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1.
Chinese Medical Journal ; (24): 2340-2352, 2021.
Article in English | WPRIM | ID: wpr-921125

ABSTRACT

BACKGROUND@#Emerging evidence indicates that the sineoculis homeobox homolog 1-eyes absent homolog 1 (SIX1-EYA1) transcriptional complex significantly contributes to the pathogenesis of multiple cancers by mediating the expression of genes involved in different biological processes, such as cell-cycle progression and metastasis. However, the roles of the SIX1-EYA1 transcriptional complex and its targets in colorectal cancer (CRC) are still being investigated. This study aimed to investigate the roles of SIX1-EYA1 in the pathogenesis of CRC, to screen inhibitors disrupting the SIX1-EYA1 interaction and to evaluate the efficiency of small molecules in the inhibition of CRC cell growth.@*METHODS@#Real-time quantitative polymerase chain reaction and western blotting were performed to examine gene and protein levels in CRC cells and clinical tissues (collected from CRC patients who underwent surgery in the Department of Integrated Traditional and Western Medicine, West China Hospital of Sichuan University, between 2016 and 2018, n = 24). In vivo immunoprecipitation and in vitro pulldown assays were carried out to determine SIX1-EYA1 interaction. Cell proliferation, cell survival, and cell invasion were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenic assay, and Boyden chamber assay, respectively. The Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen) method was used to obtain small molecules that specifically disrupted SIX1-EYA1 interaction. CRC cells harboring different levels of SIX1/EYA1 were injected into nude mice to establish tumor xenografts, and small molecules were also injected into mice to evaluate their efficiency to inhibit tumor growth.@*RESULTS@#Both SIX1 and EYA1 were overexpressed in CRC cancerous tissues (for SIX1, 7.47 ± 3.54 vs.1.88 ± 0.35, t = 4.92, P = 0.008; for EYA1, 7.61 ± 2.03 vs. 2.22 ± 0.45, t = 6.73, P = 0.005). The SIX1/EYA1 complex could mediate the expression of two important genes including cyclin A1 (CCNA1) and transforming growth factor beta 1 (TGFB1) by binding to the myocyte enhancer factor 3 consensus. Knockdown of both SIX1 and EYA1 could decrease cell proliferation, cell invasion, tumor growth, and in vivo tumor growth (all P < 0.01). Two small molecules, NSC0191 and NSC0933, were obtained using AlphaScreen and they could significantly inhibit the SIX1-EYA1 interaction with a half-maximal inhibitory concentration (IC50) of 12.60 ± 1.15 μmol/L and 83.43 ± 7.24 μmol/L, respectively. Administration of these two compounds could significantly repress the expression of CCNA1 and TGFB1 and inhibit the growth of CRC cells in vitro and in vivo.@*CONCLUSIONS@#Overexpression of the SIX1/EYA1 complex transactivated the expression of CCNA1 and TGFB1, causing the pathogenesis of CRC. Pharmacological inhibition of the SIX1-EYA1 interaction with NSC0191 and NSC0933 significantly inhibited CRC cell growth by affecting cell-cycle progression and metastasis.


Subject(s)
Animals , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Genes, Homeobox , Homeodomain Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Mice , Mice, Nude , Nuclear Proteins/genetics , Protein Tyrosine Phosphatases/genetics
2.
Int. j. morphol ; 36(3): 991-996, Sept. 2018. graf
Article in English | LILACS | ID: biblio-954220

ABSTRACT

The failure of fusion of nasal and maxillary processes results in cleft lip and palate (CLP), which is one of the most common birth defects. The morphopathogenesis of this pathology is multifactorial and still largely unclear. The aim of this study was to evaluate the presence of nestin, transcriptor factor SOX3 (Sox3) and homeobox protein DLX-4 (Dlx-4) in complete unilateral (CU) and complete bilateral (CB) CLP affected facial tissue. Oral mucosa tissue samples were obtained from 17 CUCLP and 13 CBCLP patients during surgical cleft correction and 6 unaffected control subjects. Obtained tissue sections were stained with hematoxylin and eosin and by immunohistochemistry for nestin, Sox3 and Dlx-4. The intensity of staining was graded semiquantitatively. Nestin-positive structures were detected in all CUCLP and CBCLP patients' tissue samples, varying from moderate number of nestin-positive structures to numerous. Sox3 immunoreactivity was more prominent in epithelial cells in both patient groups with frequently patchy distribution. Mainly moderate number of Dlx-4-positive cells was observed in most of tissue samples. Statistically significant moderate positive correlation was found between nestin and Sox3 factors in CUCLP patient group (Spearman's rank correlation coefficient = .517, P = .034). Increase of nestinpositive structures suggests its role in the regulation of the remodeling of tissue in both CUCLP and CBCLP affected tissue. Dominance of Sox3 positivity in cleft affected epithelium indicates its possible role in (compensatory) formation of defective oral epithelium of CUCLP and CBCLP patients. The reduced expression of Dlx-4 implicates its limited regulatory role on the craniofacial development in CUCLP and CBCLP affected tissue.


La falla en la fusión de los procesos nasal y maxilar son causante de la fisura labiopalatina (FLP), que es uno de los defectos congénitos más comunes. La morfopatogenia de esta patología es multifactorial y aún poco clara. El objetivo de este estudio fue evaluar la presencia de nestina, el factor transcriptor SOX3 (Sox3) y la proteína homeobox DLX-4 (Dlx-4) en todo el tejido facial afectado por FLP bilateral unilateral (FU) y bilateral completa (FB). Se obtuvieron muestras de tejido de mucosa oral de 17 pacientes FUFLP y 13 FBFLP durante la corrección quirúrgica de la fisura y de 6 sujetos de control no afectados. Las secciones de tejido obtenidas se tiñeron con hematoxilina y eosina y mediante inmunohistoquímica para nestina, Sox3 y Dlx-4. La intensidad de la tinción fue graduada semicuantitativamente. Se detectaron estructuras positivas para nestina en todas las muestras de tejido de pacientes FUFLP y FBFLP, variando desde un número moderado a numerosas estructuras positivas para nestina. La inmunorreactividad de Sox3 fue más prominente en las células epiteliales en ambos grupos de pacientes con distribución frecuentemente irregular. Se observó un número principalmente moderado de células Dlx-4-positivas en la mayoría de las muestras de tejido. Se encontró una correlación positiva moderada estadísticamente significativa entre los factores de nestina y Sox3 en el grupo de pacientes FUFLP (coeficiente de correlación de rangos de Spearman = 0,517, P = 0,034). El aumento de estructuras positivas para nestina sugiere su papel en la regulación de la remodelación de tejido, tanto en tejido afectado por FUFLP como FBFLP. La dominancia de la positividad de Sox3 en el epitelio afectado de la fisura, indica su posible papel en la formación (compensatoria) del epitelio oral defectuoso de pacientes FUFLP y FBFLP. La expresión reducida de Dlx-4 implica su función reguladora limitada en el desarrollo craneofacial en tejido afectado por FUFLP y FBFLP.


Subject(s)
Cleft Lip/metabolism , Cleft Palate/metabolism , Homeodomain Proteins/metabolism , SOXB1 Transcription Factors/metabolism , Nestin/metabolism , Immunohistochemistry
3.
Biol. Res ; 50: 31, 2017. graf
Article in English | LILACS | ID: biblio-950882

ABSTRACT

BACKGROUND: MiR-320 is downregulated in multiple cancers, including glioma and acts as tumor suppressor through inhibiting tumor cells proliferation and inducing apoptosis. PBX3 (Pre-B cell leukemia homeobox 3), a putative target gene of miR-320, has been reported to be upregulated in various tumors and promote tumor cell growth through regulating MAKP/ERK pathway. This study aimed to verify whether miR-320 influences glioma cells growth through regulating PBX3. METHODS: Twenty-four human glioma and paired adjacent nontumorous tissues were collected for determination of miR-320 and PBX3 expression using RT-qPCR and western blot assays. Luciferase reporter assay was performed to verify the interaction between miR-320 and its targeting sequence in the 3' UTR of PBX3 in glioma cells U87 and U251. Increased miR-320 level in U87 and U251 cells was achieved through miR-320 mimic transfection and the effect of which on glioma cells growth, proliferation, cell cycle, apoptosis and activation of Raf-1/MAPK pathway was determined using MTT, colony formation, flow cytometry and western blot assays. PBX3 knockdown was performed using shPBX3 and the influence on MAPK pathway activation was evaluated. RESULTS: MiR-320 downregulation and PBX3 upregulation was found in glioma tissues. Luciferase reporter assays identified miR-320 directly blinds to the 3' UTR of PBX3 in glioma cells. MiR-320 mimic transfection suppressed glioma cells proliferation, and induced cell cycle arrest and apoptosis. Both miR-320 overexpression and PBX3 knockdown inhibited Raf-1/MAPK activation. CONCLUSION: MiR-320 may suppress glioma cells growth and induced apoptosis through the PBX3/Raf-1/MAPK axis, and miR-320 oligonucleotides may be a potential cancer therapeutic for glioma.


Subject(s)
Humans , Brain Neoplasms/metabolism , Proto-Oncogene Proteins/metabolism , Homeodomain Proteins/metabolism , MicroRNAs/metabolism , Glioma/metabolism , Brain Neoplasms/pathology , Down-Regulation , Gene Expression Regulation, Neoplastic , Cell Differentiation/drug effects , Up-Regulation , Cell Line, Tumor , Cell Proliferation/drug effects , Glioma/pathology
5.
Int. braz. j. urol ; 39(6): 875-883, Nov-Dec/2013. tab, graf
Article in English | LILACS | ID: lil-699121

ABSTRACT

Objectives Five-alpha reductase inhibitors (5ARIs) are known as chemopreventive agents in prostate cancer with a risk of high-grade disease. This study evaluated the effects of 5ARI on androgen receptor (AR) and proteins involved in prostate cell growth such as HOXB13 expression in human prostate tissue and LNCaP prostate cancer cells. Materials and Methods We retrospectively selected 21 patients who underwent TURP between March 2007 and February 2010 for previously confirmed BPH by prostate biopsy. They were grouped into control (group 1, n = 9) and 5ARI treatment (group 2, n = 12) before TURP. AR and HOXB13 expression in prostate tissue was evaluated by immunohistochemical staining. We tested the effect of 5ARI on the expression of AR, prostate specific antigen (PSA) and HOXB13 in LNCaP cells. Cells were assessed by Western blot analysis, MTT in vitro proliferation assay, and ELISA. Results: Group 2 showed stronger reactivity for AR and HOXB13 than those of the group 1. MTT assay showed death of LNCaP cells at 25uM of 5ARI. At the same time, ELISA assay for PSA showed that 5ARI inhibited secretion of PSA in LNCaP cells. Western blot analysis showed that 5ARI did not greatly alter AR expression but it stimulated the expression of HOXB13. Conclusions These results demonstrated that 5ARI influences AR and HOXB13 expression in both LNCaP cells and human prostate tissue. In order to use 5ARI in chemoprevention of prostate cancer, we still need to clarify the influence of 5ARI in ARs and oncogenic proteins and its regulation pathway. .


Subject(s)
Aged , Humans , Male , /therapeutic use , Homeodomain Proteins/metabolism , Prostatic Hyperplasia/drug therapy , Receptors, Androgen/metabolism , Azasteroids/therapeutic use , Blotting, Western , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Prostate-Specific Antigen/blood , Prostate/chemistry , Prostate/drug effects , Prostatic Hyperplasia/metabolism , Retrospective Studies , Time Factors , Tumor Cells, Cultured , Transcription Factors/analysis
6.
Clinics ; 68(6): 887-891, jun. 2013. tab, graf
Article in English | LILACS | ID: lil-676940

ABSTRACT

OBJECTIVE: The expression of transcription factors involved in early pituitary development, such as PROP1 and POU1F1, has been detected in pituitary adenoma tissues. In this study, we sought to characterize the transcriptional profiles of PROP1, POU1F1, and TBX19 in functioning and nonfunctioning pituitary adenomas in an attempt to identify their roles in tumorigenesis and hormone hypersecretion. METHODS: RT-qPCR analyses were performed to assess the transcriptional pattern of PROP1, POU1F1, TBX19, and hormone-producing genes in tissue samples of corticotrophinomas (n = 10), somatotrophinomas (n = 8), and nonfunctioning adenomas (n = 6). RESULTS: Compared with normal pituitary tissue, POU1F1 was overexpressed in somatotrophinomas by 3-fold. PROP1 expression was 18-fold higher in corticotrophinomas, 10-fold higher in somatotrophinomas, and 3-fold higher in nonfunctioning adenomas. TBX19 expression was 27-fold higher in corticotrophinomas. Additionally, the level of TBX19 mRNA positively correlated with that of pro-opiomelanocortin (r = 0.49, p = 0.014). CONCLUSIONS: Our data demonstrate that PROP1 is overexpressed in pituitary adenomas, mainly in corticotrophinomas. Together with previously published data showing that patients who harbor PROP1 loss-of-function mutations present a progressive decline in corticotrope function, our results support a role for PROP1 in pituitary tumor development and in the maintenance of cell lineages committed to corticotrophic differentiation. .


Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , ACTH-Secreting Pituitary Adenoma/metabolism , Adenoma/metabolism , Homeodomain Proteins/metabolism , Neoplasm Proteins/metabolism , T-Box Domain Proteins/metabolism , Transcription Factor Pit-1/metabolism , ACTH-Secreting Pituitary Adenoma/genetics , ACTH-Secreting Pituitary Adenoma/pathology , Adenoma/genetics , Adenoma/pathology , Cell Differentiation , Homeodomain Proteins/genetics , Immunohistochemistry , Neoplasm Proteins/genetics , Pituitary Gland , Real-Time Polymerase Chain Reaction , RNA, Messenger/metabolism , T-Box Domain Proteins/genetics , Transcription Factor Pit-1/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
7.
Indian J Exp Biol ; 2013 Mar; 51(3): 201-207
Article in English | IMSEAR | ID: sea-147583

ABSTRACT

In the experimental group (shh inhibited group), there were significant decreases in the expression of Oct4, Nanog, Shh, GATA4, Brachyury and Goosecoid, while increases were observed for TAT and Pdx1. The expression of Sox17 did not differ between two control and experimental groups. In experimental group, the amount of GSC positive cells was somehow lower but it seems that there was no difference for Sox17. Shh inhibition induces ESCs to differentiate toward definitive endoderm by committing mesendodermal lineages.


Subject(s)
Animals , Cell Differentiation , Cell Line , Cell Lineage , DNA Primers , Dithizone/pharmacology , Embryonic Stem Cells/cytology , Endoderm/metabolism , Gene Expression Regulation, Developmental , Hedgehog Proteins/metabolism , Homeodomain Proteins/metabolism , Immunohistochemistry , Mesoderm/metabolism , Mice , Microscopy, Fluorescence , Octamer Transcription Factor-3/metabolism , Reverse Transcriptase Polymerase Chain Reaction
8.
Article in English | WPRIM | ID: wpr-44053

ABSTRACT

The genetic alterations of vitamin D receptor (VDR) are related with the growth of long bone. There were a lot of reports regarding an association of polymorphisms in the VDR promoter with many disorders, but not with idiopathic short stature (ISS). We investigated the association of them with ISS. A total of 50 subjects, including 29 ISS patients and 21 healthy controls with their heights within the normal range was recruited. We selected two single nucleotide polymorphisms (SNPs) from VDR promoter (rs11568820 at the Cdx-2 binding site upstream of exon 1e and rs4516035 at -1012 upstream of exon 1a) as candidates, respectively. In genotype analysis, the frequency of A/A genotype at the Cdx-2 binding site locus (rs11568820) upstream of exon 1e of VDR was decreased to 6.9% in ISS patients (28.6% in controls) (P = 0.027). The genetic variation at the Cdx-2 binding site of VDR promoter can be a contributing factor of growth of height.


Subject(s)
Adolescent , Alleles , Binding Sites , Child , Dwarfism/genetics , Exons , Female , Gene Frequency , Genotype , Homeodomain Proteins/metabolism , Humans , Male , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Receptors, Calcitriol/genetics
9.
Biocell ; 36(3): 127-132, Dec. 2012. ilus, graf
Article in English | LILACS | ID: lil-694713

ABSTRACT

PH domains (pleckstrin homology) are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular apartments with assistances of alternative binding partners. PH domain-containing proteins are found to be involved in a wide range of cellular events, including signalling, cytoskeleton rearrangement and vesicular trafficking. Here we showed that a novel PH domain-containing protein, PEPP2, displayed moderate phosphoinositide binding specificity. Full length PEPP2 associated with both plasma membrane and microtubules. The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells. Overexpression of PEPP2 increased membrane microviscosity, indicating a potential role of PEPP2 in regulating function of membrane and microtubules.


Subject(s)
Animals , Cell Membrane/metabolism , Cytoskeleton/metabolism , Homeodomain Proteins/metabolism , Androstadienes/pharmacology , Chlorocebus aethiops , COS Cells , Diffusion , Glutathione Transferase/metabolism , Lipids/chemistry , Microscopy, Fluorescence , Models, Biological , Microtubules/metabolism , Protein Binding , Protein Structure, Tertiary , Phosphatidylinositols/chemistry , Recombinant Fusion Proteins/chemistry , Signal Transduction , Viscosity , Wound Healing
10.
Article in Korean | WPRIM | ID: wpr-130102

ABSTRACT

Gastric hepatoid adenocarcinoma is a special type of gastric carcinoma, which produces AFP. We report a case of an metastatic gastric hepatoid adenocarcinoma mistaken for primary hepatocellular carcinoma (HCC). A 72 year-old woman was transferred to our hospital for treatment of the hepatic mass. She underwent subtotal gastrectomy for gastric cancer 2 years ago. A year ago, she was diagnosed with hepatic mass and treated with transhepatic chemoembolization under the suspicion of primary HCC in other hospital. The hepatic mass looked like primary HCC on CT, and serum AFP was elevated to 18,735 IU/mL. We did the transhepatic mass biopsy and compared it to the histology of the previous gastric cancer. The results of immunohistochemical staining between them was coincident, and so it was diagnosed as a hepatic metastasis of gastric hepatoid adenocarcinoma.


Subject(s)
Adenocarcinoma/diagnosis , Aged , Carcinoma, Hepatocellular/diagnosis , Embolization, Therapeutic , Endoscopy, Gastrointestinal , Homeodomain Proteins/metabolism , Humans , Keratin-20/metabolism , Keratin-7/metabolism , Liver Neoplasms/diagnosis , Male , Stomach Neoplasms/diagnosis , Tomography, X-Ray Computed , alpha-Fetoproteins/analysis
11.
Article in Korean | WPRIM | ID: wpr-130087

ABSTRACT

Gastric hepatoid adenocarcinoma is a special type of gastric carcinoma, which produces AFP. We report a case of an metastatic gastric hepatoid adenocarcinoma mistaken for primary hepatocellular carcinoma (HCC). A 72 year-old woman was transferred to our hospital for treatment of the hepatic mass. She underwent subtotal gastrectomy for gastric cancer 2 years ago. A year ago, she was diagnosed with hepatic mass and treated with transhepatic chemoembolization under the suspicion of primary HCC in other hospital. The hepatic mass looked like primary HCC on CT, and serum AFP was elevated to 18,735 IU/mL. We did the transhepatic mass biopsy and compared it to the histology of the previous gastric cancer. The results of immunohistochemical staining between them was coincident, and so it was diagnosed as a hepatic metastasis of gastric hepatoid adenocarcinoma.


Subject(s)
Adenocarcinoma/diagnosis , Aged , Carcinoma, Hepatocellular/diagnosis , Embolization, Therapeutic , Endoscopy, Gastrointestinal , Homeodomain Proteins/metabolism , Humans , Keratin-20/metabolism , Keratin-7/metabolism , Liver Neoplasms/diagnosis , Male , Stomach Neoplasms/diagnosis , Tomography, X-Ray Computed , alpha-Fetoproteins/analysis
12.
Article in English | WPRIM | ID: wpr-211721

ABSTRACT

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin-producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis-derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal-endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic beta-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin-induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.


Subject(s)
Animals , Biomarkers/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Proliferation/drug effects , Cell Separation , Cells, Cultured , Dermis/cytology , Diabetes Mellitus, Experimental/surgery , Female , Fibroblasts/cytology , Genitalia, Female/cytology , Glucose/metabolism , Hepatocyte Nuclear Factor 3-beta/metabolism , Homeodomain Proteins/metabolism , Humans , Insulin/pharmacology , Insulin-Secreting Cells/cytology , Islets of Langerhans Transplantation , Mesenchymal Stem Cells/cytology , Mice , Mice, Nude , Niacinamide/pharmacology , Recovery of Function , SOXF Transcription Factors/metabolism , Sodium Selenite/pharmacology , Trans-Activators/metabolism , Transferrin/pharmacology
13.
Article in Korean | WPRIM | ID: wpr-33538

ABSTRACT

Symptomatic gastro-intestinal metastasis in lung cancer is extremely rare and only a few case reports have been published. Here, we report a case with lung adenocarcinoma that presented with acute abdominal pain, nausea and vomiting due to duodenum, jejunum, and colon obstruction by the gastro-intestinal metastasis. The patient underwent colonoscopy and the pathologic report was adenocarcinoma. When there are similar histologic findings in both colon and pulmonary lesion, the question is whether both lesions are primary cancer or the colon lesions are metastases from lung cancer. Microscopic examination of a conventional pathologic section was not sufficient to make this determination. Immunohistochemistry was positive for thyroid transcription factor-1 (TTF-1) and cytokeratin 7 (CK7), and negative for cytokeratin 20 (CK20) and caudal-related homeobox transcription factor-2 (CDX-2) on colon mucosa specimen. Accordingly, we used immunohistochemical marker for differential diagnosis of primary adenocarcinoma of the lung with gastro-intestinal metastasis.


Subject(s)
Abdominal Pain , Adenocarcinoma/diagnosis , Colonoscopy , Diagnosis, Differential , Gastrointestinal Neoplasms/pathology , Homeodomain Proteins/metabolism , Humans , Immunohistochemistry , Keratin-20/metabolism , Keratin-7/metabolism , Lung Neoplasms/diagnosis , Male , Middle Aged , Nuclear Proteins/metabolism , Tomography, X-Ray Computed , Transcription Factors/metabolism
14.
Indian J Med Sci ; 2010 Sept; 64(9) 402-407
Article in English | IMSEAR | ID: sea-145560

ABSTRACT

Objective: To investigate the molecular mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) into myocardial cells induced by 5-azacytidine (5-aza), and to explore the expression and significance of DLL4-Notch signaling in this process. Materials and Methods: hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. After treatment with 5-aza for 24 h, hUCMSCs was continued to culture, the expression of GATA4 and NKx2.5 at 4 weeks after induction, DLL4 and Notch1 mRNA at 1d, 3d, 5d, 7d after induction were detected. The expression of cardiac troponin I (cTnI) after 4 weeks was determined by immunocytochemistry. Results: hUCMSCs treated with 5-aza were stained positively for cTnI 4 weeks after induction. The expression of Notch1 and DLL4 mRNA in the 5-aza-induced group was stable and significantly higher than that in the control group (mean Ct value for the Notch1 gene: 0.51 ± 0.21 in the 5-aza-induced group vs. 7.85 ± 0.35 in the control group; mean Ct value for the DLL4 gene: 1.60 ± 0.49 in the 5-aza-induced group vs. 12.42 ± 0.73 in the control group). Similar results were observed for Nkx2.5 and GATA4 genes. The expressions of Nkx2.5 and GATA4 mRNA in the 5-aza group were 4.72 ± 0.58 and 3.76 ± 0.06 times higher than that in the control group, respectively, with statistical significance. Conclusions: hUCMSCs can be differentiated into myocardial cells by 5-aza induction in vitro. 5-Aza may affect this process by regulating the expression of GATA4 and Nkx2.5 genes. The DLL4-Notch signal pathway may be involved in this process.


Subject(s)
Azacitidine/metabolism , Cell Differentiation/drug effects , GATA4 Transcription Factor/metabolism , Homeodomain Proteins/metabolism , Humans , Mesenchymal Stem Cells/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Receptor, Notch1/metabolism , Receptors, Notch/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Umbilical Cord/cytology
15.
Braz. j. med. biol. res ; 43(2): 176-185, Feb. 2010. tab, ilus, graf
Article in English | LILACS | ID: lil-538231

ABSTRACT

The molecular mechanisms and potential clinical applications of neural precursor cells have recently been the subject of intensive study. Dlx5, a homeobox transcription factor related to the distal-less gene in Drosophila, was shown to play an important role during forebrain development. The subventricular zone (SVZ) in the adult brain harbors the largest abundance of neural precursors. The anterior SVZ (SVZa) contains the most representative neural precursors in the SVZ. Further research is necessary to elucidate how Dlx5-related genes regulate the differentiation of SVZa neural precursors. Here, we employed immunohistochemistry and molecular biology techniques to study the expression of Dlx5 and related homeobox genes Er81 and Islet1 in neonatal rat brain and in in vitro cultured SVZa neural precursors. Our results show that Dlx5 and Er81 are also highly expressed in the SVZa, rostral migratory stream, and olfactory bulb. Islet1 is only expressed in the striatum. In cultured SVZa neural precursors, Dlx5 mRNA expression gradually decreased with subsequent cell passages and was completely lost by passage four. We also transfected a Dlx5 recombinant plasmid and found that Dlx5 overexpression promoted neuronal differentiation of in vitro cultured SVZa neural precursors. Taken together, our data suggest that Dlx5 plays an important role during neuronal differentiation.


Subject(s)
Animals , Rats , Cerebral Ventricles/cytology , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/metabolism , Neurogenesis/physiology , Neurons/cytology , Animals, Newborn , Cell Differentiation/physiology , Cerebral Ventricles/metabolism , Homeodomain Proteins/genetics , Immunohistochemistry/methods , Neurons/physiology , Rats, Wistar , Transfection
16.
Article in English | WPRIM | ID: wpr-635007

ABSTRACT

Pdx-1, an important transcription factor highlighting in the early pancreatic development, islet functions and pancreatic disorders, needs to be more investigated in zebrafish, and siRNA is still seldom applied in zebrafish embryo-related research. Our aim was to explore the role of pdx-1 in pancreatic development of zebrafish embryos by using siRNA approach. Microinjection, reverse transcriptase-PCR (RT-PCR), in situ hybridization and immunofluorescent staining were used in this research, and the morphology of the islet in normal zebrafish embryos, and in those treated with the siRNA specific to pdx-1 (siPDX-1) or siGFP was observed and compared. The expression of pdx-1 was detected in the stages of 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, 16-hour by RT-PCT. The in situ hybridization and immunofluorescent staining results showed that siPDX-1 disturbed the formation of the islet in zebrafish embryos. Pdx-1 played multiple roles in maintaining the phenotype of the islet during embryogenesis in zebrafish.


Subject(s)
Embryo, Nonmammalian , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Islets of Langerhans/cytology , Islets of Langerhans/embryology , Islets of Langerhans/metabolism , RNA Interference , RNA, Small Interfering/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , Zebrafish
17.
Article in English | WPRIM | ID: wpr-634879

ABSTRACT

HOXA10 gene plays an essential role in differentiation of the endometrium and in human reproduction. The aim of this study was to investigate the regulatory effect of sex steroids and HB-EGF on HOXA10 gene in Ishikawa cells. Ishikawa cells were incubated with 17-beta estradiol (10(-8) mol/L), medroxyprogesterone acetate (MPA) (10(-6) mol/L), RU486 (10(-5) mol/L) or HB-EGF (10 ng/mL) for 48 h respectively. The expression of HOXA10 gene was detected by immunofluorescence, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Our results showed that either estrogen alone, progestin alone or progestin combined with estrogen could significantly increase the expression of HOXA10 gene 48 h after the treatment (P<0.05). But estrogen combined with progestin and RU486 could inhibit the up-regulation by estrogen and progestin. HB-EGF could elevate the expression of HOXA10 gene 48 h after the treatment (P<0.05). It is concluded that both estrogen and progestin can up-regulate the expression of HOXA10 gene in Ishikawa cells, but RU486 can inhibit the effect and HB-EGF can elevate the expression level of HOXA10 gene.


Subject(s)
Cell Line, Tumor , Endometrial Neoplasms/pathology , Estradiol/pharmacology , Genes, Homeobox , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Medroxyprogesterone Acetate/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism
18.
Article in Korean | WPRIM | ID: wpr-70847

ABSTRACT

BACKGROUND/AIMS: The caudal-related homeobox transcription factor, Cdx2, plays an important role in proliferation and differentiation of intestinal epithelial cells. Its expression is confined to normal and neoplastic intestinal epithelium. We evaluated Cdx2 expression in advanced colorectal cancers to determine the correlation between Cdx2 expression and clinicopathologic characteristics. METHODS: Four hundreds twenty consecutive colorectal cancers were included in the study. Cdx2 expression was investigated by immunohistochemistry using tissue microarrays constructed from surgically resected specimens. 145 invasive breast cancers, normal tissues from gastric mucosa, liver, lung, kidney and ovary were used as control. Nuclear staining was considered to be positive and the result was divided into 3 categories. RESULTS: In the colorectal cancers, Cdx2 was expressed in 380 of 420 (90.5%) cases, and 349 of 380 (83%) cases showed strong and diffuse staining and 31 of 420 (7.5%) cases showed weakly positive staining. Forty patients (9.5%) of colorectal cancer were negative for Cdx2. All of the invasive breast cancers and all non-neoplastic control tissues except the regions of intestinal metaplasia in gastric mucosa, which showed strong Cdx2 expression, were negative for Cdx2. Loss of Cdx2 expression was observed more frequently in cases with deeper invasion (p<0.05), lymph node metastasis (p<0.05), poor histologic differentiation (p<0.001), and distant metastasis (p<0.05). CONCLUSIONS: Cdx2 could be a highly sensitive marker to detect metastasis from intestine and might be useful as a novel prognostic marker in colorectal cancers.


Subject(s)
Adult , Aged , Aged, 80 and over , Colorectal Neoplasms/metabolism , Female , Homeodomain Proteins/metabolism , Humans , Immunohistochemistry , Male , Middle Aged , Biomarkers, Tumor/analysis
19.
Article in English | WPRIM | ID: wpr-20724

ABSTRACT

BACKGROUND: CDX1 and CDX2 are members of the caudal-type homeobox gene family and control the proliferation and differentiation of intestinal mucosal cells. Their expressions are commonly reduced in colorectal cancer, but reports about the relationships between their expressions and clinicopathologic features are rare. The aim of this study was to examine the expressions of CDX1 and CDX2 mRNAs in colorectal cancers and to assess the relationships between their expressions and clinicopathologic features. METHODS: CDX1 and CDX2 mRNA expressions were analyzed by real-time polymerase chain reaction in 48 colorectal cancers and in adjacent non-tumorous normal mucosal tissue. RESULTS: CDX1 and CDX2 mRNA expressions were significantly reduced in colorectal cancer tissues versus normal mucosal tissues (p=0.001, p=0.042, respectively). As compared with paired normal mucosal tissues, colorectal tissues showed reduced CDX1 mRNA expression in 64.6% (31/48) and reduced CDX2 mRNA expression in 66.7% (32/48) of cases. A statistically significant positive correlation was found between the expressions of CDX1 mRNA and CDX2 mRNA in colorectal cancer (r=0.543, p< 0.001). However, the expressions of CDX1 and CDX2 mRNAs were not related to age, sex, cancer location, differentiation, lymphatic or vascular invasion, lymph node metastasis, stage or serum carcinoembryonic antigen level. CONCLUSIONS: CDX1 and CDX2 mRNA expressions were found to be significantly reduced in colorectal cancers, but these expressional changes were not found to be related to clinicopathologic features.


Subject(s)
RNA, Messenger/metabolism , Polymerase Chain Reaction , Middle Aged , Male , Humans , Homeodomain Proteins/metabolism , Female , Colorectal Neoplasms/metabolism
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