ABSTRACT
Idiopathic pulmonary fibrosis treatments are limited, often with severe side effects, highlighting the need for novel options. Taraxerone has diverse biomedical properties, but its mechanism remains unclear. This study investigates taraxerone's impact and the mechanisms involved in bleomycin-induced pulmonary fibrosis in mice. After establishing a pulmonary fibrosis mouse model, taraxerone was intraperitoneally injected continuously for 14-28 days. The in vivo antifibrotic and antioxidative stress effects of taraxerone were assessed. In vitro, the influence of taraxerone on transforming growth factor-ß1-induced myofibroblast transformation and oxidative stress was investigated. Subsequently, quantitative polymerase chain reaction screened the histone deacetylase and Sirtuin family, and taraxerone's effects on SIRT1 were assessed. After SIRT1 siRNA treatment, changes in myofibroblast transformation and antioxidant capacity in response to taraxerone were observed. Acetylation and phosphorylation levels of Smad3 were evaluated. We also examined the binding levels of SIRT1 with Pho-Smad3 and Smad3, as well as the nuclear localization of Smad2/3. EX527 confirmed SIRT1's in vivo action in response to taraxerone. In vitro experiments suggested that taraxerone inhibited myofibroblast differentiation by activating SIRT1 and reducing oxidative stress. We also observed a new interaction between SIRT1 and the Smad complex. Taraxerone activates SIRT1, enabling it to bind directly to Smad3. This leads to reduced Smad complex phosphorylation and limited nuclear translocation. As a result, the transcription of fibrotic factors is reduced. In vivo validation confirms taraxerone's SIRT1-mediated antifibrotic effectiveness. This suggests that targeting SIRT1-mediated inhibition of myofibroblast differentiation could be a key strategy in taraxerone-based therapy for pulmonary fibrosis.
Subject(s)
Antioxidants , Bleomycin , Mice, Inbred C57BL , Oxidative Stress , Pulmonary Fibrosis , Signal Transduction , Sirtuin 1 , Smad3 Protein , Transforming Growth Factor beta1 , Animals , Sirtuin 1/metabolism , Pulmonary Fibrosis/drug therapy , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/metabolism , Mice , Signal Transduction/drug effects , Oxidative Stress/drug effects , Smad3 Protein/metabolism , Antioxidants/pharmacology , Male , Transforming Growth Factor beta1/metabolism , Myofibroblasts/drug effects , Myofibroblasts/metabolism , Disease Models, Animal , Phosphorylation , AcetylationABSTRACT
OBJECTIVES: We aimed to investigate whether skeletal-specific H-type blood vessels exist in alveolar bone and how they function in alveolar bone remodeling. MATERIALS AND METHODS: H-type vessels with high expression of CD31 and Endomucin (CD31hi Emcnhi ) were immunostained in alveolar bone. Abundance and age-related changes in CD31hi Emcnhi endothelial cells (H-ECs) were detected by flow cytometry. Osteoprogenitors association with H-type vessels and bone mass were detected in tooth extraction model of alveolar bone remodeling by immunohistofluorescence and micro-CT, respectively. Transcription and expression of H-EC feature genes during in vitro Notch inhibition were measured by RT-qPCR and immunocytofluorescence. RESULTS: We verified that H-type vessels existed in alveolar bone, the abundance of which was highest at infancy age, then decreased but maintained a constant level during aging. In tooth extraction model, H-ECs significantly increased with concomitant perivascular accumulation of Runx2+ osteoprogenitors and gradually augmentation of bone mass. Notch inhibition of in vitro cultured H-ECs resulted in decreased expression levels of Emcn and hes1, but not Pecam1 or Kdr genes, with decreased expression levels of H-EC numbers, accordingly. CONCLUSIONS: The present study suggests that H-type vessels promote osteogenesis during alveolar bone remodeling. Notch signaling pathway regulates expression of Emcn and possibly determines fate and functions of alveolar H-ECs.
Subject(s)
Bone Remodeling , Endothelial Cells , Osteogenesis , Tooth Extraction , Animals , Mice , Platelet Endothelial Cell Adhesion Molecule-1/geneticsABSTRACT
Osteoporosis and osteoporotic fractures severely compromise quality of life in elderly people and lead to early death. Human umbilical cord mesenchymal stromal cell (MSC)-derived extracellular vesicles (hucMSC-EVs) possess considerable therapeutic effects in tissue repair and regeneration. Thus, in the present study, we investigated the effects of hucMSC-EVs on primary and secondary osteoporosis and explored the underlying mechanisms. Methods: hucMSCs were isolated and cultured. EVs were obtained from the conditioned medium of hucMSCs and determined by using transmission electron microscopy, dynamic light scattering and Western Blot analyses. The effects of hucMSC-EVs on ovariectomy-induced postmenopausal osteoporosis and tail suspension-induced hindlimb disuse osteoporosis in mouse models were assessed by using microcomputed tomography, biomechanical, histochemical and immunohistochemical, as well as histomorphometric analyses. Proteomic analysis was applied between hucMSC-EVs and hucMSCs to screen the candidate proteins that mediate hucMSC-EVs function. The effects of hucMSC-EVs on osteogenic and adipogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs), and osteoclastogenesis of the macrophage cell line RAW264.7 in vitro were determined by using cytochemical staining and quantitative real-time PCR analysis. Subsequently, the roles of the key protein in hucMSC-EVs-induced regulation on BMSCs and RAW264.7 cells were evaluated. Results: hucMSCs were able to differentiate into osteoblasts, adipocytes or chondrocytes and positively expressed CD29, CD44, CD73 and CD90, but negatively expressed CD34 and CD45. The morphological assessment revealed the typical cup- or sphere-shaped morphology of hucMSC-EVs with diameters predominantly ranging from 60 nm to 150 nm and expressed CD9, CD63, CD81 and TSG101. The systemic administration of hucMSC-EVs prevented bone loss and maintained bone strength in osteoporotic mice by enhancing bone formation, reducing marrow fat accumulation and decreasing bone resorption. Proteomic analysis showed that the potently pro-osteogenic protein, CLEC11A (C-type lectin domain family 11, member A) was very highly enriched in hucMSC-EVs. In addition, hucMSC-EVs enhanced the shift from adipogenic to osteogenic differentiation of BMSCs via delivering CLEC11A in vitro. Moreover, CLEC11A was required for the inhibitory effects of hucMSC-EVs on osteoclast formation. Conclusion: Our results suggest that hucMSC-EVs serve as a critical regulator of bone metabolism by transferring CLEC11A and may represent a potential agent for prevention and treatment of osteoporosis.
Subject(s)
Bone and Bones/metabolism , Extracellular Vesicles/metabolism , Hematopoietic Cell Growth Factors/metabolism , Lectins, C-Type/metabolism , Mesenchymal Stem Cells/metabolism , Osteoporosis/metabolism , Umbilical Cord/metabolism , Adipocytes/metabolism , Adipogenesis , Animals , Bone Marrow/metabolism , Cell Differentiation , Chondrocytes/metabolism , Disease Models, Animal , Humans , Mice , Osteoblasts/metabolism , Osteogenesis , Osteoporosis/pathology , Proteomics , RAW 264.7 Cells , Umbilical Cord/cytology , X-Ray MicrotomographyABSTRACT
The Microreader™ 23SP ID System is a novel STR kit, but there are no Mongolian data related to this kit. In this study, allelic frequencies and forensic parameters were obtained from 505 unrelated healthy Mongolians. These samples were amplified using the kit. The dataset successfully passed quality control after being submitted to STRidER (STRidER dataset reference STR000198). A total of 264 alleles were observed, with corresponding allelic frequencies ranged from 0.001 to 0.378. The combined power of discrimination (CPD) and combined probability of exclusion (CPE) of the 22 autosomal STR loci were 0.999999999999999999999999999217318 and 0.999999999776042, respectively. Furthermore, population differentiation comparisons involving previously reported groups were conducted.
Subject(s)
Ethnicity/genetics , Genetics, Population/methods , Microsatellite Repeats , Polymorphism, Genetic , Sequence Analysis, DNA , Databases, Genetic , Female , Gene Frequency , Humans , Male , Mongolia/ethnology , ProbabilityABSTRACT
Recently, researchers identified a distinct vessel subtype called type H vessels that couple angiogenesis and osteogenesis. We previously found that type H vessels are reduced in ovariectomy (OVX)-induced osteoporotic mice, and preosteoclasts are able to secrete platelet-derived growth factor-BB (PDGF-BB) to stimulate type H vessel formation and thereby to promote osteogenesis. This study aimed to explore whether harmine, a ß-carboline alkaloid, is capable of preventing bone loss in OVX mice by promoting preosteoclast PDGF-BB-induced type H vessel formation. METHODS: The impact of harmine on osteoclastogenesis of RANKL-stimulated RAW264.7 cells was verified by gene expression analysis and tartrate-resistant acid phosphatase (TRAP) staining. Enzyme-linked immunosorbent assay (ELISA) was conducted to test PDGF-BB production by preosteoclasts. A series of angiogenesis-related assays in vitro were performed to assess the pro-angiogenic effects of the conditioned media from RANKL-stimulated RAW264.7 cells treated with or without harmine. Meanwhile, the role of PDGF-BB in this process was determined. In vivo, OVX mice were intragastrically administrated with harmine emulsion or an equal volume of vehicle. 2 months later, bone samples were collected for µCT, histological, immunohistochemical and immunofluorescent analyses to evaluate bone mass, osteogenic and osteoclastic activities, as well as the numbers of type H vessels. Bone marrow PDGF-BB concentrations were assessed by ELISA. RESULTS: Exposure of RANKL-stimulated RAW264.7 cells to harmine enhanced the formation of preosteoclasts and the production of PDGF-BB. Harmine augmented the ability of RANKL-stimulated RAW264.7 cells to promote angiogenesis of endothelial cells, whereas the effect was blocked by PDGF-BB inhibition. In vivo, the oral administration of harmine emulsion to OVX mice resulted in enhanced trabecular bone mass and osteogenic responses, increased numbers of preosteoclasts, as well as reduced numbers of osteoclasts and fat cells. Moreover, OVX mice treated with harmine exhibited higher levels of bone marrow PDGF-BB and much more type H vessels in bone. CONCLUSION: Harmine may exert bone-sparing effects by suppression of osteoclast formation and promotion of preosteoclast PDGF-BB-induced angiogenesis.
Subject(s)
Bone and Bones/drug effects , Harmine/pharmacology , Neovascularization, Physiologic/physiology , Osteoporosis/physiopathology , Animals , Becaplermin/metabolism , Bone Diseases, Metabolic/drug therapy , Bone Diseases, Metabolic/metabolism , Bone Resorption/drug therapy , Bone Resorption/metabolism , Bone and Bones/metabolism , Cell Differentiation/drug effects , Cell Line , Culture Media, Conditioned/metabolism , Mice , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteogenesis/drug effects , Osteoporosis/metabolism , Ovariectomy/methods , RAW 264.7 CellsABSTRACT
Osteoporosis is a frequent complication of chronic inflammatory diseases and increases in the pro-inflammatory cytokines make an important contribution to bone loss by promoting bone resorption and impairing bone formation. Omentin-1 is a newly identified adipocytokine that has anti-inflammatory effects, but little is known about the role of omentin-1 in inflammatory osteoporosis. Here we generated global omentin-1 knockout (omentin-1-/-) mice and demonstrated that depletion of omentin-1 induces inflammatory bone loss-like phenotypes in mice, as defined by abnormally elevated pro-inflammatory cytokines, increased osteoclast formation and bone tissue destruction, as well as impaired osteogenic activities. Using an inflammatory cell model induced by tumor necrosis factor-α (TNF-α), we determined that recombinant omentin-1 reduces the production of pro-inflammatory factors in the TNF-α-activated macrophages, and suppresses their anti-osteoblastic and pro-osteoclastic abilities. In the magnesium silicate-induced inflammatory osteoporosis mouse model, the systemic administration of adenoviral-delivered omentin-1 significantly protects from osteoporotic bone loss and inflammation. Our study suggests that omentin-1 can be used as a promising therapeutic agent for the prevention or treatment of inflammatory bone diseases by downregulating the pro-inflammatory cytokines.
ABSTRACT
Nonbinary single-nucleotide polymorphisms (SNPs) are potential forensic genetic markers because their discrimination power is greater than that of normal binary SNPs, and that they can detect highly degraded samples. We previously developed a nonbinary SNP multiplex typing assay. In this study, we selected additional 20 nonbinary SNPs from the NCBI SNP database and verified them through pyrosequencing. These 20 nonbinary SNPs were analyzed using the fluorescent-labeled SNaPshot multiplex SNP typing method. The allele frequencies and genetic parameters of these 20 nonbinary SNPs were determined among 314 unrelated individuals from Han populations from China. The total power of discrimination was 0.9999999999994, and the cumulative probability of exclusion was 0.9986. Moreover, the result of the combination of this 20 nonbinary SNP assay with the 20 nonbinary SNP assay we previously developed demonstrated that the cumulative probability of exclusion of the 40 nonbinary SNPs was 0.999991 and that no significant linkage disequilibrium was observed in all 40 nonbinary SNPs. Thus, we concluded that this new system consisting of new 20 nonbinary SNPs could provide highly informative polymorphic data which would be further used in forensic application and would serve as a potentially valuable supplement to forensic DNA analysis.
Subject(s)
Fluorescence , Forensic Sciences/methods , Polymorphism, Single Nucleotide , Sequence Analysis, DNA , Animals , China , Electrophoresis/methods , Gene Frequency , Genetic Markers , Genetics, Population , Humans , Linkage Disequilibrium , Sensitivity and SpecificityABSTRACT
OBJECTIVE: To examine the expression of Twist1 in cervical cancer and to explore its biological function in the progression of cervical cancer.â© METHODS: The expressions of Twist1 in 32 cervical cancers and matched normal tissues were examined by immunohistochemistry (IHC). Cell invasive ability and the expression of invasion-related genes were determined in RNAi-based Twist1-silencing HeLa cells. The relationship between Twist1 and microRNA-33a (miR-33a) in cervical cancer was studied by Pearson correlation analysis, and the roles of miR-33a in regulation of Twist1 and cell invasiveness were studied.â© RESULTS: The positive expression rate of Twist1 was 75.0% (24/32) and 21.9% (7/32) in the cervical cancer and the matched normal tissues, respectively, with significant difference between them (P<0.05). Twist1 shRNA significantly decreased the invasiveness of HeLa cells (P<0.05). Compared with the matched normal tissues, the expression of miR-33a was increased in the cervical cancer tissues, which was negatively correlated with Twist1 (r=-0.661, P<0.05). Overexpression of miR-33a could significantly suppress Twist1 expression as well as cell invasiveness (P<0.05).â© CONCLUSION: Twist1 is critical for the invasiveness of cervical cancer cells; miR-33a, as a tumor suppressor gene, functions as an upstream regulator of Twist1 and is involved in the invasiveness of cervical cancer cell.
Subject(s)
MicroRNAs/genetics , Neoplasm Invasiveness , Nuclear Proteins/metabolism , Twist-Related Protein 1/metabolism , Uterine Cervical Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , HeLa Cells , Humans , RNA Interference , RNA, Small InterferingABSTRACT
OBJECTIVE: The objective of the present study was to explore the major risk factors of surgical complications using the Clavien-Dindo classification. METHODS: The case-control design was used. A total of 1049 patients who underwent radical gastrectomy in Hunan Cancer Hospital between October 2010 and August 2014 were retrospectively analyzed, including 122 patients (11.6%) with complications and 927 patients (88.4%) with no complications. Risk factors were evaluated. RESULTS: Following radical gastrectomy, 122 patients (11.6%) experienced a total of 151 complications. The incidence of Stages II, IIIa, IIIb, IVa, IVb and V complications was 9.6% (n = 101), 2.5% (n = 26), 1.0% (n = 11), 0.8% (n = 8), 0% (n = 0), and 0.5% (n = 5), respectively. The incidence of severe complications (Stage ≥ IIIa) was 4.8% (n = 50). Multivariate analysis showed that combined resection (Odds Ratio [OR] = 3.36, 95% confidence interval [CI]: 1.71~6.60, P < 0.01), perioperative blood transfusion (OR = 2.13, 95% CI: 1.38-3.29, P < 0.01), and BMI ≥ 25 kg/m(2) (OR = 1.98, 95% CI: 1.16-3.40, P = 0.01) were independent risk factors of complications. CONCLUSIONS: Combined resection, perioperative blood transfusion, and BMI ≥ 25 kg/m(2) are positively correlated with complications.
ABSTRACT
We previously reported that Runx2/miR-3960/miR-2861 regulatory feedback loop stimulates osteoblast differentiation. However, the effect of this feedback loop on the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) remains unclear. Our recent study showed that miR-2861 and miR-3960 expression increases significantly during ß-glycerophosphate-induced osteogenic transdifferentiation of VSMCs. Overexpression of miR-2861 or miR-3960 in VSMCs enhances ß-glycerophosphate-induced osteoblastogenesis, whereas inhibition of miR-2861 or miR-3960 expression attenuates it. MiR-2861 or miR-3960 promotes osteogenic transdifferentiation of VSMCs by targeting histone deacetylase 5 or Homeobox A2, respectively, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Furthermore, overexpression of Runx2 induces miR-2861 and miR-3960 transcription, and knockdown of Runx2 attenuates ß-glycerophosphate-induced miR-2861 and miR-3960 transcription in VSMCs. Thus, our data show that Runx2/miR-3960/miR-2861 positive feedback loop plays an important role in osteogenic transdifferentiation of VSMCs and contributes to vascular calcification.
Subject(s)
Cell Transdifferentiation/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Feedback, Physiological , MicroRNAs/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , Osteogenesis/genetics , Animals , Cell Transdifferentiation/drug effects , Feedback, Physiological/drug effects , Gene Expression Regulation/drug effects , Glycerophosphates/pharmacology , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Male , Mice, Inbred C57BL , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolism , Transcription, Genetic/drug effectsABSTRACT
Cervical cancer, the second most common type of cancer in women worldwide, is responsible for >275,100 mortalities each year and is associated with high-risk human papilloma virus (HR-HPV). HPVs have two important oncogenes, E6 and E7, which have crucial roles in malignant transformation in cervical cancer. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA originally identified in non-small cell lung cancer. Previous studies have revealed that MALAT1 is expressed in numerous tissue types, and is significant in maintaining the normal function of the body. However, it also appeared to be notably upregulated in numerous carcinoma types compared with adjacent non-cancerous tissues. In the present study, it was identified that MALAT1 expression was upregulated in cervical cancer cell lines compared with normal cervical squamous cell samples. Further study into the effect of MALAT1 on cellular phenotype revealed that MALAT1 was able to promote cell migration and proliferation. Of note, it was revealed that the expression of MALAT1 was decreased with the knockdown of HPV16 E6/E7 in CaSki cells. Furthermore, the investigations in clinical samples also revealed that MALAT1 was expressed in HPV-positive cervical squamous cells, but not in HPV-negative normal cervical squamous cells. These results indicate that HPV correlates with MALAT1 deregulation in cervical cancer.
ABSTRACT
To construct an improved biological missile, an immunoconjugate ADM-Dex-ScFv-SA3 was synthesized, which was composed of a hepatocellular carcinoma-specific, single-chain Fv antibody (ScFv-SA3) and a highly potent cytotoxic drug, adriamycin (ADM), as the warhead. Oxidized Dextran T10 (Dex-T10) was used as a linker to connect these two moieties. The 40 KD soluble anti-hepatoma human Trx-ScFv-SA3 protein was expressed in E. coli BL21 (DE3), using a prokaryotic expression vector, pET21a (+)-Trx-ScFv-SA3-His. It was purified using a His-Tag Ni-Agarose column and identified by western blot. The activity of Trx-ScFv-SA3 was verified by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to confirm that it specifically binds to the hepatocellular carcinoma cell line HepG2. To prepare ADM-Dex-ScFv-SA3, ADM was conjugated to the antibody at a molar ratio of 14.21:1. The antitumor effect of the conjugate was tested by MTT assay, plate colony formation assay and xenografts in a nude mice experimental model. In vitro experiments revealed that ADM-Dex-ScFv-SA3 could bind to tumor cells selectively and inhibit the proliferation and the colony formation ability of HepG2 cells. In vivo experiments showed that ADM-Dex-ScFv-SA3 suppressed the tumor growth and prolonged the median survival time in tumor-bearing mice. Tumor histology slides indicated a significantly slower tumor tissue proliferation in the ADM-Dex-ScFv-SA3 group. These data indicate that the targeted drug, ADM-Dex-ScFv-SA3, may be a highly potent and selective therapy for the treatment of hepatoma.
Subject(s)
Antibodies, Neoplasm/administration & dosage , Antineoplastic Agents/administration & dosage , Carcinoma, Hepatocellular/therapy , Doxorubicin/administration & dosage , Hepatocytes/drug effects , Liver Neoplasms, Experimental/therapy , Single-Chain Antibodies/administration & dosage , Animals , Antibodies, Neoplasm/chemistry , Antibodies, Neoplasm/genetics , Antineoplastic Agents/chemistry , Carcinoma, Hepatocellular/immunology , Cell Proliferation/drug effects , Doxorubicin/chemistry , Genetic Vectors/genetics , Hep G2 Cells , Hepatocytes/pathology , Humans , Liver Neoplasms, Experimental/immunology , Mice , Mice, Nude , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , Tumor Burden/drug effects , Tumor Stem Cell Assay , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Paired box 6 (PAX6), a highly conserved transcriptional factor, has been implicated in tumorigenesis. AIM: We aimed to explore the roles and molecular mechanisms of PAX6 and microRNA (miR-7) in colorectal cancer cells. METHODS: Tissue microarray immunohistochemistry and Western blot were applied to examine the PAX6 expression. Real-time RT-PCR and Western blot were performed to determine the expression of miR-7 and PAX6. Luciferase reporter assay was used to determine whether PAX6 was a target of miR-7. Effects of miR-7 and PAX6 on colorectal cell proliferation, cell cycle progression, colony formation and invasion were then investigated. Western blot was used to determine the activities of the ERK and PI3K signal pathways, as well as the protein expression of MMP2 and MMP9. RESULTS: The protein levels of PAX6 were gradually increased, while the expression of miR-7 was gradually reduced with malignancy of colorectal cancer. PAX6 was further identified as a target of miR-7, and its protein expression was negatively regulated by miR-7 in human colorectal cancer cells. Overexpression of PAX6 in Caco-2 and SW480 cells enhanced cellular proliferation, cell cycle progression, colony formation, and invasion, while miR-7 upregulation repressed these biological processes. Furthermore, the activities of ERK and PI3K signal pathways, as well as the protein levels of MMP2 and MMP9, were upregulated in PAX6-overexpressed Caco-2 and SW480 cells but deregulated in miR-7-overexpressed Caco-2 and SW480 cells. CONCLUSIONS: Our study suggests that as a novel target of miR-7, PAX6 may serve as a promising therapeutic target for colorectal cancer.
Subject(s)
Adenocarcinoma/metabolism , Biomarkers, Tumor/metabolism , Colorectal Neoplasms/metabolism , Eye Proteins/metabolism , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/metabolism , MicroRNAs/metabolism , Paired Box Transcription Factors/metabolism , Repressor Proteins/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Blotting, Western , Caco-2 Cells , Cell Proliferation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Humans , Immunohistochemistry , Neoplasm Invasiveness , PAX6 Transcription Factor , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tissue Array Analysis , Up-RegulationABSTRACT
Golgi phosphoprotein 2 (Golph2) is a type II Golgispecific membrane protein, which has been found to be overexpressed in hepatocellular carcinoma (HCC) patients. The sensitivity of diagnosis of HCC using Golph2 (76%) was markedly elevated compared with alphafetoprotein (AFP) (70%), and Golph2 is expected to be a novel and effective serum biomarker for the diagnosis of HCC. The aim of this study was to prepare monoclonal antibodies against Golph2 and to establish double-antibody sandwich enzyme-linked immunosorbent assay (s-ELISA), which will be used in diagnostics, therapeutics and as a tool in understanding the role of Golph2 in the pathogenesis of liver diseases and cancer. In this study, fusion protein TRX-Golph2 was expressed and purified using an Escherichia coli system. BALB/c mice were immunized with TRX-Golph2 recombinant protein. The hybridoma technique was used for the production of anti-Golph2 monoclonal antibody. Hybridoma clones were screened using indirect ELISA and anti-Golph2 monoclonal antibody was produced in the ascites of BALB/c mice. The specificity of anti-Golph2 monoclonal antibody was detected by western blot analysis and immunocytochemistry. s-ELISA was established using horseradish peroxidase (HRP)labeled anti-Golph2 monoclonal antibody and used to detect the antigen in the serum of HCC patients. As a result, five stable hybridoma cell clones (5C6D5, 5B7F5, 7F5F3, 8A7B4, 8C9E8) producing anti-Golph2 monoclonal antibody were established. The highest titer of anti-Golph2 monoclonal antibody (5C6D5) was 1:51,200. Western blot analysis revealed that anti-Golph2 monoclonal antibody had a high specificity for Golph2 protein. Anti-Golph2 monoclonal antibody was HRP-labeled and the optimal working concentration was found to be 1:500. The levels of antigen in a proportion of HCC patients were shown to be significantly higher compared to those found in healthy controls.
Subject(s)
Antibodies, Monoclonal , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/diagnosis , Liver Neoplasms/metabolism , Membrane Proteins/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibody Specificity/immunology , Blotting, Western , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Humans , Hybridomas/metabolism , Immunohistochemistry , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/immunology , MiceABSTRACT
HTA is a novel hepatoma associated gene screened by bioinformatic strategies in our previous study. In the present investigation, the full-length sequence of the HTA gene was cloned by 3'-rapid amplification of cDNA ends (RACE) and 5'-RACE, which was 1414 bp and the open reading frame (ORF) was constituted with 3 exons and 2 introns. There are two types of splicing of the mRNA of HTA. Northern blot analysis showed that the 1.4 kb HTA mRNA and 1.7 kb HTA mRNA transcripts were present in the hepatocellular carcinoma (HCC) cell lines HepG2 and QGY-7703 but not in the normal cell line L02 and the human umbilical vein endothelial cells (HUVECs). Overexpression of the HTA gene in the hepatic cell line QSG-7701 via stable transfection can promote its proliferation rate and colony forming ability and change the cell cycle distribution of the cell lines. These results showed that the HTA gene is a potential therapeutic target in HCC and the clarification of its gene structure and sequence information provide an essential tool for future research.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/genetics , Cloning, Molecular , Genes, Neoplasm , Liver Neoplasms/genetics , Alternative Splicing , Amino Acid Sequence , Cell Line, Tumor , Cell Proliferation , Hep G2 Cells , Human Umbilical Vein Endothelial Cells , Humans , Molecular Sequence Data , Nucleic Acid Amplification Techniques , RNA Interference , RNA, Messenger/genetics , RNA, Small InterferingABSTRACT
We present a detailed method for constructing a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma. Two novel mammalian library vectors pcDNA3-CHm and pcDNA3-CLm were constructed that contained restriction enzyme sites NheI, ClaI and antibody constant domain. Mammalian expression vector pcDNA3-CHm contains IgG heavy-chain (HC) constant region and glycosylphosphatidylinositol anchor (GPI) that could be anchored full-length antibodies on the surface of mammalian cells. GOLPH2 prokaryotic expression vector was carried out in Escherichia coli and purified by immobilized metal affinity chromatography. Variable domain of heavy-chain and variable domain of light-chain genes were respectively inserted into the vector pcDNA3-CHm and pcDNA3-CLm by ligation, and antibody libraries are displayed as whole IgG molecules on the cell surface by co-transfecting this HC-GPI with a light chain. By screening the cell library using magnetic beads and cell ELISA, the cell clone that displayed GOLPH2-specific antibodies on cell surfaces was identified. The mammalian cell-based antibody display library is a great potential application for displaying full-length functional antibodies of targeting hepatocellular carcinoma on the surface of mammalian cells. Anti-GOLPH2 display antibody was successfully isolated from the library.
Subject(s)
Antibodies, Neoplasm/immunology , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Peptide Library , Antibodies, Neoplasm/genetics , Antibodies, Neoplasm/isolation & purification , Cell Line , Cell Surface Display Techniques/methods , Gene Expression , Genetic Vectors , Humans , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purificationABSTRACT
AIM: To investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC). METHODS: Semiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Chang's liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 µmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed. RESULTS: We identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ. CONCLUSION: GABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC.
Subject(s)
Carcinoma, Hepatocellular/pathology , Gene Expression Regulation, Neoplastic , Liver Neoplasms/pathology , Receptors, GABA-A/biosynthesis , gamma-Aminobutyric Acid/metabolism , Animals , Carcinoma, Hepatocellular/metabolism , Cell Line , Cell Line, Tumor , Cell Proliferation , Cell Survival , Hep G2 Cells , Humans , Liver Neoplasms/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Biological , Neoplasm Transplantation , Polymerase Chain Reaction/methods , RNA Interference , RNA, Small Interfering/metabolism , Receptors, GABA-A/physiologyABSTRACT
UNLABELLED: Abstract Conclusion: The selected scFv antibody could specifically recognize and target nasopharyngeal carcinoma (NPC), and could be applied to clinical diagnosis and therapy. OBJECTIVE: The aim was to construct and screen fully human anti-NPC single chain Fv fusion phage libraries, and to identify the specificity of the scFv antibody. METHODS: Peripheral blood mononuclear cells of patients with NPC were immunized in vitro by NPC cells and transformed by Epstein-Barr virus. The total RNAwas used to construct the scFv libraries. By means of ELISA and immunochemistry, the positively bound scFv was selected and identified. The positive scFv was fused to EGFP, and was then expressed in E. coli strain BL21 (DE3) and purified. Furthermore, we observed the binding bioactivity. RESULTS: The fusion protein has the biological activity of binding the NPC cells and emitting green fluorescence. In targeting experiments in vivo, the results showed that the fusion protein can successfully target the NPC.
Subject(s)
Antibodies, Neoplasm/immunology , Nasopharyngeal Neoplasms/immunology , Single-Chain Antibodies/immunology , Animals , Antibodies, Neoplasm/genetics , Carcinoma , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunohistochemistry , Mice , Mice, Nude , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/diagnosis , Nasopharyngeal Neoplasms/drug therapy , Neoplasms, Experimental , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/therapeutic useABSTRACT
Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.