ABSTRACT
Adaptive response to physiological oxygen levels (physO2; 5% O2) enables embryonic survival in a low-oxygen developmental environment. However, the mechanism underlying the role of physO2 in supporting preimplantation development, remains elusive. Here, we systematically studied oxygen responses of hallmark events in preimplantation development. Focusing on impeded transcriptional upregulation under atmospheric oxygen levels (atmosO2; 20% O2) during the 2-cell stage, we functionally identified a novel role of HIF-1α in promoting major zygotic genome activation by serving as an oxygen-sensitive transcription factor. Moreover, during blastocyst formation, atmosO2 impeded H3K4me3 and H3K27me3 deposition by deregulating histone-lysine methyltransferases, thus impairing X-chromosome inactivation in blastocysts. In addition, we found atmosO2 impedes metabolic shift to glycolysis before blastocyst formation, thus resulting a low-level histone lactylation deposition. Notably, we also reported an increased sex-dimorphic oxygen response of embryos upon preimplantation development. Together, focusing on genetic and epigenetic events that are essential for embryonic survival and development, the present study advances current knowledge of embryonic adaptive responses to physO2, and provides novel insight into mechanism underlying irreversibly impaired developmental potential due to a short-term atmosO2 exposure.
Subject(s)
Gene Expression Regulation, Developmental , Hypoxia-Inducible Factor 1, alpha Subunit , Zygote , Animals , Female , Male , Mice , Blastocyst/metabolism , Embryonic Development , Histones/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Oxygen/metabolism , Transcriptome , Zygote/metabolismABSTRACT
Bone morphogenetic protein 15 (BMP15) regulates the growth and development of follicles. In particular, the long non-coding RNA H19 plays an important role in mammalian reproduction. However, the function and regulatory mechanism of the interaction of BMP15 with H19 in yak granulosa cell (GC) proliferation, autophagy, and apoptosis are poorly understood. In our study, quantitative reverse-transcription-polymerase chain reaction analysis showed that H19 were highly expressed in yak healthy follicles. H19 was induced by BMP15 protein in yak GCs. In addition, we confirmed that overexpression of H19 promoted yak GC proliferation and autophagy and inhibited apoptosis. Bioinformatic analysis and luciferase reporter assays demonstrated that H19 directly binds to miR-26b, and SMAD1 was identified as a target of miR-26b. miR-26b overexpression inhibited GC proliferation and autophagy and promoted apoptosis through decreased SMAD1 expression, which was attenuated by H19 overexpression. RNA immunoprecipitation-quantitative polymerase chain reaction and dual-luciferase assays showed that miR-26b was sponged by H19 to preserve SMAD1 expression. Furthermore, SMAD1 mRNA expression was induced and miR-26b expression was reduced after yak GCs were treated with BMP15 protein. In conclusion, our results demonstrated that the H19/miR-26b/SMAD1 axis responds to BMP15 to regulate yack GC proliferation, autophagy, and apoptosis.
Subject(s)
Bone Morphogenetic Protein 15 , MicroRNAs , RNA, Long Noncoding , Animals , Cattle , Apoptosis/genetics , Autophagy , Cell Proliferation/genetics , Mammals/genetics , Mammals/metabolism , MicroRNAs/metabolism , RNA, Long Noncoding/geneticsABSTRACT
BACKGROUND: Mycoplasma-associated pneumonia is characterized by severe lung inflammation and immunological dysfunction. However, current anti-mycoplasma agents used in clinical practice do not prevent dysfunction of alveolar macrophages caused by the high level of the cytokine tumor necrosis factor-α (TNF-α) after mycoplasma infection. Apigenin inhibits the production of TNF-α in variet inflammation associated disease. PURPOSE: This study aimed to investigate apigenin's effect on mycoplasma-induced alveolar immune cell injury and the mechanism by which it inhibits TNF-α transcription. METHODS: In this study, we performed a mouse model of Mycoplasma hyopneumoniae infection to evaluate the effect of apigenin on reducing mycoplasma-induced alveolar immune cell injury. Furthermore, we carried out transcriptome analysis, RNA interference assay, methylated DNA bisulfite sequencing assay, and chromatin immunoprecipitation assay to explore the mechanism of action for apigenin in reducing TNF-α. RESULTS: We discovered that M. hyopneumoniae infection-induced necroptosis in alveolar macrophages MH-S cells and primary mouse alveolar macrophages, which was activated by TNF-α autocrine. Apigenin inhibited M. hyopneumoniae-induced elevation of TNF-α and necroptosis in alveolar macrophages. Apigenin inhibited TNF-a mRNA production via increasing ubiquitin-like with PHD and RING finger domains 1 (Uhrf1)-dependent DNA methylation of the TNF-a promotor. Finally, we demonstrated that apigenin regulated Uhrf1 transcription via peroxisome proliferator activated receptor gamma (PPARγ) activation, which acts as a transcription factor binding to the Uhrf1 promoter and protected infected mice's lungs, and promoted alveolar macrophage survival. CONCLUTSION: This study identified a novel mechanism of action for apigenin in reducing alveolar macrophage necroptosis via the PPARγ/ Uhrf1/TNF-α pathway, which may have implications for the treatment of Mycoplasma pneumonia.
Subject(s)
Macrophages, Alveolar , Mycoplasma , Mice , Animals , Macrophages, Alveolar/metabolism , Tumor Necrosis Factor-alpha/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , Apigenin/pharmacology , Mycoplasma/metabolism , Methylation , Necroptosis , CCAAT-Enhancer-Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Yak is the livestock on which people live in plateau areas, but its fecundity is low. Follicular development plays a decisive role in yak reproductive performance. As an important regulatory factor, the expression of long non-coding RNA (lncRNAs) in yak follicular development and its regulatory mechanism remains unclear. To explore the differentially expressed lncRNAs between healthy and atretic follicular in yaks. We used RNA-seq to construct lncRNA, miRNA, and mRNA expression profiles in yak atretic and healthy follicles, and the RNA sequence results were identified by qPCR. In addition, the correlation of lncRNA and targeted mRNA was also analyzed by Starbase software. Moreover, lncRNA/miRNA/mRNA networks were constructed by Cytoscape software, and the network was verified by dual-luciferase analysis. A total of 682 novel lncRNAs, 259 bta-miRNAs, and 1704 mRNAs were identified as differentially expressed between healthy and atretic follicles. Among them, 135 mRNAs were positively correlated with lncRNA expression and 97 were negatively correlated, which may be involved in the yak follicular development. In addition, pathway enrichment analysis of differentially expressed lncRNA host genes by Kyoto Genome Encyclopedia (KEGG) showed that host genes were mainly involved in hormone secretion, granulosa cell apoptosis, and follicular development. In conclusion, we identified a series of novel lncRNAs, constructed the lncRNA ceRNA regulatory network, and provided comprehensive resources for exploring the role of lncRNAs in yak ovarian follicular development.
ABSTRACT
Mycoplasma bovis (M. bovis) is one of the important pathogens for yaks. Aminoglycosides and fluoroquinolones are frequently used medications for the treatment of M. bovis. Drug-resistant strains were inevitable with the abuse of antibiotics. The resistance of M. bovis to aminoglycosides was related to the base mutations in drug target genes. Amino acid mutations at the quinolone resistance-determining region (QRDR) in gyrA, gyrB, parC, and parE conferred resistance to fluoroquinolones. In order to investigate the resistance mechanism of M. bovis from yaks in Tibet to aminoglycosides and fluoroquinolones, six frequently used antibiotics and ten clinical M. bovis strains were administered for a drug sensitivity test for in vitro-induced highly resistant strains, a drug stable-resistance test, cross-resistance test, and analysis of target gene mutations. The results showed that the clinical strains of M. bovis from yaks in Tibet had varying degrees of resistance to fluoroquinolones and aminoglycosides. The mechanism of resistance to fluoroquinolones and aminoglycosides was identified preliminarily for M. bovis from yaks: the single-site base mutation mediated the resistance of M. bovis from yaks and both base mutations led to highly resistant strains (aminoglycosides: rrs3 and rrs4; fluoroquinolones: gyrA and parC). The active efflux system results of M. bovis showed that there was no active efflux system based on fluoroquinolones and aminoglycosides expressed in M. bovis from yaks. The research could provide a reference for clinical treatment of M. bovis.
ABSTRACT
The occurrence of diarrhea in Tibetan piglets is highly notable, but the microorganisms responsible are yet unclear. Its high incidence results in serious economic losses for the Tibetan pig industry. Moreover, the dynamic balance of intestinal microflora plays a crucial role in maintaining host health, as it is a prime cause of diarrhea. Therefore, the present study was performed to analyze the characteristics of bacterial microbiota structure in healthy, diarrheal and treated weaned piglets in Tibet autonomous region for providing a theoretical basis to prevent and control diarrhea. The study was based on the V3-V4 region of the 16S rRNA gene and gut microbiota functions following the metagenome analysis of fresh fecal samples (n = 5) from different groups. The Shannon and Simpson indices differed substantially between diarrheal and treated groups (p < 0.05). According to our findings, the beta diversities, especially between healthy and diarrheal groups, were found different. Firmicutes, Bacteroidetes and Proteobacteria were the dominant phyla in three groups. Furthermore, the abundance of Fusobacteria in the diarrheal group was higher than the other groups. The dominant genera in the diarrheal group were Fusobacterium, Butyricimonas, Sutterella, Peptostreptococcus, and Pasteurella. Moreover, Lactobacillus, Megasphaera and Clavibacter were distinctly less abundant in this group. It is noteworthy that the specific decrease in the abundance of pathogenic bacteria after antibiotic treatment in piglets was noticed, while the level of Lactobacillus was evidently increased. In conclusion, fecal microbial composition and structure variations were discovered across the three groups. Also, the ecological balance of the intestinal microflora was disrupted in diarrheal piglets. It might be caused by a reduction in the relative number of beneficial bacteria and an increase in the abundance of pathogenic bacteria. In the context of advocating for non-resistant feeding, we suspect that the addition of probiotics to feed may prevent early-weaning diarrhea in piglets. Moreover, our findings might help for preventing diarrhea in weaned Tibetan piglets with a better understanding of microbial population dynamics.
ABSTRACT
Terminalia bellirica (Gaertn.) Roxb. (TB) is a traditional herbal combination used in Chinese medicine for the treatment of a broad range of diseases. In this study, thirty KM mice were randomly divided into control (N), infection group (NS), and the TB protection group (HS). Based on its digestive feature, intestinal physical barrier, immunological barrier and gut microbiota effects in vivo on challenged with S.typhimurium mice were investigated after oral administration of 600 mg/kg b.wt of TB for 13 days. The results show that the extract could improve the level of serum immunoglobulins (IgA and IgG), decrease the intestinal cytokine secretion to relieve intestinal cytokine storm, reinforce the intestinal biochemical barrier function by elevating the sIgA expression, and strengthen the intestinal physical barrier function. Simultaneously, based on the V3-V4 region of the 16S rRNA analyzed, the results of the taxonomic structure of the intestinal microbiota demonstrated that the TB prevention effect transformed the key phylotypes of the gut microbiota in S. Typhimurium-challenged mice and promoted the multiplication of beneficial bacteria. Furthermore, the abundance of Firmicutes and Deferribacteres increased, while that of Bacteroidetes and Actinobacteria decreased. At the genus level, the abundance of Ruminococcus and Oscillospira was substantially enhanced, while the other dominant genera showed no significant change between the vehicle control groups and the TB prevention groups. In summary, these results provide evidence that the administration of TB extract can prevent S. Typhimurium infection by alleviating the intestinal physical and immunological barriers and normalizing the gut microbiota, highlighting a promising application in clinical treatment. Thus, our results provide new insights into the biological functions of TB for the preventive effect of intestinal inflammation.
Subject(s)
Salmonella typhimurium , Terminalia , Animals , Mice , Bacteria/genetics , Intestines , Plant Extracts/pharmacology , Plant Extracts/chemistry , RNA, Ribosomal, 16S , Salmonella typhimurium/genetics , Terminalia/chemistry , Terminalia/geneticsABSTRACT
Mycoplasma bovis (M. bovis) is one of the most important pneumonia pathogens in yaks. It may result in more economic losses due to the cold and anoxia condition at Qinghai Tibetan plateau. However, to date, limited information on M. bovis infection in yaks is available in China. For this purpose, the seroprevalence of M. bovis was investigated in yaks living in the mentioned area through commercial ELISA kits. A total of 959 yaks were incorporated into this study. The prevalence of the disease in yaks was 48.70%. The serological results revealed a relatively high prevalence of M. bovis infection in yaks. The present study may greatly contribute to the prevention of this disease. More importance should be given to the potential threat caused by M. bovis in the special plateau.
Subject(s)
Cattle Diseases/epidemiology , Mycoplasma Infections/epidemiology , Mycoplasma bovis , Animals , Cattle , Cattle Diseases/microbiology , China/epidemiology , Enzyme-Linked Immunosorbent Assay , Female , Male , Mycoplasma Infections/microbiology , Prevalence , Seroepidemiologic Studies , Tibet/epidemiologyABSTRACT
Mycoplasma bovis (M. bovis) is one of the important pathogens which may cause bovine respiratory disease syndrome (BRDS), and results in huge economic losses for yaks (Bos gaurus) breeding industry. However, there is limited information about M. bovis in yaks. In our study, 145 nasal mucus samples from yaks with pneumonia were collected to clarify. Bacteriological determination was carried out through biochemical identification and Polymerase Chain Reaction (PCR) detection. And ten strains of Mycoplasma bovis (M. bovis) were found from collected samples. Then, the growth curve of isolated strains was determined by the change of optical density (OD630), pH value and Color Change Cnit (CCU). K-B disk method was also used for antimicrobial susceptibility testing. Results of colony morphology and biochemical testing were consistent with the biological characters of M. bovis. The nucleotide sequences of uvrC specific gene and 16S rRNA gene among the 10 strains were highly homologous. The growth curve assay showed that the isolates cultured in PPLO medium were in lag phase for 24 h, entered stable period in 42 h, and entered decline phase after 78 h. The isolates were found resistant to macrolides, aminoglycosides and lincomycin at various degrees, but they were sensitive or moderately sensitive to doxycycline and kanamycin under antimicrobial susceptibility analysis. In conclusion, the results provided certain reference for the follow-up research and guiding for the treatment of M. bovis in yaks.
Subject(s)
Cattle Diseases , Mycoplasma Infections , Mycoplasma bovis , Animals , Anti-Bacterial Agents/pharmacology , Cattle , Macrolides/pharmacology , Mycoplasma Infections/veterinary , Mycoplasma bovis/genetics , RNA, Ribosomal, 16S/geneticsABSTRACT
The intronic microRNA, miR-125b, plays a vital role in promyelocytic and hematopoietic stem cells, and in the development and apoptosis of cancer cells. In this study, we showed that miR-125b regulates granulosa cell (GC) apoptosis in the yak ovary. Bioinformatic analyses and luciferase reporter assays demonstrated that bone morphogenetic protein receptor type 1B (BMPR1B) is an miR-125b target. miR-125b overexpression induced apoptosis in yak GC, and affected the mRNA and protein expression of BMPR1B and the ratio of Bcl2/Bax. Silencing of miR-125b decreased the rate of yak GC apoptosis and increased the ratio of Bcl2/Bax. In addition, the effects of an miR-125b inhibitor were overturned by cotransfection with siRNA-BMPR1B2 (siRNA-299) in yak GC. Together, these results demonstrated that miR-125b regulates GC apoptosis in the yak ovary by targeting BMPR1B.
Subject(s)
Apoptosis/genetics , Bone Morphogenetic Protein Receptors, Type I/genetics , Granulosa Cells/metabolism , MicroRNAs/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , bcl-2-Associated X Protein/genetics , Animals , Antagomirs/genetics , Antagomirs/metabolism , Base Sequence , Binding Sites , Bone Morphogenetic Protein Receptors, Type I/antagonists & inhibitors , Bone Morphogenetic Protein Receptors, Type I/metabolism , Cattle , Female , Gene Expression Regulation , Granulosa Cells/cytology , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , Primary Cell Culture , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
To explore the expression profile of the cellular miRNAs in bovine ovarian granulosa cells responding to transforming growth factor-ß1 (TGF-ß1), the effect of TGF-ß1 on cell proliferation was firstly investigated by CCK-8 method and the results showed that there was a significant inhibitory effect on bovine granulosa cell proliferation treated with 5/10â¯ng/mL human recombinant TGF-ß1 for 24â¯h compared to the control (Pâ¯<â¯0.05). Then, we performed high-throughput sequencing of two small RNA libraries prepared from cultured bovine granulosa cells stimulated with or without 10â¯ng/mL human recombinant TGF-ß1. A total of 13,257,248 and 138,726,391 clean reads per library were obtained from TGF-ß1 and control groups, respectively. There were 498 and 499 bovine-specific exist miRNAs (exist miRNAs), 627 and 570 conserved known miRNAs (known miRNAs), and 593 and 585 predicted novel miRNAs in TGF-ß1 and control groups, respectively. A total of 78 miRNAs with significant differential expression, including 39 up-regulated miRNAs and 39 down-regulated miRNAs were identified in the TGF-ß1 group compared with the control. Real-time quantitative PCR analyses of bta-miR-106a and bta-miR-1434-5p showed that their up-expressions were interrupted by SB431542, an inhibitor that blocks TGFß1/Smad signaling, which supported the sequencing data. GO analysis showed involvement of the predicted genes of the differentially expressed miRNAs in a broad spectrum of cell biological processes, cell components, and molecular functions. KEGG pathway analysis of the predicted miRNA targets further indicated that these differentially expressed miRNAs are involved in various signaling pathways, such as Wnt, MAPK, and TGF-ß signaling, which might be involved in follicular development. These results provide valuable information on the composition, expression, and function of miRNAs in bovine granulosa cells responding to TGF-ß1, and will aid in understanding the molecular mechanisms of TGF-ß1 in granulosa cells.
Subject(s)
Granulosa Cells/cytology , High-Throughput Nucleotide Sequencing/methods , MicroRNAs/genetics , Sequence Analysis, RNA/methods , Transforming Growth Factor beta1/pharmacology , Animals , Cattle , Cell Proliferation/drug effects , Cells, Cultured , Female , Gene Expression Regulation/drug effects , Gene Regulatory Networks , Granulosa Cells/drug effects , High-Throughput Nucleotide Sequencing/veterinary , Humans , Sequence Analysis, RNA/veterinary , Signal TransductionABSTRACT
In many cells, mRNAs containing inverted repeated Alu elements (IRAlus) in their 3' untranslated regions (UTRs) are inefficiently exported to the cytoplasm. Such nuclear retention correlates with paraspeckle-associated protein complexes containing p54(nrb). However, nuclear retention of mRNAs containing IRAlus is variable, and how regulation of retention and export is achieved is poorly understood. Here we show one mechanism of such regulation via the arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). We demonstrate that disruption of CARM1 enhances the nuclear retention of mRNAs containing IRAlus. CARM1 regulates this nuclear retention pathway at two levels: CARM1 methylates the coiled-coil domain of p54(nrb), resulting in reduced binding of p54(nrb) to mRNAs containing IRAlus, and also acts as a transcription regulator to suppress NEAT1 transcription, leading to reduced paraspeckle formation. These actions of CARM1 work together synergistically to regulate the export of transcripts containing IRAlus from paraspeckles under certain cellular stresses, such as poly(I:C) treatment. This work demonstrates how a post-translational modification of an RNA-binding protein affects protein-RNA interaction and also uncovers a mechanism of transcriptional regulation of the long noncoding RNA NEAT1.
Subject(s)
Cell Nucleus/metabolism , Protein-Arginine N-Methyltransferases/metabolism , RNA, Long Noncoding/metabolism , RNA, Messenger/metabolism , Alu Elements/genetics , Animals , Cell Line , DNA Methylation , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Inverted Repeat Sequences/genetics , Mice , Protein Binding , Protein Processing, Post-Translational , Protein TransportABSTRACT
Many long noncoding RNAs (lncRNAs) are constrained to the nucleus to exert their functions. However, commonly used vectors that were designed to express mRNAs have not been optimized for the study of nuclear RNAs. We reported recently that sno-lncRNAs are not capped or polyadenylated but rather are terminated on each end by snoRNAs and their associated proteins. These RNAs are processed from introns and are strictly confined to the nucleus. Here we have used these features to design expression vectors that can stably express virtually any sequence of interest and constrain its accumulation to the nucleus. Further, these RNAs appear to retain normal nuclear associations and function. SnoVectors should be useful in conditions where nuclear RNA function is studied or where export to the cytoplasm needs to be avoided.
Subject(s)
Cell Nucleus/genetics , Genetic Vectors/chemistry , RNA, Long Noncoding/metabolism , RNA, Small Nucleolar/metabolism , Cell Line , Cell Nucleus/metabolism , HeLa Cells , Humans , RNA Isoforms/metabolism , RNA Processing, Post-TranscriptionalABSTRACT
The Hu sheep is world-famous for its hyper-prolificacy and the bone morphogenetic protein (BMP)/Smad pathway and several other closely related molecules (GDF9, TGF-betaRI) have been shown to have a close relationship with reproduction in sheep. In order to investigate the mechanism of high fecundity in Hu sheep and its relationship with the BMP/Smad pathway, 147 Hu sheep were blood sampled for detection of the FecB mutation (A746G) in the BMPRIB gene by PCR-SSCP, and sixteen adult Hu ewes classified as either high-fecundity (HF) or low-fecundity (LF) animals were sacrificed for tissue and antral follicle sampling. The tissue distribution patterns of mRNAs encoding BMP/Smad pathway molecules including BMPs (BMP2, BMP4, BMP6, BMP7 and BMP15), BMP receptors (BMPRIA, BMPRIB and BMPRII), intracellular transducers (Smad1, Smad5 and Smad4) and closely related molecules (GDF9 and TGF-betaRI) were detected by RT-PCR and the gene expression levels in antral follicles were investigated by real-time PCR. The results showed that all experimental animals were homozygous for the BMPRIB (A746G) mutation, and all detected genes related to the BMP/Smad pathway and GDF9 and TGF-betaRI were expressed in the ovary. In addition, BMP4, BMPRIB, BMPRII, Smad4, GDF9 and TGF-betaRI mRNAs were more abundant in the antral follicles of HF animals than those of LF animals (P<0.05), but BMP15 mRNA was less abundant (P<0.01). This suggests that there could be an unidentified genetic mutation in BMPRIB, or other unidentified genes and unknown factors, which controls ovarian number by changing the expression patterns of genes known to regulate ovulation rate via the BMP/Smad pathway and closely related molecules (GDF9 and TGF-betaRI).
Subject(s)
Bone Morphogenetic Proteins/genetics , Fertility/genetics , Ovarian Follicle/metabolism , Sheep/genetics , Smad Proteins/genetics , Animals , Base Sequence , Bone Morphogenetic Protein Receptors, Type I/genetics , Bone Morphogenetic Protein Receptors, Type I/metabolism , Bone Morphogenetic Proteins/metabolism , DNA Mutational Analysis , Female , Fertility/physiology , Gene Expression Regulation , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Infertility, Female/genetics , Infertility, Female/metabolism , Infertility, Female/veterinary , Mutation/physiology , Ovarian Follicle/cytology , Ovarian Follicle/physiology , Polymorphism, Restriction Fragment Length/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Sheep/metabolism , Sheep/physiology , Signal Transduction/genetics , Smad Proteins/metabolism , Tissue DistributionABSTRACT
Recombinant human endostatin (rhEndostatin) has been shown to inhibit tumor growth, but the variable antitumor activity of different rhEndostatin preparations has necessitated the development of an accurate, reproducible in vivo bioassay for evaluating the rhEndostatin activity. To assess the in vivo antitumor efficacy of rhEndostatin, H22 tumor-bearing mice received three doses of rhEndostatin and the potency of rhEndostatin preparations in inhibiting tumor growth was determined by ED(50)-potency assay and validated by dose-response parallel-line assay. There was a consistent and highly reproducible linear regression relationship between rhEndostatin dosage and tumor growth inhibition rate. The ED(50) values were determined from dose-response regression lines for seven rhEndostatin preparations with high reproducibility. On the basis of the current study, the potency of rhEndostatin preparations was assigned a value of 6.09 x 10(5) U/ampoule and a 95% confidence limit of 5.96 x 10(5)-6.22 x 10(5). We consider that this procedure can be served as a potential candidate pharmacopoeial method for potency measurement of different rhEndostatin preparations.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor/methods , Endostatins/pharmacology , Liver Neoplasms, Experimental/drug therapy , Analysis of Variance , Animals , Antineoplastic Agents/administration & dosage , Confidence Intervals , Disease Models, Animal , Dose-Response Relationship, Drug , Endostatins/administration & dosage , Humans , Linear Models , Male , Mice , Pharmacopoeias as Topic , Random Allocation , Reproducibility of ResultsABSTRACT
Bcl-2 is an anti-apoptotic protein. If the level of Bcl-2 protein can be reduced sufficiently in tumors using RNA interference (RNAi) to target the gene message, the apoptosis of tumor cells may be promoted. In this study, we synthesized 19 nucleotides (nts) small interference RNA (siRNA) constructs suppressing bcl-2 gene expression in human tumor cells (HeLaB2 and BGC-823 cell lines) in vitro. The bcl-2 gene expression levels were significantly reduced when these siRNA were transfected into experimental two tumor cells for 72 hours. The apoptosis process was also examined in the tumor cells. Here we synthesized siRNA from a DNA template under the control of the RNA polymerase III promoter in transfected tumor cells. Using this DNA vector-based approach, we found that the siRNA efficiently and specifically inhibited the synthesis of protein encoded by the bcl-2 gene in HeLaB2 and BGC-823 tumor cells. Tumor growth was inhibited by 66.5% with 2mg/kg pSilencer 3.1H1-bcl-2 in mouse liver tumor-bearing BALB/c mice. This approach may prove to be a valuable clinical technique for the analysis of specific gene functions and gene therapy of malignant tumors that utilize the bcl-2 gene via RNA interference.
Subject(s)
Gene Expression , Genes, bcl-2/genetics , Genetic Therapy/methods , Neoplasms/therapy , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , RNA Interference , Animals , Apoptosis , Cell Line, Tumor , Down-Regulation , Genetic Vectors/genetics , Humans , Male , Mice , Mice, Inbred BALB C , Neoplasms/chemistry , Neoplasms/genetics , Plasmids/genetics , Proto-Oncogene Proteins c-bcl-2/analysis , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Small Interfering/genetics , Silencer Elements, Transcriptional/genetics , Xenograft Model Antitumor AssaysABSTRACT
In order to establish the methods of high-performance liquid chromatography (HPLC) for determining the purity of recombinant human endostatin (rhEndostatin) and in vitro or in vivo activity of rhEndostatin, two columns were firstly used in HPLC analysis for determining the purity of rhEndostatin, including Waters Symmetry 300C4 (4.6 mm x 250 mm, 5 microm) and the Superdex75 HR 10/30. Cell lines, bovine capillary endothelial cells (BCEs) or human umbilical vein endothelial cells (HUVECs) expression human vascular endothelial growth factor (hVEGF) were used in method MTT or LDH as substrate, respectively. The bioactivity in vivo was assayed by the anti-tumor proliferation rate in H22 liver tumor-bearing mice. The results showed that the retention time of rhEndostatin sample was stable at 19.066 min or 11.506 min in reverse phase HPLC (RP-HPLC) or gel filtering HPLC (GF-HPLC). The stableness, repeat and recovery rates were over 99% in both methods and there was no statistical difference between these two methods (p > 0.05). In nonserum culture medium, rhEndostatin can sensitively and stably inhibit the proliferation of the HUVEC cells that were transfected with plasmid encoding hVEGF. LDH substrate methods is the most sensitive and stable method. The anti-tumor activity in H22 tumor-bearing mice was also highly repeatable and had an inhibition rate over 50% at 20 mg kg(-1) weight. As a conclusion, the RP-HPLC and GF-HPLC set up in this paper are highly repeatable, accurate and sensitive for detecting the purity of rhEndostatin. The bioactivity of rhEndostatin can be measured through detection the proliferation-inhibition on HUVECs transfectants with hVEGF in vitro or on H22 liver tumor in vivo.