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1.
Cancer Biol Ther ; 17(11): 1149-1157, 2016 11.
Article in English | MEDLINE | ID: mdl-27686621

ABSTRACT

This study aimed to understand the exact function and potential mechanism of miR-4500 in colorectal cancer (CRC). In this study, the expression of miR-4500 was decreased in both CRC cells and tissues, and downregulated miR-4500 indicated advanced tumor stage and poor survival. By bisulfite sequencing analysis, we found that the CpG island in the promoter region of miR-4500 was hypermethylated in CRC cells and tissues compared with normal control cells and non-tumor tissues, respectively. Functionally, gain- and loss-of-function analyses indicated the tumor suppressor role of miR-4500: it suppressed cell proliferation, cell cycle progression, migration, and invasion. Predictive algorithms and experimental analyses identified HMGA2 as a direct target of miR-4500. Reintroducing HMGA2 impaired the inhibitory effects of miR-4500 on cell growth and motility. Clinically, higher HMGA2 protein expression in CRC tissues was associated with advanced tumor stage and poor survival. An inverse correlation was found between miR-4500 levels and HMGA2 protein expression. Taken together, this study provides the first evidence that miR-4500 functions as a novel tumor suppressor in the miR-4500/HMGA2 axis in colorectal carcinogenesis, and restoring miR-4500 expression might represent a promising therapeutic strategy for CRC.


Subject(s)
Colorectal Neoplasms/genetics , HMGA2 Protein/genetics , MicroRNAs/genetics , Animals , Cell Proliferation/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , DNA Methylation , Down-Regulation , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , HCT116 Cells , HMGA2 Protein/metabolism , Heterografts , Humans , Mice , Mice, Nude , MicroRNAs/biosynthesis , Prognosis
2.
World J Gastroenterol ; 22(12): 3451-9, 2016 Mar 28.
Article in English | MEDLINE | ID: mdl-27022227

ABSTRACT

AIM: To study differences in the visceral sensitivity of the colonic mucosa between patients with diarrhea-predominant irritable bowel syndrome (IBS-D) and those with ulcerative colitis (UC) in remission and to relate these differences with changes in the 5-hydroxytryptophan (5-HT) signaling pathway. METHODS: Gastrointestinal symptoms were used to determine the clinical symptom scores and rectal visceral sensitivity of patients with IBS-D and patients with UC in remission. Blood levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) were measured using an HPLC-electrochemical detection system. The levels of 5-HT 3 receptor (3R), 4R, and 7R mRNAs in colonic biopsy samples were detected using reverse transcription-polymerase chain reaction. The protein expression of TPH1 was analyzed by Western blot and immunohistochemistry. RESULTS: Abdominal pain or discomfort, stool frequency, and the scores of these symptoms in combination with gastrointestinal symptoms were higher in the IBS-D and UC groups than in the control groups. However, no significant differences were observed between the IBS-D and UC remission groups. With respect to rectal visceral sensitivity, the UC remission and IBS-D groups showed a decrease in the initial perception threshold, defecating threshold and pain threshold. However, these groups exhibited significantly increased anorectal relaxation pressure. Tests examining the main indicators of the 5-HT signaling pathway showed that the plasma 5-HT levels, 5-HIAA concentrations, TPH1 expression in the colonic mucosa, and 5-HT3R and 5-HT5R expression were increased in both the IBS-D and the UC remission groups; no increases were observed with respect to 5-HT7R expression. CONCLUSION: The IBS-D and UC groups showed similar clinical symptom scores, visceral sensitivity, and levels of serotonin signaling pathway indicators in the plasma and colonic mucosa. However, the pain threshold and 5-HT7R expression in the colonic mucosa were significantly different between these groups. The results reveal that (1) IBS-D and UC are related to visceral sensitivity pathogenesis and the clinical manifestations of these conditions and (2) the observed differences in visceral hypersensitivity are possibly due to differences in levels of the 5-HT7 receptor, a component of the 5-HT signaling pathway.


Subject(s)
5-Hydroxytryptophan/blood , Colitis, Ulcerative/blood , Colon/metabolism , Diarrhea/blood , Intestinal Mucosa/metabolism , Irritable Bowel Syndrome/blood , Signal Transduction , Abdominal Pain/blood , Abdominal Pain/etiology , Adult , Colitis, Ulcerative/complications , Colitis, Ulcerative/diagnosis , Colitis, Ulcerative/physiopathology , Colon/innervation , Defecation , Diarrhea/diagnosis , Diarrhea/etiology , Diarrhea/physiopathology , Female , Humans , Hydroxyindoleacetic Acid/blood , Intestinal Mucosa/innervation , Irritable Bowel Syndrome/complications , Irritable Bowel Syndrome/diagnosis , Irritable Bowel Syndrome/physiopathology , Male , Middle Aged , Pain Perception , Pain Threshold , Prospective Studies , Receptors, Serotonin/genetics , Receptors, Serotonin/metabolism , Remission Induction , Serotonin Plasma Membrane Transport Proteins/metabolism , Tryptophan Hydroxylase/metabolism , Young Adult
3.
Zhongguo Zhong Xi Yi Jie He Za Zhi ; 35(4): 419-24, 2015 Apr.
Article in Chinese | MEDLINE | ID: mdl-26043563

ABSTRACT

OBJECTIVE: To explore the intervention of baicalin on signal transduction and activating transcription factor expression of ulcerative colitis (UC) patients. METHODS: Recruited were UC patients at Outpatient Department of Digestive Disease, Inpatient Department of Digestive Disease, Center for Digestive Endoscopy of College City Branch, Guangdong Provincial Hospital of Traditional Chinese Medicine, and Southern Hospital affiliated to Southern Medical University from June 2010 to January 2011. They were assigned to the UC group (33 cases) and the diarrhea-predominant irritable bowel syndrome (IBS-D) group (30 cases). Another 30 healthy subjects were recruited as a healthy control group. Peripheral blood mononuclear cells (PBMCs) in vitro intervened by different concentrations baicalin were taken from UC patients. IL23R gene expressions in vitro intervened by different concentrations baicalin were detected using Q-PCR. Expressions of signal transducer and activator of transcription 4 (STAT4) , STAT6, phosphorylated-STAT4 (p-STAT4), and p-STAT6 were detected using Western blot. Serum levels of IFN-γ, IL-4, IL-6, and IL-10 were measured by ELISA. Effects of different concentrations baicalin on expressions of PBMCs, and levels of IFN-γ, IL-4, IL-10 of UC patients were also detected. RESULTS: Compared with the negative control group, 40 µmol baicalin obviously decreased IL23R gene expression of UC patients (P <0. 01). Compared with the healthy control group and the IBS-D group, p-STAT4/STAT4 ratios increased, p-STAT6/STAT6 ratios decreased, levels of IFN-γ, IL-4, IL-10 all increased in the US group (all P <0. 05). Compared with the negative control, 5 and 10 µmol baicalin groups, 20 and 40 moL baicalin obviously decreased p-STAT4/STAT4 ratios (all P <0. 05); 20 and 40 µmoL baicalin obviously increased p-STAT6/STAT6 ratios (all P <0. 05); 20 and 40 µmoL baicalin obviously lowered levels of IFN-γ and IL-4, and elevated IL-10 levels (all P <0. 05). CONCLUSION: 40 µmoL baicalin could in vitro inhibit p-STAT4/STAT4 ratios, adjust p-STAT6/STAT6 ratios and related cytokines, thereby balancing the immunity and relieving inflammatory reactions of UC.


Subject(s)
Activating Transcription Factors/metabolism , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Colitis, Ulcerative/drug therapy , Flavonoids/therapeutic use , Blotting, Western , Colitis, Ulcerative/metabolism , Cytokines/metabolism , Humans , Interleukin-10/metabolism , Interleukin-4/metabolism , Interleukin-6/metabolism , Irritable Bowel Syndrome/drug therapy , Irritable Bowel Syndrome/metabolism , Leukocytes, Mononuclear , Medicine, Chinese Traditional , Phosphorylation , STAT6 Transcription Factor/metabolism , Signal Transduction
4.
World J Gastroenterol ; 20(41): 15299-309, 2014 Nov 07.
Article in English | MEDLINE | ID: mdl-25386078

ABSTRACT

AIM: To evaluate the role of baicalin in ulcerative colitis (UC) with regard to the CD4(+)CD29(+) T helper cell, its surface markers and serum inflammatory cytokines. METHODS: Flow cytometry was used to detect the percentage of CD4(+)CD29(+) cells in patients with UC. Real time polymerase chain reaction was used to detect expression of GATA-3, forkhead box P3, T-box expressed in T cells (T-bet), and retinoic acid-related orphan nuclear hormone receptor C (RORC). Western blotting was used to analyze expression of nuclear factor-κB (NF-κB) p65, phosphorylation of NF-κB (p-NF-κB) p65, STAT4, p-STAT4, STAT6 and p-STAT6. The concentrations of interferon-γ (IFN-γ), interleukin (IL)-4, IL-5, IL-6, IL-10 and TGF-ß in serum were determined by ELISA assay. RESULTS: The percentages of CD4(+)CD29(+) T cells were lower in treatment with 40 and 20 µmol/L baicalin than in the treatment of no baicalin. Treatment with 40 or 20 µmol/L baicalin significantly upregulated expression of IL-4, TGF-ß1 and IL-10, increased p-STAT6/STAT6 ratio, but downregulated expression of IFN-γ, IL-5, IL-6, RORC, Foxp3 and T-bet, and decreased ratios of T-bet/GATA-3, p-STAT4/STAT4 and p-NF-κB/NF-κB compared to the treatment of no baicalin. CONCLUSION: The results indicate that baicalin regulates immune balance and relieves the ulcerative colitis-induced inflammation reaction by promoting proliferation of CD4(+)CD29(+) cells and modulating immunosuppressive pathways.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Colitis, Ulcerative/immunology , Flavonoids/pharmacology , Immunosuppressive Agents/pharmacology , Integrin beta1/blood , Lymphocyte Activation/drug effects , T-Lymphocytes, Helper-Inducer/drug effects , Adolescent , Adult , Biomarkers/blood , Case-Control Studies , Cell Proliferation/drug effects , Cells, Cultured , Colitis, Ulcerative/blood , Colitis, Ulcerative/genetics , Cytokines/blood , Dose-Response Relationship, Drug , Female , Gene Expression Regulation , Humans , Inflammation Mediators/blood , Integrin beta1/immunology , Male , Middle Aged , Signal Transduction/drug effects , Signal Transduction/genetics , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Time Factors , Young Adult
5.
J Cell Biochem ; 115(3): 596-603, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24142406

ABSTRACT

Pyk2 and Src phosphorylation is initiated by CCL18, which promotes breast cancer metastasis via its functional G protein-coupled receptor PITPNM3. However, the function of Pyk2 and Src in CCL18-induced breast cancer metastasis is poorly understood. Quantitative reverse-transcription polymerase chain reactions (qRT-PCRs), Western blot, boyden chamber assay, and adherence assay were performed to delineate the consequences of Pyk2/Src in CCL18-induced breast cancer cells. Co-immunoprecipitation and immunofluorescence were performed to analyze the interaction of proteins. Upon the binding of CCL18 to PITPNM3, Pyk2 translocates from the cytoplasm to the plasma membrane to form a stable complex with PITPNM3, subsequently activating Src kinase. Moreover, upon stimulation with CCL18, Pyk2 and Src become essential for integrin alpha5/beta1 clustering-dependent adherence, migration, and invasion. Pyk2 and Src are important in CCL18-induced breast cancer metastasis.


Subject(s)
Breast Neoplasms/genetics , Chemokines, CC/genetics , Focal Adhesion Kinase 2/metabolism , src-Family Kinases/metabolism , Breast Neoplasms/pathology , Calcium-Binding Proteins/metabolism , Cell Line, Tumor , Female , Focal Adhesion Kinase 2/genetics , Humans , Membrane Proteins/metabolism , Neoplasm Metastasis , src-Family Kinases/genetics
6.
Sci Transl Med ; 4(130): 130ra48, 2012 Apr 18.
Article in English | MEDLINE | ID: mdl-22517885

ABSTRACT

A major obstacle to developing small interfering RNAs (siRNAs) as cancer drugs is their intracellular delivery to disseminated cancer cells. Fusion proteins of single-chain fragmented antibodies (ScFvs) and positively charged peptides deliver siRNAs into specific target cells. However, the therapeutic potential of ScFv-mediated siRNA delivery has not been evaluated in cancer. Here, we tested whether Polo-like kinase 1 (PLK1) siRNAs complexed with a Her2-ScFv-protamine peptide fusion protein (F5-P) could suppress Her2(+) breast cancer cell lines and primary human cancers in orthotopic breast cancer models. PLK1-siRNAs transferred by F5-P inhibited target gene expression, reduced proliferation, and induced apoptosis of Her2(+) breast cancer cell lines and primary human cancer cells in vitro without triggering an interferon response. Intravenously injected F5-P/PLK1-siRNA complexes concentrated in orthotopic Her2(+) breast cancer xenografts and persisted for at least 72 hours, leading to suppressed PLK1 gene expression and tumor cell apoptosis. The intravenously injected siRNA complexes retarded Her2(+) breast tumor growth, reduced metastasis, and prolonged survival without evident toxicity. F5-P-mediated delivery of a cocktail of PLK1, CCND1, and AKT siRNAs was more effective than an equivalent dose of PLK1-siRNAs alone. These data suggest that F5-P could be used to deliver siRNAs to treat Her2(+) breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/therapy , Cell Cycle Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Receptor, ErbB-2/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Cell Line, Tumor , Cell Proliferation , Female , Humans , Mice , RNA, Small Interfering/genetics , RNA, Small Interfering/physiology , Single-Chain Antibodies/administration & dosage , Single-Chain Antibodies/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Polo-Like Kinase 1
7.
Nan Fang Yi Ke Da Xue Xue Bao ; 29(2): 213-6, 2009 Feb.
Article in Chinese | MEDLINE | ID: mdl-19246281

ABSTRACT

OBJECTIVE: To study the BRCA1 mutations in patients with early-onset breast cancer and their affected relatives in Guangdong province and explore the relationship between BRCA1 mutation and the expressions of estrogen receptor(ER), progesterone receptor(PR), HER2 and ALN. METHODS: From 58 patients with early-onset breast cancer and their affected relatives, the genomic DNA was extracted from the peripheral blood mononuclear cells and the coding regions of the BRCA1 gene was amplified using polymerase chain reaction. BRCA1 gene mutations were screened by denaturing high performance liquid chromatography (DHPLC) and subsequent direct DNA sequencing. The expression of ER, PR, HER2 and ALN were detected with immunohistochemistry and their relations with the gene mutation were analyzed. RESULTS: Disease-related BRCA1 mutations were detected in 2 of the 58 patients, who were younger than 35 years old, including 1 with a novel splice-site mutation (IVS5-1 G-->A). No association was found between this novel mutation and the expressions of ER, PR, HER2 and ALN. CONCLUSION: The incidence of BRCA1 mutation is significantly lower in patients with early-onset breast cancer and their affected relatives in Guangdong province than in the Western populations. The novel mutation identified in BRCA1 gene may represent a mutation characteristic of the patients in Guangdong province. BRCA1 gene mutations may not have any relation with the expression of ER, PR, HER2 and ALN.


Subject(s)
Breast Neoplasms/genetics , Genes, BRCA1 , Mutation , Adult , Age of Onset , Base Sequence , China , DNA Mutational Analysis , Female , Genotype , Humans , Molecular Sequence Data , Receptor, ErbB-2/genetics , Receptors, Estrogen/genetics , Receptors, Progesterone/genetics
8.
Nan Fang Yi Ke Da Xue Xue Bao ; 26(5): 570-2, 2006 May.
Article in Chinese | MEDLINE | ID: mdl-16762851

ABSTRACT

OBJECTIVE: To study the feasibility of vector-mediated RNA interference for HER-2-positive breast cancer therapy. METHODS: A plasmid vector capable of mediating HER-2 RNA interference was constructed, and HER-2-positive breast cancer cell line SKBR-3 was transfected with this constructed vector. The expression of HER-2 mRNA and protein was analyzed by RT-PCR and Western blotting, and the growth and apoptosis of SKBR-3 cells was analyzed after transfection. RESULTS: The expressions of HER-2 mRNA and HER-2 protein was downregulated in response to vector-mediated HER-2 RNA interference, which also resulted in tumor cell growth inhibition and increased number apoptotic cells. CONCLUSION: HER-2 is a good target for RNA interference and RNA interference targeting HER-2 can lead to HER-2 breast cancer cell apoptosis and growth inhibition.


Subject(s)
RNA Interference , RNA, Small Interfering/genetics , Receptor, ErbB-2/metabolism , Apoptosis , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Female , Genetic Vectors , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, ErbB-2/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection
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