OBJECTIVE: To investigate the molecular mechanism of Wnt5a and Epstein-Barr virus latent membrane protein 1 (LMP1) aberrant expression in the nasopharyngeal carcinogenesis and to estimate if it can act as a molecular marker for nasopharyngeal cancer (NPC). METHODS: Immunohistochemistry combined with previously made tissue microarrays were used to study the expression of Wnt5a and LMP1 in the nasopharyngeal carcinogenesis tissues. We investigated the role of over expression of Wnt5a and LMP1 in the development and progression of NPC and their relation with the clinicopathological features of NPC and whether they could act as molecular markers in benign and malignant NPC. RESULTS: The positive percentage of Wnt5a and LMP1 protein expression in the NPC was significantly increased as compared with that in atypically hyperplastic nasopharyngeal epithelium, hyperplastic nasopharyngeal epithelium and histologically normal nasopharyngeal epithelium (P<0.05, P<0.01, and P<0.01). Wnt5a and LMP1 proteins were significantly higher in atypically hyperplastic nasopharyngeal epithelium than those in the hyperplastic nasopharyngeal epithelium and normal nasopharyngeal epithelium (P<0.05 and P<0.01). The positive expression of Wnt5a and LMP1 proteins in clinical T3 and T4 staged NPC was higher than that in clinical T1 and T2 staged NPC (P<0.01 and P<0.05). The positive expression of Wnt5a protein in the NPC with lymph node metastasis was higher than that in the NPC without lymph node metastasis (P<0.01). The positive percentage of LMP1 protein was significantly increased in non-keratinizing carcinoma compared with undifferentiated carcinoma and keratinizing carcinoma (P<0.05 and P<0.05). The expression of Wnt5a protein in the NPC had significant positive correlation with LMP1 (r=0.354, P<0.001). Combined molecular phenotype of both Wnt5a and LMP1 expression was a good marker to distinguish NPC from non-cancerous nasopharyngeal epithelium. CONCLUSION The expression of Wnt5a and LMP1 protein in the NPC is positively correlated, and both wnt5a and LMP1 protein play important roles in the nasopharyngeal carcinogenesis either together or successively promoting the malignant transformation of nasopharyngeal epithelium and the development and progression of NPC. Both Wnt5a and LMP1 positive expression may act as good markers for NPC differential diagnosis.
Biomarkers, Tumor ; genetics ; metabolism ; Carcinogenesis ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Oncogene Proteins, Viral ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Tissue Array Analysis ; Viral Matrix Proteins ; genetics ; metabolism ; Wnt Proteins ; genetics ; metabolism ; Wnt-5a Protein

OBJECTIVETo study the effect of miR-21 down-regulation on the radiosensitivity of TE-1 cells in vitro.

METHODSTE-1 cells were transfected via lentivirus with a vector containing the antisense oligonucleotides of miR21, and the subclones with stable down-regulation of miR21 expression were selected with puromycin and designated as TE-1-miR21(-), whose expression level of miR21 was determined using real-time quantitative PCR. The radiosensitivity of TE-1 and TE-1-miR21(-) cells were evaluated with colony formation assay, and the expressions of β-catenin was determined using Western blotting and RT-PCR. Flow cytometry was used to analyze the proportion of p75NTR(+) cells in TE-1 and TE-1-miR21(-) cells.

RESULTSA cell subclone stably expressing a low level of miR21 was obtained and verified by real-time quantitative PCR. Colony formation assay showed an enhanced the radiosensitivity of TE-1-miR21(-) cells compared to parental TE-1 cells. RT-PCR revealed no significant changes in β-catenin mRNA expression in TE-1-miR21(-) cells, whereas its β-catenin protein expression was markedly suppressed by high-dose (8 and 10 Gy) irradiation. Flow cytometry assay showed a decreased proportion of p75NTR(+) cells in TE-1-miR21(-) cells compared to that in TE-1 cells.

CONCLUSIONDown-regulation of miR21 can enhance the radiosensitivity of TE-1 cells, which might result from the inactivation of wnt/β-catenin signal pathway and a decreased p75NTR(+) cell proportion.

Cell Line, Tumor ; Down-Regulation ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; MicroRNAs ; genetics ; metabolism ; Radiation Tolerance ; Wnt Proteins ; metabolism ; Wnt Signaling Pathway ; beta Catenin ; metabolism

OBJECTIVE: To investigate the expression and significance of miRNA-324-3p and its target gene WNT2B in tissue specimens of nasopharyngeal carcinoma (NPC) specimens. METHOD: qRT-PCR was used to detect the expression of miRNA-324-3p and WNT2B mRNA, and Western blot was applied to assay the expression of WNT2B protein in 39 cases of NPC specimens and 21 cases of non-carcinoma epithelium. The relationship between their expression levels and clinicopathological characteristics and their correlation with clinical pathological parameters was analyzed. RESULT: The expression of miRNA-324-3p was significantly down-regulated decreased but WNT2B mRNA/protein increased obviously in NPC specimens (P < 0.01). A negative correlation between miRNA-324-3p and WNT2B was spotted (P < 0.05). The expression levels of these markers were closely correlated with T stage, clinic stage and cervical lymph node metastasis (P < 0.05). CONCLUSION The loss of miRNA-324-3p and ectopic WNT2B might co-induce the initiation and progression of NPC.
Carcinoma ; Glycoproteins ; genetics ; metabolism ; Humans ; Lymphatic Metastasis ; MicroRNAs ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; Neoplasm Proteins ; metabolism ; RNA, Messenger ; metabolism ; Wnt Proteins ; genetics ; metabolism

OBJECTIVETo investigate whether the suppression of Wnt10b by siRNA could prevent the development of hair follicle in the cultured rat embryonic skin.

METHODSsiRNA-Wnt10b was synthesized by chemosynthesis method. The dorsal skin of SD rat at embryos were cultured in DMEM in the presence of different percentage of interfering RNA targeting Wnt10b. Wnt10b/beta-catenin expression was analyzed by real-time PCR everyday and by Western blot on the third day. The cultured embryonic skin underwent paraffin embedding, section, HE staining on the third day,in which the number of de novo hair follicle was calculated and statistically analyzed.

RESULTSWnt10b gene in the cultured embryonic skin could be knocked down with the siRNA-based method. Beta-catenin mRNA was not greatly influenced by the downregulation of Wnt10b mRNA. The number of de novo hair follicle placode in cultured embryonic skin decreased, along with the downregulation of Wnt10b and beta-catenin proteins expression.

CONCLUSIONSThe downregulation of Wnt10b mRNA and protein by siRNA reduces the number of de novo hair follicle placode in the cultured rat embryonic skin. Wnt10b may control cytoplasm beta-catenin concentration at the protein level.

Animals ; Hair Follicle ; embryology ; metabolism ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Rats ; Skin ; embryology ; metabolism ; Tissue Culture Techniques ; Wnt Proteins ; genetics ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo investigate methylation status of Wif-1 and β-catenin expression in colorectal serrated lesions.

METHODSVarious colorectal lesions were collected including 52 cases of hyperplastic polyps, 41 cases of sessile serrated adenoma, 23 cases of traditional serrated adenoma, 24 cases of colorectal cancer and 24 cases of normal mucosa. All specimens were subject to immunohistochemical staining of β-catenin.SYBR Green PCR analysis of Wif-1 promoter methylation was performed in 29 cases of hyperplastic polyps, 29 cases of sessile serrated adenoma, 19 cases of traditional serrated adenoma, 14 cases of colorectal cancer and 16 cases of normal mucosa.

RESULTSAbnormal expression rates of β-catenin in normal mucosa, hyperplastic polyps, sessile serrated adenoma, traditional serrated adenoma and colorectal cancer were 12.5% (3/24), 59.6% (31/52), 63.4% (26/41), 73.9% (17/23) and 100.0% (24/24), respectively. The corresponding methylation rates of Wif-1 promoter were 2/16, 10/29 (34.5%), 16/29 (55.2%), 15/19 and 13/14 (P < 0.05), respectively. Abnormal β-catenin expression was positively correlated with Wif-1 promoter methylation in traditional serrated adenomas (r = 0.536, P < 0.05).

CONCLUSIONSAbnormal β-catenin expression and methylation rate of Wif-1 promoter are significantly higher in colorectal serrated lesions. Methylation of Wif-1 promoter may be related to the abnormal expression of β-catenin through activation of Wnt/β-catenin signaling pathway, which may contribute to the development of colorectal serrated lesions.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Adenoma ; genetics ; metabolism ; pathology ; Carcinoma ; genetics ; metabolism ; pathology ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; DNA Methylation ; Humans ; Hyperplasia ; Immunohistochemistry ; Intestinal Mucosa ; metabolism ; Intestinal Polyps ; pathology ; Repressor Proteins ; genetics ; metabolism ; Wnt Signaling Pathway ; beta Catenin ; metabolism

Child ; Child, Preschool ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Infant ; Male ; Wilms Tumor ; genetics ; metabolism ; Wnt Proteins ; genetics ; metabolism ; beta Catenin ; genetics ; metabolism

This study was aimed to investigate the expression level of Wnt5a gene in some hematologic diseases and leukemic cell lines so as to provide a basis for further research of Wnt5a role and its mechanism in hematologic malignancies. The mononuclear cells of peripheral blood and bone marrow were isolated by human lymphocytic isolation solution. The expression of Wnt5a gene in specimen of 31 cases and three leukemic cell lines (Jurkat, K562, HL-60) were detected by RT-PCR. The results showed that in four out of five AML cases, negative or weak positive expressions were observed and negative expressions were observed also in K562 and HL-60 cells. Only in one AML case with complete remission and Jurkat cells the strong positive expressions were observed. The negative expression was observed in all six CML cases. In three out of four ALL cases, the expression was positive or weak positive and one negative. The expressions in two CLL cases were negative. Out of two MM cases, the expression in one was weak positive and in other was negative. Out of three lymphoma cases, the expression in one case was weak positive and in other two cases were negative. There were positive or weak positive expressions in two cases of AA, two cases of IDA, three cases of ITP, one cases of PV and ET cases. It is concluded that there have obvious down-regulated or lost expression of Wnt5a gene in 31 cases of hematologic disease and myelocytic leukemic cell lines except ALL samples. Nevertheless there have general positive expression of Wnt5a in cases of non-malignant hematologic diseases. These results suggest that the genesis of myelocytic leukemia is related to the down-regulated expression of Wnt5a.
Adolescent ; Adult ; Aged ; Child ; Down-Regulation ; Gene Expression Regulation, Leukemic ; Hematologic Neoplasms ; genetics ; metabolism ; Humans ; Middle Aged ; Proto-Oncogene Proteins ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured ; Wnt Proteins ; metabolism ; Wnt-5a Protein ; Young Adult

This study was purposed to investigate the effect of RUNX1 on transcription activity of WNT5A promoter in mouse bone marrow derived mesenchymal stem cells (MSC), and to explore the mechanism by which bone marrow environments regulate MSC. RT-PCR was used to detect the expression of RUNX1 in MSC isolated from mouse bone marrow and cultured in vitro; the chromatin immunoprecipitation (ChIP) was used to investigate the direct in vivo interaction between the RUNX1 and WNT5A promoter; retrovirus system was utilized to introduce the RUNX1 gene into MSC to detect the regulation of RUNX1 on the transcription activity of WNT5A promoter. The results showed that mouse bone marrow derived MSC was positive for Oil Red O, van Kossa and toluidine blue staining respectively and RUNX1 expressed in MSC. WNT5A promoter could be bound by RUNX1, and the expression level of WNT5A was enhanced with the increase of RUNX1. It is concluded that RUNX1 expresses in mouse bone marrow derived MSC, WNT5A is a direct target gene of RUNX1 and its transcriptional activity is regulated by RUNX1.
Animals ; Bone Marrow Cells ; metabolism ; Cell Differentiation ; Cells, Cultured ; Chromatin Immunoprecipitation ; Core Binding Factor Alpha 2 Subunit ; genetics ; Mesenchymal Stromal Cells ; metabolism ; Mice ; Mice, Inbred C57BL ; Transcription, Genetic ; Wnt Proteins ; genetics ; Wnt-5a Protein

Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by beta-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca+2/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled.
Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Animals ; Blastomeres/cytology/*metabolism ; Cell Line ; Embryonic Development/genetics ; *Feedback, Physiological ; Frizzled Receptors/genetics/metabolism ; GTP-Binding Proteins/genetics/*metabolism ; Gene Expression Regulation, Developmental ; Humans ; Mutation ; Phosphoproteins/genetics/*metabolism ; Protein Binding ; RNA, Small Interfering/genetics ; Repressor Proteins/genetics/metabolism ; Transfection ; Wnt Proteins/*genetics/metabolism ; Xenopus ; Xenopus Proteins/*genetics/metabolism

As a member of the hox gene family, hoxB4 gene encodes a class of DNA-dependent homeobox domain nucleoprotein, which is a specific transcription factor, playing an important role in regulating the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Therefore, it is important to understand the mechanisms involved in regulating expression of hoxB4 in the HSC. Previous studies have suggested that some hoxB4 upstream regulatory factors, such as USF-1 (upstream activating factor -1), USF-2 (upstream activating factor -2) and NF-Y complex, as well as hematopoietic cytokines, such as platelet growth factor (TPO) and Wnt3a protein, play important regulatory roles in the expression of hoxB4 in hematopoietic stem cells. In this review the structure and biological characteristics of hoxB4, mechanisms involved in regulating expression of hoxB4 in the HSC are summarized.
CCAAT-Binding Factor ; metabolism ; Gene Expression Regulation ; Genes, Homeobox ; genetics ; physiology ; Hematopoietic Stem Cells ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; physiology ; Humans ; Transcription Factors ; genetics ; metabolism ; physiology ; Upstream Stimulatory Factors ; metabolism ; Wnt Proteins ; metabolism ; Wnt3 Protein ; Wnt3A Protein