RITA selectively inhibits proliferation of BAP1-deficient cutaneous melanoma cells in vitro / 南方医科大学学报

OBJECTIVE@#To screen for small molecular compounds with selective inhibitory activity against cutaneous melanoma cells with BAP1 deletion.@*METHODS@#Cutaneous melanoma cells expressing wild-type BAP1 were selected to construct a BAP1 knockout cell model using CRISPR-Cas9 system, and small molecules with selective inhibitory activity against BAP1 knockout cells were screened from a compound library using MTT assay. Rescue experiment was carried out to determine whether the sensitivity of BAP1 knockout cells to the candidate compounds was directly related to BAP1 deletion. The effects of the candidate compounds on cell cycle and apoptosis were detected with flow cytometry, and the protein expressions in the cells were analyzed with Western blotting.@*RESULTS@#The p53 activator RITA from the compound library was shown to selectively inhibit the viability of BAP1 knockout cells. Overexpression of wild-type BAP1 reversed the sensitivity of BAP1 knockout cells to RITA, while overexpression of the mutant BAP1 (C91S) with inactivated ubiquitinase did not produce any rescue effect. Compared with the control cells expressing wild-type BAP1, BAP1 knockout cells were more sensitive to RITA-induced cell cycle arrest and apoptosis (P < 0.0001) and showed an increased expression of p53 protein, which was further increased by RITA treatment (P < 0.0001).@*CONCLUSION@#Loss of BAP1 results in the sensitivity of cutaneous melanoma cells to p53 activator RITA. In melanoma cells, the activity of ubiquitinase in BAP1 is directly related to their sensitivity to RITA. An increased expression of p53 protein induced by BAP1 knockout is probably a key reason for RITA sensitivity of melanoma cells, suggesting the potential of RITA as a targeted therapeutic agent for cutaneous melanoma carrying BAP1-inactivating mutations.

Doublecortin-like kinase 1 activates Hippo pathway to promote migration, invasion and proliferation of pancreatic cancer cells / 中华肿瘤杂志

Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.

Differential Expression of Long Non-coding RNA Cancer Susceptibility Candidate 2 and Imprinted Gene H19 in Extrahepatic Cholangiocarcinoma / 中国医学科学院学报

Objective To investigate the expression and the potential roles of long non-coding RNA(lncRNA)cancer susceptibility candidate 2(CASC2)and imprinted gene H19 in extrahepatic cholangiocarcinoma(ECC). Methods Four samples from patients with ECC were collected for high-throughput sequencing which was conducted to reveal the transcriptomic profiles of lncRNA CASC2 and H19.Bioinformatics tools were employed to predict the potential roles of the two genes.Another 22 ECC tissue samples and the cholangiocarcinoma cell lines(RBE,QBC939,HuH-28,and HuCCT1)with different degrees of differentiation were selected for validation.The para-carcinoma tissue and normal human intrahepatic biliary epithelial cell(HIBEC)were used as the control groups.The expression levels of lncRNA CASC2 and H19 in carcinoma tissue,para-carcinoma tissue,and cell lines were determined by real-time quantitative polymerase chain reaction(qRT-PCR).The correlation analysis was carried out for the clinical indicators of patients with the expression levels of the target genes. Results The two target genes showed significantly different expression between carcinoma tissue and para-carcinoma tissue(all P<0.05).Specifically,CASC2 had higher expression level in the carcinoma tissue than in the para-carcinoma tissue(t=1.262,P=0.025),whereas the expression of H19 showed an opposite trend(t=1.285,P=0.005).The expression levels of CASC2 in QBC939(t=8.114,P=0.015)and HuH-28(t=9.202,P=0.012)cells were significantly higher than that in the control group.The expression levels of H19 were significantly lower in RBE(t=-10.244,P<0.001),QBC939(t=-10.476,P<0.001),HuH-28(t=-19.798,P<0.001),and HuCCT1(t=-16.193,P=0.004)cells than in the control group.Bioinformatics analysis showed that CASC2 was mainly involved in the metabolic process and H19 in the development of multicellular organisms.Both CASC2 and H19 were related to catalytic activity.The expression level of lncRNA CASC2 was correlated with pathological differentiation(χ 2=6.222,P=0.022)and lymph node metastasis(χ2=5.455,P=0.020),and that of lncRNA H19 with pathological differentiation(χ2=1.174,P=0.029)and tumor size(χ2=-0.507,P=0.037). Conclusions In the case of ECC,lncRNA CASC2 and H19 have transcription disorders.lncRNA CASC2 is generally up-regulated in the carcinoma tissue,while H19 is down-regulated.Both genes have the potential to become new molecular markers for ECC.

Effect of crocin on PKHD1 and KLLN genes expression in kidney tissue of male rats treated with cadmium / Efecto de la crocina sobre la expresión de los genes PKHD1 y KLLN en tejido renal de ratas macho tratadas con cadmio

According to data from studies, antioxidant herbal compounds are, likely to have a useful role in reducing the harmful effects of environmental pollutants and toxic chemicals that most people are exposed to. Cadmium is one of the toxic elements that accumulate in many organs, especially in kidneys. The aim of this study was to investigate the effect of crocin on the expression of PKHD1 and KLLN genes in cadmium-treated rats.In this experimental study, 40 adults male Wistar rats (200-250 g) were randomly divided into the following groups: control group received normal saline, cadmium group (15mg/kg), crocin group (20mg/kg) and cadmium group daily fed with crocin at a dose of 20 mg/kg.After eight weeks of treatment, rats were dissected, and kidney tissues were removed for evaluation of PKHD1 and KLLN gene expression by real time method. The data were analyzed using one-way ANOVA and significant difference between groups was P<0.05.Our results showed an increase in PKHD1 gene expression and a decrease in KLLN gene expression in kidney tissue in the cadmium group compared to the control group (P <0.001).Also, a significant decrease in PKHD1 gene expression (P <0.001) and an increase in KLLN gene expression P <0.05) were observed in the tissues of all cadmium-treated rats compared to cadmium.Crocin consumption can have a protective effect against the impaired expression of PKHD1 and KLLN cadmium-induced apoptotic pathway.

Diversos estudios sugieren que compuestos antioxidantes de hierbas tienen un papel útil en la reducción de los efectos nocivos de los contaminantes ambientales y los químicos tóxicos a los que está expuesta la mayoría de las personas. El cadmio es uno de los elementos tóxicos que se acumulan en muchos órganos, especialmente en los riñones. El objetivo de este estudio fue investigar el efecto de la crocina en la expresión de los genes PKHD1 y KLLN en ratas tratadas con cadmio.En este estudio experimental, 40 ratas Wistar macho adultas (200-250 g) se dividieron aleatoriamente en los siguientes grupos: el grupo de control recibió solución salina normal, el grupo de cadmio (15 mg / kg), el grupo de crocina (20 mg / kg) y el grupo de cadmio alimentado diariamente con crocina a una dosis de 20 mg / kg.Después de ocho semanas de tratamiento, se disecaron las ratas y se extrajeron los tejidos renales para evaluar la expresión de los genes PKHD1 y KLLN mediante un método en tiempo real. Los datos se analizaron mediante ANOVA de una vía y la diferencia significativa entre los grupos fue P <0,05.Nuestros resultados mostraron un aumento en la expresión del gen PKHD1 y una disminución en la expresión del gen KLLN en el tejido renal en el grupo de cadmio en comparación con el grupo de control (P <0,001).Además, se observó una disminución significativa en la expresión del gen PKHD1 (P <0,001) y un aumento en la expresión del gen KLLN P <0,05) en los tejidos de todas las ratas tratadas con cadmio en comparación con el cadmio.El consumo de crocina puede tener un efecto protector contra la expresión alterada de la vía apoptótica inducida por cadmio PKHD1 y KLLN.

The Molecular Mechanism of LncRNA-CASC2/miR-155-5p/APC Axis Regulating the Progression of Non-Hodgkin Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of lncRNA-CASC2 (CASC2) /miR-155-5p/APC axis to the progression of non-Hodgikn lymphoma (NHL).@*METHODS@#The expression level of CASC2 and miR-155-5p in NHL cell lines were examined by qRT-PCR. Dual-luciferase reporter gene assay was used to verify the relationship between miR-155-5p, CASC2 and APC. The effects of CASC/miR-155-5p/APC axis to the proliferation, invasion and apoptosis of NK-92 cells were detected by MTT, Transwell assay and flow cytometry assay, respectively.@*RESULTS@#CASC2 was downregulated in NHL cell lines. Overexpression of CASC2 could inhibit the proliferation and invasion of NK-92 cells, and promote its apoptosis. Dual-luciferase reporter gene assay confirmed that there was a targeting relationship between miR-155-5p, CASC2 and APC. The restoration experiments proved that knockdown of both miR-155-5p and CASC2 or APC could restore the inhibitory effect of miR-155-5p silencing to the biological behavior of NK-92 cells.@*CONCLUSION@#Overexpression of CASC2 suppresses the proliferation and invasion of NK-92 cells, promote the apoptosis of NK-92 cells via targeting miR-155-5p and upregulating APC expression.

MicroRNA-663 facilitates the growth, migration and invasion of ovarian cancer cell by inhibiting TUSC2

BACKGROUND: MicroRNAs (miRNAs) have emerged as the critical modulators of the tumorigenesis and tumor progression. METHODS: The levels of miR-663 in ovarian cancer cell lines and clinical tissues were detected using qRT-PCR assays. The Transwell invasion and wound healing assay were conducted to assess the roles of miR-663 in the migration and invasion of ovarian cancer cell in vitro. Rescue assays were carried out to confirm the contribution of tumor suppressor candidate 2 (TUSC2) in the aggressiveness of cancer cell which was regulated by miR-663. RESULTS: The levels of miR-663 were up-regulated in ovarian cancer tissues in comparison with the corresponding normal tissues. Up-regulation of miR-663 increased the proliferation, colony formation, migration and invasion of ovarian cancer SKOV3 cell. Additional, over-expression of miR-663 increased the tumor growth of SKOV3 in xenograft model. Bioinformatics analysis and luciferase reporter assay identified that miR-663 decreased the level of TUSC2 via binding to the 3'-UTR of TUSC2 gene. Finally, the expression of TUSC2 was inversely associated with the level of miR-663 in ovarian carcinoma tissue and over-expression of TUSC2 inhibited the migration and invasion abilities of SKOV3 that was promoted by miR-663. CONCLUSION: Altogether, these results indicate that miR-663 acts as a potential tumor-promoting miRNA through targeting TUSC2 in ovarian cancer.

Novel tumor metastasis suppressorgene LASS2/TMSG1 S248A mutant promotes invasion of prostate cancer cells through increasing ATP6V0C expression / 北京大学学报(医学版)

OBJECTIVE@#LASS2/TMSG1 gene is a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by Department of Pathology,Peking University of Basic Medical Sciences. It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of vacuolar-ATPase (ATP6V0C). In this study, we explored the effect of LASS2/TMSG1 and its mutants on proliferation, migration and invasion of human prostate cancer cells and its molecular mechanism.@*METHODS@#We constructed four LASS2/TMSG1 mutants and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential. The stable transfectants were identified by qPCR and Western blot through analyzing the expression of LASS2/TMSG1 and ATP6V0C, the cell biology functions of LASS2/TMSG1 and its four mutants were studied using growth curve,MTT assay, soft agar colony formation assay, wound migration assay, Matrigel invasion study and flow cytometry. Furthermore, immunofluorescence was used to analysis the interaction of LASS2/ TMSG1 mutants and ATP6V0C.@*RESULTS@#LASS2/TMSG1 mRNA and protein in LASS2/TMSG1 group and Mut1-Mut4 groups were higher than that in Vector group; Western blot showed that ATP6V0C protein in LASS2/TMSG1 wild group was lower than that in Vector group, but ATP6V0C protein in LASS2/TMSG1 S248A group was obviously higher than that in Vector group. MTT test and growth curve assay showed growth ability in LASS2/TMSG1 S248A group was increasing compared with other groups from day 5. Soft Agar colony formation experiment showed anchor independent growth ability in LASS2/TMSG1 S248A group was higher than those in the other groups (P<0.05), Cell migrations (from 35.3%±3.2% to 70.3%±3%) in LASS2/TMSG1 S248A group was increasing compared with LASS2/TMSG1 wild group (P<0.01), and more cells passed through Matrigel in LASS2/TMSG1 S248A group compared with LASS2/TMSG1 wild group (from 50±3.2 to 203±6.5, P<0.01), the apoptosis rate in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 7% to 15.1%, P<0.05), and the G0/G1 ratio in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 51.0% to 85.4%). Furthermore, double immunofluorescent staining observed the colocalization between ATP6V0C and LASS2/TMSG1 protein and its mutations, the expression of ATP6V0C in LASS2/TMSG1 S248A group increased significantly compared with the other groups.@*CONCLUSION@#LASS2/TMSG1 S248A promotes proliferation, migration and invasion of prostate cancer cells through increasing ATP6V0C expression, suggesting that aa248-250 is an important function site for LASS2/TMSG1 in invasion suppression of prostate cancer cells.

Association of Ugrp2 gene polymorphisms with adenoid hypertrophy in the pediatric population / Associação de polimorfismos do gene Ugrp2 com hipertrofia de adenoide na população pediátrica

Abstract Introduction: Adenoid hypertrophy is a condition that presents itself as the chronic enlargement of adenoid tissues; it is frequently observed in the pediatric population. The Ugrp2 gene, a member of the secretoglobin superfamily, encodes a low-molecular weight protein that functions in the differentiation of upper airway epithelial cells. However, little is known about the association of Ugrp2 genetic variations with adenoid hypertrophy. Objective: The aim of this study is to investigate the association of single nucleotide polymorphisms in the Ugrp2 gene with adenoid hypertrophy and its related phenotypes. Methods: A total of 219 children, comprising 114 patients suffering from adenoid hypertrophy and 105 healthy patients without adenoid hypertrophy, were enrolled in this study. Genotypes of the Ugrp2 gene were determined by DNA sequencing. Results: We identified four single nucleotide polymorphisms (IVS1-189G>A, IVS1-89T>G, c.201delC, and IVS2-15G>A) in the Ugrp2 gene. Our genotype analysis showed that the Ugrp2 (IVS1-89T>G) TG and (c.201delC) CdelC genotypes and their minor alleles were associated with a considerable increase in the risk of adenoid hypertrophy compared with the controls (p = 0.012, p = 0.009, p = 0.013, and p = 0.037, respectively). Furthermore, Ugrp2 (GTdelCG, GTdelCA) haplotypes were significantly associated with adenoid hypertrophy (four single nucleotide polymorphisms ordered from 5′ to 3′; p = 0.0001). Polymorfism-Polymorfism interaction analysis indicated a strong interaction between combined genotypes of the Ugrp2 gene contributing to adenoid hypertrophy, as well as an increased chance of its diagnosis (p < 0.0001). In addition, diplotypes carrying the mutant Ugrp2 (c.201delC) allele were strongly associated with an increased risk of adenoid hypertrophy with asthma and with allergies (p = 0.003 and p = 0.0007, respectively). Conclusion: Some single nucleotide polymorphisms and their combinations in the Ugrp2 gene are associated with an increased risk of developing adenoid hypertrophy. Therefore, we tried to underline the importance of genetic factors associated with adenoid hypertrophy and its related clinical phenotypes.

Resumo Introdução: A adenoide ou hipertrofia de tonsila faríngea é uma condição que se apresenta como o aumento crônico de tecidos linfoides na rinofaringe e é frequentemente observada na população pediátrica. O gene Ugrp2, um membro da superfamília da secretoglobina, codifica uma proteína de baixo peso molecular que funciona na diferenciação das células epiteliais das vias aéreas superiores. No entanto, pouco se sabe sobre a associação de variações genéticas do Ugrp2 com hipertrofia de tonsila faríngea. Objetivo: Investigar a associação de polimorfismos de nucleotídeos únicos no gene Ugrp2 com hipertrofia de tonsila faríngea e seus fenótipos relacionados. Método: Foram incluídos no estudo 219 crianças, 114 pacientes com hipertrofia de tonsila faríngea e 105 saudáveis. Os genótipos do gene Ugrp2 foram determinados por sequenciamento de DNA. Resultados: Identificamos quatro polimorfismos de nucleotídeo único (IVS1-189G>A, IVS1-89T>G, c.201delC, e IVS2-15G>A) no gene Ugrp2. Nossa análise genotípica mostrou que os genótipos Ugrp2 (IVS1-89T>G) TG e (c.201delC) CdelC e seus alelos menores foram associados a um aumento considerável no risco de HA em comparação com os controles (p = 0,012, p = 0,009, p = 0,013 e p = 0,037, respectivamente). Além disso, os haplótipos Ugrp2 (GTdelCG, GTdelCA) foram significativamente associados com hipertrofia de tonsila faríngea (quatro polimorfismos de nucleot' ordenados de 5' a 3'; p = 0,0001). A análise de interação polimorfismo-polimorfismo indicou uma forte interação entre genótipos combinados do gene Ugrp2 que contribuiu para hipertrofia de tonsila faríngea, bem como uma chance maior de seu diagnóstico (p < 0,0001). Além disso, os diplótipos que transportam o alelo mutante Ugrp2 (c.201delC) foram fortemente associados a um risco aumentado de hipertrofia de tonsila faríngea com asma e com alergias (p = 0,003 e p = 0,0007, respectivamente). Conclusão: Alguns polimorfismos de nucleotídeo único e suas combinações no gene Ugrp2 estão associados a um risco aumentado de desenvolver hipertrofia de tonsila faríngea. Portanto, tentamos enfatizar a importância dos fatores genéticos e fenótipos clínicos associados a essa hipertrofia.

Immunohistochemical WWOX expression and association with angiogenesis, p53 expression, cell proliferation and clinicopathological parameters in cervical cancer / Avaliação da expressão do gene WWOX por avaliação imunohistoquímica, sua associação com marcador de angiogênese, expressão do p53, proliferação celular e parâmetros clinicopatológicos no câncer de colo uterino

Abstract Objective The current study evaluated the expression of WW domain-containing oxidoreductase (WWOX), its association with clinicopathological features and with p53, Ki-67 (cell proliferation) and CD31 (angiogenesis) expression in patients with invasive cervical squamous cell carcinoma (ICSCC). To the best of our knowledge, no other study has evaluated this association. Methods Women with IB stage-ICSCC (n = 20) and women with uterine leiomyoma (n = 20) were prospectively evaluated. Patients with ICSCC were submitted to type BC1 radical hysterectomy and pelvic lymphadenectomy. Patients in the control group underwent vaginal hysterectomy. Tissue samples were stained with hematoxylin and eosin for histological evaluation and protein expression was detected by immunohistochemistry studies. Results The WWOX expression was significantly lower in the tumor compared with the expression in thebenign cervix (p = 0.019). TheWWOXexpressionwas inversely associated with the CD31 expression in the tumor samples (p = 0.018). There was no association betweentheWWOXexpression with the p53 expression (p = 0.464)or the Ki-67expression (p = 0.360) in the samples of invasive carcinoma of the cervix. There was no association between the WWOX expression and tumor size (p = 0.156), grade of differentiation (p = 0.914), presence of lymphatic vascular invasion (p = 0.155), parametrium involvement (p = 0.421) or pelvic lymph node metastasis (p = 0.310) in ICSCC tissue samples. Conclusion The results suggested that WWOX may be involved in ICSCC carcinogenesis, and this marker was associated with tumor angiogenesis.

Resumo Objetivo O presente estudo avaliou a expressão do WWOX, sua associação com características clinicopatológicas e com a expressão do p53, ki-67 (proliferação celular) e CD31 (angiogênese) em pacientes com carcinoma invasivo de células escamosas do colo uterino, ou simplesmente câncer do colo uterino (CCE). Métodos Foram avaliadas prospectivamente pacientes com CCE no estágio IB (n = 20) e mulheres com mioma uterino, no grupo controle (n = 20). As pacientes com CCE foram submetidas à histerectomia radical e à linfadenectomia pélvica do tipo B-C1. As mulheres no grupo-controle foram submetidas à histerectomia vaginal. As amostras de tecido foramcoradas comhematoxilina e eosina para avaliação histológica e a expressão das proteínas foi detectada por imuno-histoquímico. Resultados A expressão do WWOX foi significativamente menor no tumor quando comparada com sua expressão no colo do útero benigno (p = 0,019). A expressão tumoral de CD31 foi inversamente associada à expressão de WWOX (p = 0,018). Sua expressão não foi associada à expressão tumoral de p53 e Ki-67 em pacientes com CCE (p = 0,464 e p = 0,360, respectivamente). Não houve associação entre a expressão de WWOX e o tamanho do tumor (p = 0,156), grau de diferenciação (p = 0,914), presença de invasão vascular linfática (p = 0,155), comprometimento do paramétrio (p = 0,421) ou metástase dos linfonodos pélvicos (p = 0,310) em pacientes com CCE. Conclusão Os resultados sugeriram que o WWOX pode estar envolvido na carcinogênese do CICECU e esse marcador foi associado à angiogênese tumoral.

Nueva mutación del gen TSC2 en un paciente pediátrico con diagnóstico clínico de esclerosis tuberosa / Novel mutation in TSC2 gene in pediatric patient with clinical diagnosis of tuberous sclerosis

El complejo de esclerosis tuberosa es un desorden neurocutáneo autosómico dominante causado por mutaciones en los genes TSC1 o TSC2. El diagnóstico se basa en criterios clínicos o el criterio genético. La presentación clínica es altamente variable y las manifestaciones de la enfermedad pueden desarrollarse durante toda la vida. Se reporta el caso de un niño que cumple criterios clínicos para el diagnóstico de esclerosis tuberosa y cuyo estudio molecular identificó una variante nueva del gen TSC2. Se trata de una mutación sin sentido, esporádica, no reportada previamente (c.583_586dupATCG) localizada en el exón 6, que provoca un codón de parada temprano y altera la estructura de la proteína. Puede considerarse una variante patogénica por el tipo de mutación y permite ampliar el espectro de variantes del gen TSC2 como causa del complejo de esclerosis tuberosa.

Tuberous sclerosis complex (TSC) is a neurocutaneous autosomal dominant disorder that results from mutations within either the TSC1 gene or the TSC2 gene. Diagnosis is based on well-established clinical criteria or genetic criteria. Clinical features are highly variable and could be developing over the life. We present a case of TSC with a molecular test that identified a novel variant in TSC2 gene. It is a sporadic missense mutation which has not been previously reported in the literature. It is caused by premature termination of protein translation and results in the production of truncated and non-functional proteins. This mutation is considered as a pathogenic variant and allows to broaden the spectrum of variants of TSC2 gene as a cause of TSC.

Estudio clínico genético en pacientes con complejo de esclerosis tuberosa / Clinical and genetic study patients with tuberous sclerosis complex

El complejo de esclerosis tuberosa (CET) es una enfermedad autosómica dominante multisistémica producida por mutaciones en los genes supresores de tumores TSC1 o TSC2. Objetivo: Caracterizar clínica y genéticamente pacientes pediátricos con diagnóstico de CET. Pacientes y Método: Estudio descriptivo de registros clínicos de 42 pacientes pediátricos controlados en un servicio de neuropsiquiatría infantil con diagnóstico de CET y estudio genético en 21 de ellos. Se amplificó por reacción en cadena de la polimerasa y secuenció el exón 15 del gen TSC1 y los exones 33, 36 y 37 del gen TSC2. Se analizó la relación entre las mutaciones encontradas con la severidad y evolución clínica. Resultados: En el 61,9% de los pacientes las manifestaciones comenzaron antes de los 6 meses de edad. Las manifestaciones iniciales de CET más frecuentes fueron las crisis convulsivas (73,8%) y el hallazgo de rabdomiomas cardiacos (16,6%). Durante su evolución, todos los pacientes presentaron compromiso neurológico; el 92,9% presentó epilepsia. Todos los pacientes presentaron máculas hipomelanóticas, 47,6% pangiofibromas faciales, 23,8% parches de Shagreen, 47,6% rabdomiomas cardiacos y 35,7% hamartomas retinianos. El estudio genético realizado a 21 pacientes identificó 2 mutaciones heterocigotas patogénicas en TSC1 y una en TSC2. Este último paciente presentaba un fenotipo clínico más severo. Conclusiones: Las manifestaciones neurológicas y dermatológicas fueron las más frecuentes en los pacientes con CET. Se identificaron 2 mutaciones patogénicas en el gen TSC1 y una en el gen TSC2. La mutación en TSC2 se manifestó en un fenotipo clínico más severo.

Tuberous sclerosis complex (TSC) is a multisystem autosomal dominant disease caused by mutations in the tumor suppressor genes TSC1 or TSC2. Objective: To characterize clinically and genetically patients diagnosed with TSC. Patients and Method: Descriptive study of clinical records of 42 patients from a pediatric neuropsychiatry department diagnosed with TSC and genetic study in 21 of them. The exon 15 of TSC1 gene and exons 33, 36 and 37 of TSC2 gene were amplified by polymerase chain reaction and sequenced. The relationship between the mutations found with the severity and clinical course were analyzed. Results: In 61.9% of the patients the symptoms began before 6 months of age. The initial most frequent manifestations of TSC were new onset of seizures (73.8%) and the detection of cardiac rhabdomyomas (16.6%). During the evolution of the disease all patients had neurological involvement; 92.9% had epilepsy. All patients presented hypomelanotic spots, 47.6% facial angiofibromas, 23.8% Shagreen patch, 47.6 heart rhabdomyomas and 35.7% retinal hamartomas. In the genetic study of 21 patients two heterozygous pathogenic mutations in TSC1 and one in TSC2 genes were identified. The latter had a more severe clinical phenotype. Conclusions: Neurological and dermatological manifestations were the most frequent ones in patients with TSC. Two pathogenic mutations in TSC1 and one in TSC2 genes were identified. The patient with TSC2 mutation manifested a more severe clinical phenotype.

Limites e possibilidades do ensino à distância (EaD) na educação permanente em saúde: revisão integrativa / Limits and possibilities of distance learning in continuing education in health: integrative review

Trata-se de um estudo sobre o uso do ensino a distância (EaD) como uma estratégia de ensino na educação permanente em saúde (EPS), que teve como objetivo identificar e analisar os limites e possibilidades do uso da EaD na EPS. Estudo de revisão integrativa. O resultado aponta que a EaD é uma estratégia inovadora possível e potencial para a EPS, facilitando o desenvolvimento da aprendizagem dentro ou fora da instituição de saúde, porém é evidente a escassez de pesquisas na área. As limitações para a realização dos programas estão relacionadas à variável tempo, preparação para lidar com as tecnologias e importância do tutor como facilitador da aprendizagem. Conclui-se que o uso da EaD tem tido uma importante contribuição para o desenvolvimento dos recursos humanos em saúde, seja no processo de formação e/ou no processo contínuo de conhecimento.

This is a study on the use of distance learning (EaD, in Portuguese) as a teaching strategy in continuing health education (EPS, in Portuguese), which aimed to identify and analyze the limits and posibilities of using EaD in the EPS. Integrative Review Study. The result shows that EaD is an innovative, possible and potential strategy for EPS, facilitating the development of learning within or outside the health institution, although is evident the lack of research in the area. The limitations for the implementation of the programs are related to the time variable, preparation for dealing with the technologies and the importance of the tutor as a facilitator of learning. It concludes that the use of EaD has an important contribution to the development of human resources in health, is in the process of training and/or in the continuous knowledge process.

Bap1 y neoplasias melanocíticas / Bap1 and melanocytic neoplasms

El melanoma maligno cutáneo (MMC) es un cáncer genéticamente heterogéneo, en cuya patogénesis participarían varios genes. Algunos de estos activan la vía MAP kinasa (BRAF, NRAS, KIT, NF1), mientras que otros confieren una mayor susceptibilidad a melanoma familiar, como CDKN2A, CDK4, MITF y BAP1. BAP1 (BRCA1-associated-protein 1) ha sido descrito como una proteína que se une a BRCA1 para inhibir el crecimiento celular. Actualmente se sabe que es producto de un gen supresor de tumores (denominado BAP1) y que actúa como una enzima con actividad deubiquitinasa, la cual se asocia a varios complejos de proteínas, regulando diversas vías celulares relacionadas con el ciclo celular, diferenciación y muerte celular, así como también gluconeogénesis y respuesta a daño del ADN. Tanto su actividad deubiquitinasa como su localización nuclear son relevantes para su función en la supresión de tumores.

Malignant cutaneous melanoma (MMC) is a genetically heterogeneous cancer and various genes participate in its pathogenesis. Some of these genes activate the MAP kinase pathway (BRAF, NRAS, KIT, NF1) and others are related to a higher susceptibility to familial melanoma like CDKN2A, CDK4, MITF y BAP1. BAP1 (BRCA1-associated –protein 1) has been described as a BRCA1-binding protein inhibiting cell growth. This protein is a product of a gene with tumor suppressor activity, the protein being a deubiquitinase associated to multiple protein complexes regulating various cellular pathways, including the cell cycle, differentiation and cell death, as well as gluconeogenesis and DNA damage response. Both deubiquitinase activity and location to the nucleus are relevant to its tumor suppressor function.

miR-205 promotes proliferation and invasion of laryngeal squamous cell carcinoma by suppressing CDK2AP1 expression

BACKGROUND: The aberrant expression of microRNAs (miRNAs) has been found in various types of cancer. miR-205 was reported to be upregulated in laryngeal squamous cell carcinoma (LSCC) tissues, however, the mechanisms by which miR-205 functions as a regulator of LSCC are largely unknown. RESULTS: In this study, Real-time qPCR and Western blot assay showed that expression of miR-205 was upregulated and expression of cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) was downregulated in LSCC tissues. The expression levels of miR-205 were negatively related to those of CDK2AP1 in LSCC tissues and cell lines. Moreover, we found that miR-205 was the upstream regulator of CDK2AP1 and could suppress the CDK2AP1 expression in LSCC cells. 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide assays and transwell invasion assay were performed to test the proliferation and invasion of LSCC cells. Gelatin zymography was used to detect the activity of MMP2 and MMP9. CDK2AP1, c-Myc and CyclinD1 expression in cells was assessed with Western blotting. We found that miR-205 was the upstream regulator of CDK2AP1 and could suppress the expression of CDK2AP1 in LSCC cells. In addition, miR-205 significantly induced cell proliferation and invasion by suppressing CDK2AP1 expression. Consistent with miR-205 inhibitors, overexpressed CDK2AP1 suppressed the activity of MMP2 and MMP9 and c-Myc and CyclinD1 expression in LSCC cells. CONCLUSION: These findings help us to better elucidate the molecular mechanisms of LSCC progression and provide a new theoretical basis to further investigate miR-205 as a potential biomarker and a promising approach for LSCC treatment.

Association between Promoter Polymorphisms of TFF1, TFF2, and TFF3 and the Risk of Gastric and Diffuse Gastric Cancers in a Korean Population

Gastric cancer is one of the most common cancers in the world. The aims of this study were to evaluate the association between polymorphisms in TFF gene family, TFF1, TFF2, and TFF3 and the risk of gastric cancer (GC) and GC subgroups in a Korean population via a case-control study. The eight polymorphisms in TFF gene family were identified by sequencing and genotyped with 377 GC patients and 396 controls by using TaqMan genotyping assay. The rs184432 TT genotype of TFF1 was significantly associated with a reduced risk of GC (odds ratio, [OR) = 0.45; 95% confidence interval, [CI] = 0.25-0.82; P = 0.009), more protective against diffuse-type GC (OR = 0.20; 95% CI = 0.05-0.89; P = 0.035) than GC (OR = 0.34; 95% CI = 0.14-0.82; P = 0.017) in subjects aged < 60 yr, and correlated with lymph node metastasis negative GC and diffuse-type GC (OR = 0.44; 95% CI = 0.23-0.86; P = 0.016 and OR = 0.20; 95% CI = 0.05-0.87; P = 0.031, respectively). In addition, a decreased risk of lymph node metastasis negative GC and diffuse-type GC was observed for rs225359 TT genotype of TFF1 (OR = 0.46, 95% CI = 0.24-0.88; P = 0.020 and OR = 0.21, 95% CI = 0.05-0.88; P = 0.033, respectively). These findings suggest that the rs184432 and rs225359 polymorphisms in TFF1 have protective effects for GC and contribute to the development of GC in Korean individuals.

Histone Deacetylases and Their Regulatory MicroRNAs in Hepatocarcinogenesis

A growing body of evidence suggests that epigenetic modifications are promising potential mechanisms in cancer research. Among the molecules that mediate epigenetic mechanisms, histone deacetylases (HDACs) are critical regulators of gene expression that promote formation of heterochromatin by deacetylating histone and non-histone proteins. Aberrant regulation of HDACs contributes to malignant transformation and progression in a wide variety of human cancers, including hepatocellular carcinoma (HCC), gastric cancer, lung cancer, and other cancers. Thus, the roles of HDACs have been extensively studied because of their potential as therapeutic targets. However, the underlying mechanism leading to deregulation of individual HDACs remains largely unknown. Some reports have suggested that functional microRNAs (miRNAs) modulate epigenetic effector molecules including HDACs. Here, we describe the oncogenic or tumor suppressive functions of HDAC families and their regulatory miRNAs governing HDAC expression in hepatocarcinogenesis.

Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer.

Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O6-methyguanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O6-position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC) tissue samples, 14 low malignant potential (LMP) tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP) after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression.

Effect of a Surfactant on the Antimicrobial Activity of Sodium Hypochlorite Solutions

The objective of the present study was to evaluate the antimicrobial activity of sodium hypochlorite (NaOCl) associated with a surfactant. Seventy single-rooted extracted human teeth were inoculated with Enterococcus faecalis, and incubated for 21 days (37 °C). The groups were distributed according to the irrigation solution used during root canal preparation: 5%, 2.5% and 1% NaOCl; 5%, 2.5% and 1% Hypoclean(r), a solution containing a surfactant (cetrimide) associated with NaOCl. Three microbiological samples were collected from each tooth: S1 - before instrumentation; S2 - immediately after instrumentation; and S3 - after a seven-day period. Data were submitted to ANOVA and Tukey test with 5% significance level. The results showed that immediately after root canal preparation (S2), E. faecalis was eliminated in all the experimental groups. However, after 7 days (S3), only the groups in which Hypoclean was used, remained contamination-free, including Hypoclean associated with 1% NaOCl, while the root canals irrigated with 1% NaOCl only, presented the highest percentage of bacterial growth. In conclusion, the addition of surfactant increased the antimicrobial activity of 1% NaOCl to levels similar to 5% NaOCl.

O objetivo da presente pesquisa foi avaliar a atividade antimicrobiana de hipoclorito de sódio (NaOCl), associado a um tensoativo. Setenta dentes humanos monorradiculares extraídos foram inoculados com Enterococcus faecalis e incubados durante 21 dias (37 °C). Os grupos foram distribuídos de acordo com a solução irrigadora utilizada no preparo do canal: hipoclorito de sódio a 5%, 2,5% e 1%; Hypoclean(r) a 5%, 2,5% e 1% - uma solução contendo um surfactante (cetrimida) associado com NaOCl. Três amostras microbiológicas foram coletadas de cada dente: S1 - antes de instrumentação; S2 - imediatamente após a instrumentação; e S3 - após um período de sete dias. Os dados foram submetidos à análise de variância e teste de Tukey com 5% de nível de significância. Os resultados mostraram que imediatamente após o preparo do canal radicular (S2), o E. faecalis foi eliminado em todos os grupos experimentais. No entanto, após 7 dias (S3), apenas os grupos em que se utilizou Hypoclean permaneceram livres de contaminação, incluindo Hypoclean 1%, enquanto que os canais radiculares irrigados apenas com hipoclorito de sódio 1% apresentaram a mais elevada percentagem de crescimento bacteriano. Em conclusão, a adição de surfactante aumentou a atividade antimicrobiana de 1% de NaOCl a níveis semelhantes aos do NaOCl 5% .

Birt-Hogg-Dubé syndrome / Sindrome de Birt-Hogg-Dube

A 45-year-old woman with a history of renal carcinoma was observed for facial, cervical and truncal flesh-colored papules. Relatives had similar skin findings and a brother had repeated episodes of pneumothorax. The computerized tomography scan revealed multiple cysts on both lungs. A skin biopsy revealed a perifollicular fibroma. The clinical diagnosis of Birt-Hogg-Dubé syndrome (BHDS) was corroborated by identification of a novel frameshift c.573delGAinsT (p.G191fsX31) mutation in heterozygosity on exon 6 of the folliculin gene. The presence of multiple and typical benign hair follicle tumors highlights the role of the dermatologist in the diagnosis of this rare genodermatosis that is associated with an increased risk of renal cell cancer and pulmonary cysts, warranting personal and familial follow-up and counseling.

Uma mulher de 45 anos com história de carcinoma renal foi observada por pápulas cor da pele, faciais, cervicais e tronculares. Referia história familiar de achados cutâneos semelhantes e irmão com episódios repetidos de pneumotórax. Identificaram-se múltiplos quistos pulmonares por tomografia computorizada. Uma biópsia cutânea revelou fibroma perifolicular. O diagnóstico clínico de síndrome de Birt-Hogg-Dubé (BHDS) foi contudo corroborado pela identificação de uma nova mutação frameshift c.573delGAinsT (p.G191fsX31) em heterozigotia no exão 6 do gene da foliculina. A presença de múltiplos e típicos tumores benignos do folículo piloso, realça o papel do dermatologista no diagnóstico desta rara genodermatose, que está associada a um risco aumentado de tumores de células renais e cistos pulmonares, exigindo seguimento e aconselhamento pessoal e familiar.