RESUMO
Biliary atresia (BA) is a destructive bile duct disease occurring in newborn children within a few weeks after birth. In this study, the effect of miR-29c and miR-129-5p on epithelial-mesenchymal transition (EMT) in experimental BA was explored by constructing BA mouse models via Rhesus rotavirus vaccine infection. miR-29c and miR-129-5p expression was analyzed by real-time quantitative polymerase chain reaction. EMT was established by induction with transforming growth factor (TGF)-ß1. miR-29c and miR-129-5p were overexpressed and inhibited, respectively, by Lipofectamine transfection. EMT-related protein (formin-like 2, FMNL2; E-cadherin; vimentin; and cytokeratin-19, CK-19) expression was analyzed by western blot and immunofluorescent assay. The results indicated that miR-29c and miR-129-5p were downregulated and upregulated in BA mice. TGF-ß1 induction caused a time-dependent decrease and increase in miR-29c and miR-129-5p, respectively. Additionally, TGF-ß1 induced an increase in FMNL2 and vimentin expression and a decrease in E-cadherin and CK-19 expression (P < 0.05). Overexpression or suppression of miRNA-29c or miR-129-5p, respectively, induced the inhibition of FMNL2 and vimentin, and promotion of E-cadherin and CK-19 expression, in the test groups compared to the non-intervention group (P < 0.05). However, the FMNL2, vimentin, E-cadherin, and CK- 19 expression did not differ between the control and non-intervention groups (P > 0.05). Thus, miR-29c upregulation or miR-129-5p downregulation effectively prevented EMT in BA by regulating the expression of EMT pathway-related proteins. Therefore, miR-29c and miR-129-5p could be utilized as therapeutic targets for BA in the future.
Assuntos
Atresia Biliar/genética , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , MicroRNAs/genética , Fator de Crescimento Transformador beta1/farmacologia , Animais , Animais Recém-Nascidos , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Atresia Biliar/etiologia , Atresia Biliar/metabolismo , Atresia Biliar/patologia , Caderinas/genética , Caderinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Forminas , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratina-19/genética , Queratina-19/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , Cultura Primária de Células , Vacinas contra Rotavirus/administração & dosagem , Vacinas contra Rotavirus/efeitos adversos , Transdução de Sinais , Vimentina/genética , Vimentina/metabolismoRESUMO
The aim of the current study was to investigate survivin expression in congenital choledochal cysts (CCCs), and its associations with clinical parameters of CCCs. In total, 121 children with CCCs were included in this study as the case group, and their cysts were staged according to the Todani classification system. Additionally, 49 normal gallbladder specimens from healthy children were included as the control group. Survivin detection was conducted using immunohistochemical staining. Associations between positive survivin expression and clinical parameters of CCCs were then analyzed. Positive survivin expression was observed in the cytoplasm, and was seen as granular with yellow or dark brown staining. In the case group, positive survivin expression was detected in most tissues. Specifically, compared to that of normal tissues, the cystic-shaped and fusiform-shaped CCC tissues had significantly higher positive survivin expression rates (all P < 0.05). Importantly, positive survivin expression was also shown to be significantly associated with gender and histological type (both P < 0.05). In conclusion, increased survivin expression was observed in CCC tissues, and was correlated with certain clinical parameters of CCCs, suggesting a possible prognostic value of survivin for CCC progression.
Assuntos
Cisto do Colédoco/genética , Proteínas Inibidoras de Apoptose/genética , Biomarcadores/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Cisto do Colédoco/metabolismo , Cisto do Colédoco/patologia , Feminino , Vesícula Biliar/metabolismo , Humanos , Lactente , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , SurvivinaRESUMO
Alveolar bone osteoblasts are widely used in dental and related research. They are easily affected by systemic diseases such as diabetes. However, the mechanism of diabetes-induced alveolar bone absorption remains unclear. This study systematically explored the changes in human alveolar bone cell-related gene expression and biological pathways, which may facilitate the investigation of its mechanism. Alveolar bone osteoblasts isolated from 5 male diabetics and 5 male healthy adults were cultured. Total RNA was extracted from these cells and subjected to gene microarray analysis. Differentially expressed genes were screened, and a gene interaction network was constructed. An enrichment pathway analysis was simultaneously performed on differentially expressed genes to identify the biological pathways associated with changes in the alveolar bone cells of diabetic humans. In total, we identified 147 mRNAs that were differentially expressed in diabetic alveolar bone cells (than in the normal cells; 91 upregulated and 36 downregulated mRNAs). The constructed co-expression network showed 3 pairs of significantly-expressed genes. High-enrichment pathway analysis identified 8 pathways that were affected by changes in gene expression; three of the significant pathways were related to metabolism (inositol phosphate metabolism, propanoate metabolism, and pyruvate metabolism). Here, we identified a few potential genes and biological pathways for the diagnosis and treatment of alveolar bone cells in diabetic patients.
Assuntos
Processo Alveolar/metabolismo , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Processo Alveolar/citologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/citologia , Osteoblastos/metabolismoRESUMO
The aim of this study was to construct the recombinant adenovirus carrying human TIMP-1shRNA gene expression system for preliminary identification to lay the foundation for the further study of gene therapy. Using the Adeno-X system, the recombinant adenovirus plasmid pAdeno-X green fluorescent protein (GFP)-tissue inhibitor of metalloprotease (TIMP)-1 small hairpin (1shRNA) was constructed by including the target gene fragment of the TIMP-1shRNA shuttle plasmid pShuttle2-GFP-TIMP-1shRNA and the backbone plasmid pAdeno-X by homologous recombination in Escherichia coli. Recombinant plasmids were transfected into HEK293A cells to package the recombinant adenovirus rvAdeno-XGFP-TIMP-1shRNA. The recombinant adenovirus was identified by polymerase chain reaction, and the viral titer and infection efficiency were detected using GFP. Polymerase chain reaction and restriction endonuclease digestion demonstrated that rvAdeno-XGFP-TIMP-1shRNA had been successfully constructed, which has a strong ability to infect the kidney. The TIMP-1shRNA adenovirus expression vector was successfully constructed using homologous recombination methods.
Assuntos
Adenoviridae/genética , RNA Interferente Pequeno/metabolismo , Recombinação Genética , Inibidor Tecidual de Metaloproteinase-1/genética , Animais , Forma Celular , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Reação em Cadeia da Polimerase , Ratos , TransfecçãoRESUMO
Proline accumulation is involved in plant osmotic stress tolerance. Given that DNA methylation is related to almost all metabolic processes through regulation of gene expression, we suspected that this epigenetic modification and proline biosynthesis are probably related. To test this, we investigated whether osmotic stress-induced proline accumulation is associated with DNA methylation modifications in rice. We assessed DNA methylation and expression of 3 key genes (P5CR, P5CS, and δ-OAT) involved in proline biosynthesis, and measured proline content under both osmotic stress (15% polyethylene glycol) and control conditions. After osmotic stress, selfed progenies of osmotic-stressed plants accumulated higher concentrations of proline in leaves under both normal conditions and under osmotic stress than the unstressed control plants. Concomitantly, under osmotic stress, the selfed progeny plants showed higher expression levels of P5CS and δ-OAT than the control. This up-regulated expression was stably inherited by the subsequent generation. Methylation-sensitive Southern blotting indicated that 2 of the 3 genes, P5CS and δ-OAT, had greater DNA demethylation in the selfed progenies than in the control. Apparently DNA demethylation facilitated proline accumulation by up-regulating expression of the P5CS and δ-OAT genes in response to osmotic stress.