Medicated effect of CDKN1B on promoting proliferation of islet β cells in diabetes mellitus / 中国综合临床

The serious decrease in the number of functional β cells is one of the main features in the pathogenesis of diabetes mellitus. CDKN1B is a new kind of regulatory protein, which can bind and inactivate cyclin and cyclin-dependent kinase complex to control the process of cell cycle. It was suggested that down-regulation or deletion of CDKN1B in islet β cells could accelerate the proliferation of islet β cells, thus increasing the number of islet β cells, which is of great significance for treatments of diabetes.

Construction and expression of the eukaryotic expression vector of wild-type p27 and lacking the nuclear localization signal p27 / 西安交通大学学报(医学版)

【Objective】 To construct the eukaryotic expression vector carrying the human wild-type p27 and lacking nuclear localization signal p27△NLS coding sequences， and the express them in HEK293T cells， which may contribute to investigating the different locations and roles of p27 in the cytoplasm and nucleus. 【Methods】 Total RNA was prepared from human breast cancer MCF7 cells， and cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR）. After amplification of the p27 CDs and non-NLS fragments by PCR， full length p27WT （CDKN1B， NM_004064.5） and p27△NLS coding regions were obtained. PCR products were then subcloned into the eukaryotic expression vector pCMV-Blank. After identification with bacterial PCR， double restriction enzyme digestion and sequencing， they were defined officially as pCMV-p27WT and pCMV-p27△NLS， respectively. The recombinant plasmids were transfected into HEK293T cells by electroporation. After 48 h， the levels of p27 protein in the cytoplasm and nucleus were detected by Western blotting. 【Results】 The sequencing results showed that the sequences of p27WT and p27△NLS inserted into the plasmids were both correctly consistent with that of NM_004064.5. After transfection with pCMV-p27WT， total p27 protein expression was increased and distributed in both the cytoplasm and nucleus of HEK293T cells. After transfection with pCMV-p27△NLS， p27 protein was significantly increased and almost entirely localized in the cytoplasm of HEK293T cells. 【Conclusion】 The eukaryotic expression plasmids of human p27WT and p27△NLS coding sequences were successfully constructed and overexpressed in HEK293T cells. This research may lay a foundation for investigating the biological function of p27 in the cell cycle progression of tumor cells.

Anti-micro rna-221 a promising genetic therapy of oral squamous cell carcinoma (scc-25)

Abstract Micro-RNA-221(miR-221) is one of oncogenic miRNAs that plays a vital role in the development and progression of oral cancers. The aim of this study is to introduce a new gene therapy for oral squamous cell carcinoma by blocking the expression of oncogenic miR-221 by its inhibitor. The present work was performed on squamous cell carcinoma cell line SCC-25 and anti-miR-221 was delivered to the cells using an ultrasound micro bubbles. Assessment of the effect of miR-221 inhibitor on SCC-25 cells was done using MTT assay, cell cycle analysis and apoptosis detection. In addition, reverse transcription-polymerase chain reaction was also used to detect the expression -miR-221 and its target genes. Using ANOVA, statistical analysis of the results showed significant inhibition of cell viability with and induction of cell apoptosis of SCC-25 cell line after transfection. Moreover, the expression of miR-221, Epidermal growth factor receptor (EGFR) and CDKNIB/p27 were downregulated without significant difference. Transfection of SCC-25 by inhibitor of miR-221 resulting in blockage of its expression leading to arresting of tumor growth. These results proved the effective role of micro-RNA inhibitors as novel therapeutic agent for oral cancers.

Resumo Micro-RNA-221 (miR-221) é um dos miRNAs oncogênicos que desempenham um papel vital no desenvolvimento e progressão de carcinomas orais. O objetivo deste estudo é apresentar uma nova terapia gênica para o carcinoma epidermóide oral por meio do bloqueio da expressão do miR-221 oncogênico por seu inibidor. O presente trabalho foi realizado na linhagem de células de carcinoma de células escamosas SCC-25 e o anti-miR-221 foi administrado às células usando micro-bolhas de ultrassom. A avaliação do efeito do inibidor miR-221 em células SCC-25 foi feita usando ensaio de MTT, análise do ciclo celular e detecção de apoptose. Além disso, a reação em cadeia da polimerase com transcrição reversa também foi usada para detectar a expressão -miR-221 e seus genes-alvo. Usando ANOVA, a análise estatística dos resultados mostrou inibição significativa da viabilidade celular e indução da apoptose celular da linhagem celular SCC-25 após a transfecção. Além disso, a expressão de miR-221, receptor do fator de crescimento epidérmico (EGFR) e CDKNIB/p27 foram regulados para baixo sem diferença significativa. A transfecção de SCC-25 por inibidor de miR-221 resultou no bloqueio de sua expressão, levando à interrupção do crescimento do tumor. Esses resultados comprovaram o papel eficaz dos inibidores de micro-RNA como novo agente terapêutico para carcinomas orais.

Neoplasia endocrina múltiple tipo 1 con mutación negativa y fenocopias / Multiple endocrine neoplasia type 1 with negative mutation and phenocopies

Introducción: existen 4 tipos de neoplasias endocrinas múltiples, las cuales se caracterizan por la aparición de tumores en 2 o más glándulas endocrinas. La prevalencia de neoplasia endocrina múltiple 1 es aproximadamente 2 por 100 000, y constituyen una enfermedad poco frecuente. Objetivo: descartar, ante la sospecha de una neoplasia endocrina múltiple 1 con mutación negativa, otras enfermedades para poder diagnosticarla como tal. Presentación del caso clínico: mujer de 36 años, con diagnóstico de macroprolactinoma e hiperparatiroidismo primario normocalcémico (neoplasia endocrina múltiple 1 clínica), hallazgos clínicos que justificaron el estudio genético. Inicialmente para neoplasia endocrina múltiple 1, resultó negativo. En pacientes con neoplasia endocrina múltiple 1 clínica -o alta sospecha de neoplasia endocrina múltiple 1 en los que no se identifica mutación- hay que considerar que se trate de una fenocopia y ampliar el estudio genético: CDC73, CDKN1B, CaSR y AIP. También se analizaron estos genes, y fueron negativos. Otra entidad a considerar sería el hiperparatiroidismo aislado familiar. Conclusiones: llegar al diagnóstico de neoplasia endocrina múltiple 1 a veces no es tan simple, como identificar una mutación positiva. Es importante descartar fenocopias, para poder diagnosticar correctamente al paciente, pues esto determinará el seguimiento en búsqueda de otros posibles tumores, lo que -en último término- puede condicionar el pronóstico(AU)

Introduction: there are four types of multiple endocrine neoplasias which are characterized by occurrence of tumors in two or more endocrine glands. The prevalence rate of multiple endocrine neoplasia type 1 is 2 per 100 000 patients approximately and it is a rare disease. Objective: to rule out the existence of any other disease in order to properly diagnose a suspected multiple endocrine neoplasia type 1 with negative mutation. Clinical case presentation: a 36 years-old woman diagnosed with macroprolactinoma and primary normocalcemic hyperparathyroidism (clinical multiple endocrine neoplasia type 1) and clinical findings supporting the performance of a genetic study. The study initially yielded negative results for the above-mentioned disease. However, in those patients with clinical multiple endocrine neoplasia type 1- or high suspicious of multiple endocrine neoplasia type 1 with no identified mutation- it must be considered that there is a phenocopy and the genetic study must be extended to include CDC 73, CDKN1B, CaSR and AIP. These genes were also analyzed with negative results. Another disease to be considered would be isolated family hyperparathyroidism. Conclusions: making the diagnosis of a multiple endocrine neoplasia type 1 is not sometimes as simple as identifying a positive mutation. It is important to rule out possible phenocopies to be able to adequately diagnose a patient, since this will determine the search for other probable tumors which may ultimately influence this prognosis(AU)

MENX and MEN4

Multiple endocrine neoplasias are autosomal dominant disorders characterized by the occurrence of tumors in at least two endocrine glands. Two MEN syndromes have long been known and are well characterized: the MEN type 1 (MEN1) and type 2 (MEN2). These syndromes are caused by germline mutations in the MEN1 and RET genes, respectively, and have a different tumor spectrum. Recently, a variant of the MEN syndromes arose spontaneously in a rat colony and was named MENX. Affected animals consistently develop multiple endocrine tumors, with a spectrum that shares features with both MEN1 and MEN2 human syndromes. Genetic studies identified a germline mutation in the Cdkn1b gene, encoding the p27 cell cycle inhibitor, as the causative mutation for MENX. Capitalizing on these findings, heterozygous germline mutations in the human homologue, CDKN1B, were searched for and identified in patients with multiple endocrine tumors. As a consequence of this discovery, a novel human MEN syndrome, named MEN4, was recognized, which is caused by mutations in p27. Altogether, these studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. Here we review the characteristics of the MENX and MEN4 syndromes and we briefly address the main function of p27 and how they are affected by MENX/4-associated mutations.