its amplitude by AP-Q. These findings demonstrated that neuron voltage-gated Na(+) channels are also targets of AP-Q. Overall, the present results suggested that AP-Q induced calcium influx via Na(+)-dependent activation of voltage-gated sodium channels (VGSCs), VGCCs and reverse operation of the Na(+)/Ca(2+)exchanger.

miR-499A>G rs3746444 and miR-146aG>C expression and hepatocellular carcinoma risk in the Chinese population.

We conducted a case-control study of a possible association of miR-499A>G rs3746444 and miR-146aG>C rs2910164 with risk of hepatocellular carcinoma. Samples from 172 hepatocellular carcinoma patients and 185 cancer-free controls were collected from October 2008 to December 2011. PCR-RFLP analysis was performed to determine the polymorphisms in each individual. The MAFs of miR-146aG>C and miR-499A>G in controls were similar to that known from the SNP database, and frequencies of genotypes in controls were in line with Hardy-Weinberg equilibrium. We found that miR-499 AG was significantly associated with decreased risk for hepatocellular carcinoma when compared with miR-499 AA genotype (adjusted odds ration = 0.74, 95% confidence interval = 0.24-0.96). However, subjects carrying miR-146a GG had a non-significant 0.62-fold decreased risk of hepatocellular carcinoma. We did not find a significant association of miR-146aG>C rs2910164 and miR-499A>G rs3746444 polymorphisms with hepatocellular carcinoma risk in the Chinese population. Further investigations are warranted to clarify the relationship between miRNA polymorphisms and susceptibility to hepatocellular carcinoma risk in various ethnic populations.

DNA methyltransferases 1 and 3B are required for hepatitis C virus infection in cell culture.

DNA methyltransferases (DNMTs) are responsible for establishing and maintaining DNA methylation, which are dysregulated in hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC). In this report, using lentivirus-mediated shRNA interference technology, we identified DNMT1 and DNMT3B as host factors involved in HCV propagation. Our results demonstrated that down-regulation of DNMT1 or DNMT3B expression in Huh7.5.1 cells severely impaired cell culture-produced HCV (HCVcc) infection. Furthermore, knockdown of DNMT1 or DNMT3B did not affect HCV entry and internal ribosome entry site (IRES)-directed translation but did inhibit subgenomic replication. In contrast, knockdown of DNMT3A had no significant effect on HCV infection, entry, translation, or replication, which suggested that DNMT3A did not play a significant role in HCV life cycle. Moreover, we showed that DNMT inhibitors 5-Aza-C and 5-Aza-dC significantly suppressed HCVcc infection, viral RNA replication, and protein expression. These results suggest that DNMTs are critical for HCV replication and may represent potent targets for the treatment of HCV infection.

Expression of putative stem marker nestin and CD133 in advanced serous ovarian cancer.

It is hypothesized that "cancer stem cells" are responsible for the resistance to chemotherapy of cancer cells in ovarian cancers. The objective of the studies was to explore if the stem cell biomarkers could be used to predict the tumor chemotherapy-resistance in serous ovarian cancer patients. Expression of two putative stem cell markers CD133 and nestin, and vascular epithelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) were detected in 123 cases of advanced serous ovarian cancer specimens by immunohistochemistry. To estimate intra-tumoral microvessel density (MVD), CD34 immunostaining was also performed. CD133 and nestin were defined to be positive in 35.0% and 32.5% of the serous ovarian carcinoma tissues, respectively. It was observed that overexpression of nestin but not CD133 was associated with the cisplatin-based chemotherapy resistance and shorter overall survival of the patients, and nestin was found to be an independent prognostic factor. Moreover, positive nestin expression also correlated to increased expression of EGFR and VEGF, and elevated MVD in tumors. The results of this study suggest that serous ovarian cancers with high expression level of nestin represent an aggressive malignant phenotype associated with poor prognosis, and treatment targeted the nestin positive cancer cells might be a promising therapeutic strategy for this subgroups.

Expression of CD147 in advanced non-small cell lung cancer correlated with cisplatin-based chemotherapy resistance.

CD147, a widely expressed cell surface glycoprotein in cancer, is associated with tumor invasiveness and chemotherapy resistance. Recently, CD147 is also regarded as a potential therapeutic target for cancer therapy. The aim of the study was to investigate CD147 expression in non-small cell lung cancer (NSCLC), and evaluate its correlation with cisplatin-based chemotherapy resistance. In this study, we examined immunohistochemically the expression of CD147 in 118 advanced NSCLC cases treated with cisplatin-based chemotherapy, and then the association of CD147 expression with clinicopathological characteristics was analyzed. Furthermore, RNA interference approach was used to silence CD147 expression in a cisplatin-resistant human lung cancer cell line A549/DDP, and the inhibition effect of cisplatin on tumor cells was assayed by MTT. In the overall series, positive CD147 expression was observed in 101/118 (85.6%) cases. A membranous CD147 pattern was identified in 76/101 (75.2%) of CD147 positive tumors. CD147 membranous expression,but not the overall CD147 expression, was associated with poor response to cisplatin-based chemotherapies and a poor prognosis in advanced NSCLC patients. In vitro results showed that silencing CD147 increased the proliferation inhibitory effect of cisplatin to A549/DDP cells. In conclusion, our study indicated that membranous CD147 expression is a predictive factor of the response to cisplatin-based chemotherapies, and the use of CD147-targeted therapeutic adjuvants might be considered in the treatment of advanced NSCLC patients.

Inflammation in subcutaneous adipose tissue: relationship to adipose cell size.

AIMS/HYPOTHESIS: Inflammation is associated with increased body mass and purportedly with increased size of adipose cells. We sought to determine whether increased size of adipose cells is associated with localised inflammation in weight-stable, moderately obese humans. METHODS: We recruited 49 healthy, moderately obese individuals for quantification of insulin resistance (modified insulin suppression test) and subcutaneous abdominal adipose tissue biopsy. Cell size distribution was analysed with a multisizer device and inflammatory gene expression with real-time PCR. Correlations between inflammatory gene expression and cell size variables, with adjustment for sex and insulin resistance, were calculated. RESULTS: Adipose cells were bimodally distributed, with 47% in a 'large' cell population and the remainder in a 'small' cell population. The median diameter of the large adipose cells was not associated with expression of inflammatory genes. Rather, the fraction of small adipose cells was consistently associated with inflammatory gene expression, independently of sex, insulin resistance and BMI. This association was more pronounced in insulin-resistant than insulin-sensitive individuals. Insulin resistance also independently predicted expression of inflammatory genes. CONCLUSIONS/INTERPRETATION: This study demonstrates that among moderately obese, weight-stable individuals an increased proportion of small adipose cells is associated with inflammation in subcutaneous adipose tissue, whereas size of mature adipose cells is not. The observed association between small adipose cells and inflammation may reflect impaired adipogenesis and/or terminal differentiation. However, it is unclear whether this is a cause or consequence of inflammation. This question and whether small vs large adipose cells contribute differently to inflammation in adipose tissue are topics for future research. TRIAL REGISTRATION: ClinicalTrials.gov NCT00285844.

Nephrogenic systemic fibrosis with a spectrum of clinical and histopathological presentation: a disorder of aberrant dermal remodeling.

BACKGROUND: Nephrogenic fibrosing dermopathy (NFD) has emerged as a clinicopathologic entity since 1997 and recently renamed as nephrogenic systemic fibrosis (NSF). The etiology and pathogenesis remain uncertain. Characteristic clinical presentation is described as diffuse thickening and hardening of the skin occurring in patients with renal insufficiency. Typical histological features include proliferation of CD34 positive fibrocytes, increased thick collagen bundles and mucin deposition, without significant inflammatory infiltrate. Variations in clinical presentations have been reported, including papular and plaque-like skin lesions, focal lesion only, as well as systemic involvement. Histological changes can be subtle and non-specific, overlapping with other disease processes and harboring features including calcification and osteoclast-like giant cells with osseous metaplasia. METHODS: We reviewed patients with NSF that presented to our dermatology clinic by chart review, clinical examination and histological examination. Skin biopsy specimens were obtained from all cases. Histopathology evaluations were carried out by three dermatopathologists (AD, BS and GK) independently and the features were compared among all the cases. Special stains and immunohistochemistry study were also performed to highlight the histological features. RESULTS: Seven cases of NSF presented with a spectrum of clinical manifestations, from classic diffuse hardening of the skin to localized linear plaques. On histological examination, proliferation of CD34-positive fibrocytes ranged from sparse to dense, collagen bundles ranged from thin to thick, and the interstitial dermal mucin accumulation ranged from scant-patchy to abundant. In addition, the lesion displayed various degrees of vascular proliferation, inflammatory infiltrates and intensities of CD68 and Factor XIIIa staining. Two cases showed extensive dermal calcification and ossification. CONCLUSION: NSF may present with a spectrum of clinical abnormalities, and exhibit overlapping histopathological features resembling cicatrix and other dermal reparative/regenerative processes. NSF may in fact to be a disorder of aberrant extracellular matrix remodeling in patients with renal insufficiency.

Naevus comedonicus: a spectrum of body involvement.

Naevus comedonicus (NC) is a rare developmental anomaly, with < 200 cases reported in the literature. It usually occurs on the face, neck or chest, appearing as groups of closely arranged dilated follicular openings with keratin plugs. Several associations have been made in the literature. We review the current literature, emphasizing the clinical features, associated conditions and therapeutic options.