TGIF1 splicing variant 8 is overexpressed in oral squamous cell carcinoma and is related to pathologic and clinical behavior.

OBJECTIVE: Possible differences in splicing variants of TGIF1 in oral squamous cell carcinoma (OSCC) have not yet been reported. This study analyzed the expression levels of different splicing variants of the TGIF1 gene in OSCC compared with nontumoral epithelium (NT) and the relationship with clinical-pathologic features of tumors. STUDY DESIGN: Forty-eight frozen samples of OSCC and 17 of NT were analyzed using quantitative reverse transcription polymerase chain reaction. RESULTS: TGIF1v2 and v8 are overexpressed in OSCC, whereas TGIF1v5 is underexpressed when compared with NT. Low TGIF1v8 expression was correlated with lower cellular differentiation, positive blood vascular invasion, advanced pathologic stage, and positive vascular lymphatic invasion of OSCC. TGIF1v8 is also related to overall survival over time, with lower values associated with an increased risk of cancer-related death. CONCLUSIONS: These data suggest that alternative splicing of TGIF1 is deregulated in OSCC, with overexpression of some splicing variants, especially TGIF1v8, which is associated with advanced stages of OSCC.

Detection of TGIF1 homeobox gene in oral squamous cell carcinoma according to histologic grading.

OBJECTIVE: TGIF1 homeobox gene involvement in oral cancer has not yet been investigated. This study analyzed the expression of TGIF1 transcripts and protein in oral squamous cell carcinoma (OSCC). STUDY DESIGN: Snap-frozen samples from 16 patients were taken from both OSCC and nontumoral adjacent epithelium (NT) for in situ hybridization (ISH). Forty-six paraffin-embedded samples of OSCC were submitted to immunohistochemistry (IHC). A descriptive analysis of the transcript signal detection was accomplished, and TGIF1 immunoexpression was carried out considering protein levels, localization, and cellular differentiation. RESULTS: ISH reactions showed TGIF1 transcripts with a signal that was frequently intense in NT, and generally weak in OSCC, and that had stronger transcript signal in well-differentiated areas of OSCC when compared with poorly differentiated ones. IHC reactions had poorly differentiated cases associated with TGIF1 protein expression in both the nucleus and cytoplasm (P = .05, Fisher test). CONCLUSIONS: TGIF1 gain or loss of function might possibly play a role in oral cancer cell differentiation.

Hibridização in situ com sonda não-radioativa para mRNA: princípios e aplicações em patologia / In situ hybridization with non-radioactive riboprobes: principles and applications in pathology

A técnica de hibridização in situ (ISH) tem sido usada para identificar mRNA (ou DNA) em amostras de tecido de material humano e animal. Embora uma série de protocolos para essa técnica seja utilizada, as descrições não são bem detalhadas. O objetivo deste trabalho é descrever a reação de hibridização in situ em tecido fresco e sua aplicação em patologia, tornando mais compreensível essa técnica tão importante, que possibilita observar a localização tecidual e a expressão temporal e espacial dos transcritos de um determinado gene (mRNA). Resultados de reações com as ribossondas PITX1, SHH e WNT-5A, realizadas em amostras de tecido congelado, são apresentados.

In situ hybridization (ISH) has been used to identify mRNA (or DNA) in fresh tissue samples of humans and animals. Several protocols describing this technique are available, although its description is not usually detailed enough. The present work describes in situ hybridization reaction on fresh tissue in a way to make understandable this important technique, which allows verifying the cellular localization, and the spatial and temporal expression, of gene transcripts (mRNA). Results with PITX1, TGIF, SHH and WNT-5A riboprobes, in fresh tissue samples, are presented.

Evaluation of the genomic DNA extracted from formalin-fixed, paraffin-embedded oral samples archived for the past 40-years

BACKGROUND: The most common human archival specimens are formalin-fixed, paraffin-embedded tissues (PETs). DNA can be extracted from PETs, but sometimes, it is unsuitable for molecular techniques as slow degradation of DNA occurs with time. OBJECTIVE: The aim of this study was to verify and discuss if samples of oral PETs archived for the past 40-years are possible substrates for molecular biology studies, using PCR. Methods: The samples were submitted to phenol-chloroform extraction method. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis and amplification by PCR. RESULTS: It was observed a weak positive correlation between genomic DNA yield and specimen age. The agarose gel electrophoresis demonstrated that genomic DNA length was more frequently composed of small fragments. The 268-bp fragments of the beta-globin gene was amplified in 55 percent of cases and preferentially in more recent ones, which showed strong amplification if compared with older samples. WAF1 gene with 149-bp presented weak but detectable amplification in 75 percent of cases. The 536-bp fragment of beta-globin gene was detected in 25 percent of samples. The amplification was intense in genomic DNA extracted from recent cases and weak in older ones. CONCLUSION: This study shown that, despite degradation, it is possible to use genomic DNA obtained from PETs, archived for the past forty years, in PCR amplification of small DNA products, being large DNA fragments more difficult to amplificate.

INTRODUÇÃO: O material fixado em formol e embebido em parafina constitui hoje a maior fonte de tecido humano arquivado. O DNA extraído de tecido parafinado por vezes não é adequado para as técnicas de biologia molecular, visto que se apresenta parcialmente degradado. OBJETIVOS: O objetivo desse estudo foi verificar se tecido humano de boca parafinado e arquivado pelos últimos 40 anos pode ser usado para a reação em cadeia da polimerase (PCR). MÉTODOS: Amostras foram submetidas à técnica de extração de DNA pelo método do fenol-clorofórmio. Para quantificação e qualificação do DNA foram realizadas análise em espectrofotômetro, eletroforese em gel de agarose e amplificação pela técnica da PCR. RESULTADOS: Foi observada fraca correlação positiva entre a quantidade de DNA obtida e a idade das amostras. A eletroforese em gel de agarose demonstrou que a maioria do DNA obtido foi constituída de fragmentos pequenos. O fragmento de 268-pb do gene da beta-globina foi amplificado em 55 por cento dos casos, preferencialmente nos casos mais recentes. O fragmento de 149-pb do gene WAF1 apresentou amplificação fraca, mas presente em 75 por cento dos casos. O fragmento de 536-bp do gene da beta-globina foi detectado em somente 25 por cento dos casos e também preferencialmente nos casos mais recentes. CONCLUSÕES: Esse estudo mostrou que, apesar de o DNA estar degradado, é possível usar DNA genômico extraído de tecido parafinado arquivado pelos últimos 40 anos em reações de PCR de produtos pequenos. A amplificação de produtos maiores, entretanto, é mais difícil.