RESUMO
Objective: To investigate the effect of long-chain non-coding RNA Fez family zinc finger protein 1 antisense RNA1 (lncRNA FEZF1-AS1) on the biological function of hepatocellular carcinoma (HCC). Methods: SMMC771 and BEL-7402 cells were transfected with sh-FEZF1-AS1 and OE-FEZF1-AS1, respectively. The expression of lncRNA FEZF1-AS1 was detected by real-time quantitative PCR. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8), and apoptosis was detected by flow cytometry. The effects of lncRNA FEZF1-AS1 on invasion and migration were detected by Transwell and wound healing assays. The expression levels of adhesion molecules were detected by Western blot. The effect of lncRNA FEZF1-AS1 on the in vivo growth was verified by nude mice xenograft experiments. Results: The silencing or ectopic expression of lncRNA FEZF1-AS1 inhibited or promoted the proliferation of hepatocellular carcinoma cells. CCK-8 assay showed that the proliferation abilities of SMMC7721 and BEL-7402 cells in sh-FEZF1-AS1 transfection group significantly decreased, achieving (35.43±4.06)% and (34.68±3.97)%, respectively, on the fifth day. There were significant differences between sh-FEZF1-AS1 group and sh-NC group [52.21±8.46)% and (53.76±7.64)%] (all P<0.05). In contrast, the proliferation ability of SMMC7721 and BEL-7402 cells transfected with OE-FEZF1-AS1 was significantly increased, achieving (83.49±6.92)% and (80.31±3.13)%, respectively, on the fifth day. There were significant differences between OE-FEZF1-AS1 and OE-NC group [53.03±8.84)% and (55.11±7.09)%] (all P<0.05). The subsequent flow cytometry results showed that cell apoptotic rates of SMMC7721 and BEL-7402 cells transfected with sh-FEZF1-AS1 were (13.02±1.38)% and (11.88±1.29)%, respectively, which were significantly higher than those in sh-NC groups [(5.57±1.46)% and (8.06±1.42)%, respectively, all P<0.05]. In contrast, the apoptotic rates of SMMC7721 and BEL-7402 cells transfected with OE-FEZF1-AS1 were (3.01±0.39)% and (3.22±0.43)%, which were significantly lower than those in OE-NC groups [(6.68±0.96)% and (6.63±0.45)%, all P<0.05]. In addition, knockdown or overexpression of lncRNA FEZF1-AS1 expression inhibited or enhanced the migration and invasion abilities as well as the levels of adhesion molecules in hepatocellular carcinoma cells. After 30 days of feeding under the same conditions, the tumor volumes of sh-FEZF1-AS1 and sh-NC SMMC7721 cells xenograft mice models were (0.26±0.03) cm(3) and (0.63±0.06) cm(3), respectively, showing significant difference (P<0.05). The tumor volumes of sh-FEZF1-AS1 and sh-NC BEL-7402 cells were (0.31±0.02) cm(3) and (0.72±0.08) cm(3), and the difference was statistically significant (P<0.05). Conclusion: lncRNA FEZF1-AS1 may strengthen the growth, migration and invasion of hepatocellular carcinoma cells.
Assuntos
Carcinoma Hepatocelular , Movimento Celular , Proliferação de Células , Neoplasias Hepáticas , RNA Longo não Codificante , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Proteínas do Tecido Nervoso , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras , Fatores de TranscriçãoRESUMO
Objective: To explore the role and its molecular mechanism of miR-328 during the differentiation of embryonic stem cells (ESCs) into insulin-producing cells (IPCs) in vitro. Method: Mouse embryonic stem cell line-mESCs-Nanog-GFP was induced in conditioned medium and divided into negative control group, miR-328 agomir transfected group, miR-328 antagomir transfected group and transforming growth factor ß2 (TGF-ß2) siRNA transfected group. The function of IPCs was identified by real-time quantitative PCR (qPCR) detecting system and immunofluorescence in above-mentioned groups. Methods of qPCR, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) were used to detect effects of overexpression and inhibition of miR-328 on differentiation of multilineage precursor cells. We predicted the binding sites of miR-328 and TGF-ß2 by performing the bioinformatics analysis. Dual luciferase reporter gene and Western blotting were employed to identify the regulatory relationship between miR-328 and TGF-ß2. Results: mESCs could be transfected with miR-328 agomir, with an efficacy of 70%-80%. Up-regulated miR-328 in MPCs reduced the RNA expression of several key transcription factors which were crucial for early pancreatic development. Additionally, the insulin released by IPCs decreased in response to glucose stimulation (all P<0.05). However, overexpression of miR-328 led to the decrease of protein level of insulin and Nkx6.1 (all P<0.05). Transfection of miR-328 antagomir had the opposite effects (P<0.05). The dual luciferase reporter gene assay revealed that miR-328 functioned via binding to the 3' non-coding region (3'-UTR) of the TGF-ß2. Western blotting indicated that miR-328 regulated protein expression. After knockdown of miR-328, the relative expression of TGF-ß2 was 1.00±0.01. After co-transfection of miR-328 antagomir and TGF-ß2 siRNA, the relative expression of TGF-ß2 was 0.80±0.03. After downregulating TGF-ß2, the relative expression of TGF-ß2 was 0.20±0.01. Knockdown of TGF-ß2 down-regulated the expression of early pancreatic transcription factors (P<0.05) and inhibited Pdx1(+)cell differentiation. Conclusion: miR-328 can inhibit the differentiation of ESCs into IPCs via binding to 3' UTR of TGF-ß2, and provide a new regulatory pathway for the treatment of diabetes with stem cells.
Assuntos
Células Secretoras de Insulina , Animais , Diferenciação Celular , Camundongos , MicroRNAs , Células-Tronco Embrionárias Murinas , Fator de Crescimento Transformador beta2RESUMO
Objective: To investigate the expression and clinical significance of KIAA1199 in primary hepatocellular carcinoma. Methods: A total of 136 cases of primary hepatocellular carcinoma tissues and paired adjacent tissues were collected. Immunohistochemistry and Western blot were used to detect the expression of KIAA1199 in primary hepatocellular carcinoma tissues and paired adjacent tissues. The relationship between KIAA1199 and clinicopathological parameter of primary hepatocellular carcinoma was analyzed. Results: The positive rate of KIAA1199 in primary hepatocellular carcinoma was 82.3% (112/136), which was higher than that in paired para-cancerous tissues (14.7%, 20/136). High expression of KIAA1199 was significantly correlated with age, cirrhosis history, tumor size, tumor number, degree of differentiation, TNM staging and microvenous invasion (MVI) (P<0.05), but without gender, drinking alcohol hobby, hepatitis history, family genetic history, tumor location (P>0.05). The Kaplan-Meier survival curves indicated that high KIAA1199 expression was associated with poor survival (P<0.01). In addition, Cox proportional hazards model showed that the expression of KIAA1199 was related to age, cirrhosis history, tumor size, tumor number, degree of differentiation, TNM staging and MVI (P<0.05). Conclusion: The expression of KIAA1199 is up-regulated in primary hepatocellular carcinoma, which is significantly correlated with the clinicopathological features and prognosis, high expression of KIAA1199 increased the risk of death in patients with primary hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas/análise , Biomarcadores Tumorais , Humanos , Hialuronoglucosaminidase , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos ProporcionaisRESUMO
Objective: The intact rat liver decellularized scaffolds were preparedand repopulated hepatocytes by continuous perfusion technology.Toprovideexperimental support for the application of decellularized liver scaffolds in liver engineering. Method: Decellularized liver scaffolds were obtained by perfusing method. The composition and structure was examined by HE, Masson, Sirius red stain and immunofluorescence. The ultrastructure was examined by scanning electron microscope (SEM). DNA content was used to confirm the effect of decellularization. The circulation perfusion device was established. Hepatocytes were recellularized into the scaffolds by multiposition parenchymal injection method and infusion method, thenthe scaffolds were cultured in the circulation perfusion device in vitro. After cultivation, HE staining, immunofluorescence and SEM were conducted to observe the growth situation of hepatocytes in the scaffolds. Results: The rat decellularized liver scaffolds were successfully obtained by perfusion method. Histological staining demonstrated the remove of cellular component and the reservation of extracellular cellmatrix. Immunofluorescence staining demonstrated the retention of collagen I. SEM showed that the ultrastructure of the extracellular cell matrix presented thereticular structure. DNA content of the scaffolds was 47.5±18.1 ng/mg. The circulation perfusion device was composed of a peristaltic pump, oxygenator, chamber and the convey tubes. The multipositional parenchymal injection method resulted in a better engraftment rate. HE staining, immunofluorescence and SEM revealed that the growth and function of hepatocytes were goodin the scaffold. Conclusion: The decellularized rat liver scaffolds have favorable biochemical properties. The liver decellularized scaffolds applied with the circulation perfusion device could provide a well 3D plat form for culture of hepatocytes.
Assuntos
Fígado , Engenharia Tecidual , Alicerces Teciduais , Animais , Técnicas de Cultura de Células , Matriz Extracelular , Hepatócitos , Perfusão , RatosRESUMO
The aim of this study was to identify key genes related to invasive ductal carcinoma (IDC) of the breast by analyzing gene expression data with bioinformatic tools. Microarray data set GSE31138 was downloaded from Gene Expression Omnibus, including 3 breast cancer tissue samples and 3 normal controls. Differentially expressed genes (DEGs) between breast cancer and normal control were screened out (FDR < 0.05 and |logFC| > 2). Coexpression between genes was examined with String, and a network was then constructed. Relevant pathways and diseases were retrieved with KOBAS. A total of 56 DEGs were obtained in the IDC of the breast compared with normal controls. A gene coexpression network including 27 pairs of genes was constructed and all the genes in the network were upregulated. Further study indicated that most of the genes in the coexpression network were enriched in ECM-receptor interaction (COL4A2, FN1, and HMMR) and nucleotide excision repair (CETN2 and PCNA) pathways, and that the most significantly related disease was autoimmune lymphoproliferative syndromes. A number of DEGs were acquired through comparative analysis of gene expression data. These findings are beneficial in promoting the understanding of the molecular mechanisms in breast cancer. More importantly, some key genes were revealed via gene coexpression network analysis, which could be potential biomarkers for IDC of the breast.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal/genética , Genes Neoplásicos , Predisposição Genética para Doença , Feminino , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Microarray data were collected from bile duct samples from subjects with malignant biliary strictures by endoscopic retrograde cholangiopancreatography to screen for key genes associated with this disease. A predicted interaction network was constructed for these genes to interpret their functions. The gene expression dataset GSE34166 (10 samples: 6 malignant and 4 benign control samples) was downloaded from the Gene Expression Omnibus database. R package scripts were used to process the data and screen for differentially expressed genes. Genes identified were uploaded to the analysis tool String 8.3 to generate a gene interaction network. A hub gene was identified by calculating the node degree. The interaction network of the hub gene with other genes in the human genome was constructed and screened (score >0.9), and pathway-enrichment analysis was performed to elucidate the hub gene function. In total, 377 differentially expressed genes were identified and a network comprising 209 pairs of interactions was constructed. The most critical hub gene was identified as GSTA1, and a GSTA1-based interaction network was constructed consisting of 25 genes (containing the differentially expressed gene GSTA3). The cytochrome P450 (CYP450)-metabolic pathway displayed the most significant enrichment. Additionally, 4 transcription factors and their binding sites were also identified. In conclusion, we have identified the differentially expressed genes GSTA1 (a hub gene) and GSTA3, which may cause abnormal gene expression and tumorigenesis through CYP450-metabolic pathways. The transcription factors and their binding sites in the promoter of the hub gene provide potential directions for future drug design.
Assuntos
Neoplasias dos Ductos Biliares/genética , Proteínas ELAV/biossíntese , Glutationa Transferase/biossíntese , Redes e Vias Metabólicas , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Biologia Computacional , Proteína Semelhante a ELAV 2 , Regulação Neoplásica da Expressão Gênica , Glutationa Transferase/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Mapas de Interação de Proteínas/genéticaRESUMO
The crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin, has been determined to a resolution of 2.8 A by the isomorphous replacement method and preliminary refinement. The structure closely resembles that of the cleaved form of alpha-1-proteinase inhibitor, with some important exceptions. The disposition of the new carboxyl chain terminus liberated by proteolysis is different with respect to the central beta-sheet A in the structures of these two molecules. In alpha-1-proteinase inhibitor, the new chain terminus inserts in beta-sheet A to add a middle strand to the sheet. In plakalbumin, this strand remains free near the site at which the cleavage occurs. A structural basis for this difference in behavior is proposed from the structures and sequences of these two molecules and other members of the serpin family. The structures and positions of the putative signal peptide of ovalbumin, the several post-translational modifications, and the relationship of the intron-exon patterns of plakalbumin and alpha-1-proteinase inhibitor to their protein structures are also described.