ABSTRACT
[ Abstract] Objective To investigate the protective effect of withaferin A (WA) against high fat diet induced obesity and its associated mechanism. Methods C57BL / 6 J mice at 8-week of age were divided into two groups. The mice were fed with high fat diet (HFD) and were given an intraperitoneal injection of WA or DMSO (solvent control) . The body weight and food intake of the mice were monitored. One week later, inguinal white adipose tissue (iWAT), interscapular brown adipose tissue (BAT), epididymal white adipose tissue (eWAT) and retroperitoneal white adipose tissue (rWAT) were collected and weighed. Expression levels of the genes associated with white adipose browning were detected in iWAT. HE staining was applied to observe the morphological changes of iWAT. Results The data showed that body weight and fat weight in WA group were significantly lower than those in the control group, and the food intake was not changed significantly. Real-time PCR analysis showed that the expression level of browning related genes in iWAT of the WA group was significantly increased. The result from Western blotting analysis showed that the protein levels of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) increased significantly in iWAT of the WA group. The typical morphological change of adipose browning, such as the multilocular adipocytes was observed in inguinal white adipose tissue of the mice treated with WA by using HE staining and mmunofluorescence assay. Conclusion Taken together, these observations indicate that withaferin A can protect the mice from high fat diet induced obesity by promoting white adipose tissue browning.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the anti-myeloma effect of suberoylanilide hydroxamic acid (SAHA) and on mouse myeloma cell line SP2/0 in vitro and in vivo and its mechanism.</p><p><b>METHODS</b>The inhibitory effect of SAHA on SP2/0 cells was measured by CCK-8 assay,and the apoptosis and cell cycle were analyzed by flow cytometry FACS. The protein expression of Caspase-3 and p53 of SP2/0 cells treated with SAHA were examined by Western blot. Annexin V/7-AAD double staining was performed to detect the apoptosis of SP2/0 induced by SAHA in vitro. SP2/0 cells (1×10) resuspended in 200 µl PBS were inoculated subcutaneously and intravenously into BALB/c mice, so as to establish aggressive or non-aggressive myeloma-bearing mouse models respectively. On day 3 after modeling, mice received SAHA or vehicle control treatment by intraperitoneal injection. The dose of SAHA was 60 mg/kg·d, 5 times a week for 3 weeks.</p><p><b>RESULTS</b>In SAHA-treated SP2/0 cells, the proliferation inhibition rate and apoptotic cells increased in a dose dependent manner. Also, SAHA significantly increased the ratio of cells in G phase and decreased in S phase. Molecular mechanisms of apoptosis and cell cycle arrest of SP2/0 induced by SAHA partly correlated with up-regulating the expression level of Caspase-3 and p53. In the non-aggressive myeloma-bearing mice, SP2/0 cells disappeared in peripheral blood after SAHA treatment. In the aggressive myeloma-bearing mice, inhibition of tumor growth and prolongation of the cell survival were observed after SAHA treatment.</p><p><b>CONCLUSION</b>SAHA inhibited SP2/0 cell proliferation, this effect associates with inducing apoptosis and cell cycle arrest, the mechanism of SAHA ralates partly with activating Caspase-3 and p53 pathway.</p>
Subject(s)
Animals , Mice , Antineoplastic Agents , Apoptosis , Cell Line, Tumor , Cell Proliferation , Histone Deacetylase Inhibitors , Hydroxamic Acids , Mice, Inbred BALB C , Multiple MyelomaABSTRACT
MTUS1 (microtubule-associated tumor suppressor 1) has been identified that can function as a tumor suppressor gene in many malignant tumors. However, the function and mechanisms underlying the regulation of MTUS1 are unclear. In the present study, we reported that miR-19a and miR-19b (miR-19a/b) promote proliferation and migration of lung cancer cells by targeting MTUS1. First, MTUS1 was proved to function as a tumor suppressor in lung cancer and was linked to cell proliferation and migration promotion. Second, an inverse correlation between miR-19a/b expression and MTUS1 mRNA/protein expression was noted in human lung cancer tissues. Third, MTUS1 was appraised as a direct target of miR-19a/b by bioinformatics analysis. Fourth, direct MTUS1 regulation by miR-19a/b in lung cancer cells was experimentally affirmed by cell transfection assay and luciferase reporter assay. Finally, miR-19a/b were shown to cooperatively repress MTUS1 expression and synergistically regulate MTUS1 expression to promote lung cancer cell proliferation and migration. In conclusion, our findings have provided the first clues regarding the roles of miR-19a/b, which appear to function as oncomirs in lung cancer by downregulating MTUS1.
Subject(s)
Female , Humans , Male , A549 Cells , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Lung Neoplasms , Genetics , Metabolism , Pathology , MicroRNAs , Genetics , Metabolism , RNA, Neoplasm , Genetics , Metabolism , Tumor Suppressor Proteins , GeneticsABSTRACT
Programmed cell death 4 (PDCD4) is a RNA-binding protein that acts as a tumor suppressor in many cancer types, including colorectal cancer (CRC). During CRC carcinogenesis, PDCD4 protein levels remarkably decrease, but the underlying molecular mechanism for decreased PDCD4 expression is not fully understood. In this study, we performed bioinformatics analysis to identify miRNAs that potentially target PDCD4. We demonstrated miR-181b as a direct regulator of PDCD4. We further showed that activation of IL6/STAT3 signaling pathway increased miR-181b expression and consequently resulted in downregulation of PDCD4 in CRC cells. In addition, we investigated the biological effects of PDCD4 inhibition by miR-181b both in vitro and in vivo and found that miR-181b could promote cell proliferation and migration and suppress apoptosis in CRC cells and accelerate tumor growth in xenograft mice, potentially through targeting PDCD4. Taken together, this study highlights an oncomiR role for miR-181b in regulating PDCD4 in CRC and suggests that miR-181b may be a novel molecular therapeutic target for CRC.
Subject(s)
Animals , Humans , Male , Mice , Apoptosis Regulatory Proteins , Genetics , Metabolism , Caco-2 Cells , Cell Proliferation , Colorectal Neoplasms , Genetics , Metabolism , Pathology , Heterografts , Mice, Nude , Mice, SCID , MicroRNAs , Genetics , Metabolism , Neoplasm Proteins , Genetics , Metabolism , Neoplasm Transplantation , RNA, Neoplasm , Genetics , Metabolism , RNA-Binding Proteins , Genetics , MetabolismABSTRACT
Osteosarcoma is the most common primary sarcoma of bone, and it is a leading cause of cancer death among adolescents and young adults. However, the molecular mechanism underlying osteosarcoma carcinogenesis remains poorly understood. Recently, cyclin-dependent kinase 6 (CDK6) was identified as an important oncogene. We found that CDK6 protein level, rather than CDK6 mRNA level, is much higher in osteosarcoma tissues than in normal adjacent tissues, which indicates a post-transcriptional mechanism involved in CDK6 regulation in osteosarcoma. MiRNAs are small non-coding RNAs that repress gene expression at the post-transcriptional level and have widely been shown to play important roles in many human cancers. In this study, we investigated the role of miR-29b as a novel regulator of CDK6 using bioinformatics methods. We demonstrated that CDK6 can be downregulated by miR-29b via binding to the 3'-UTR region in osteosarcoma cells. Furthermore, we identified an inverse correlation between miR-29b and CDK6 protein levels in osteosarcoma tissues. Finally, we examined the function of miR-29b-driven repression of CDK6 expression in osteosarcoma cells. The results revealed that miR-29b acts as a tumor suppressor of osteosarcoma by targeting CDK6 in the proliferation and migration processes. Taken together, our results highlight an important role for miR-29b in the regulation of CDK6 in osteosarcoma and may open new avenues for future osteosarcoma therapies.
Subject(s)
Animals , Humans , Mice , Rats , 3' Untranslated Regions , Base Sequence , Bone Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cyclin-Dependent Kinase 6 , Genetics , Metabolism , MicroRNAs , Metabolism , Osteosarcoma , Metabolism , Pathology , RNA Interference , RNA, Messenger , Metabolism , RNA, Small Interfering , Metabolism , Sequence Alignment , Up-RegulationABSTRACT
The BCL6 (B-Cell Lymphoma 6) gene is a proto-oncogene that is often expressed in diffuse large B-cell lymphomas (DLBCLs). BCL6 loss of function can kill DLBCL cells, demonstrating that BCL6 is necessary for the survival of DLBCL cells and could be a therapeutic target. In this study, we found that BCL6 protein levels were consistently upregulated in DLBCL tissues, whereas its mRNA levels varied randomly in tissues, suggesting that a post-transcriptional mechanism was involved in BCL6 regulation. We used bioinformatics analysis to search for miRNAs, which potentially target BCL6, and identified specific targeting sites for miR-10a in the 3'-untranslated region (3'-UTR) of BCL6. We further identified an inverse correlation between miR-10a levels and BCL6 protein levels, but not mRNA levels, in DLBCL tumor tissue samples. By overexpressing or knocking down miR-10a in DLBCL cells, we experimentally validated that miR-10a directly recognizes the 3'-UTR of the BCL6 transcript and regulated BCL6 expression. Furthermore, we demonstrated that negatively regulating BCL6 by miR-10a suppressed the proliferation and promoted apoptosis of DLBCL cells.
Subject(s)
Humans , 3' Untranslated Regions , Apoptosis , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Lymphoma, Large B-Cell, Diffuse , Genetics , Metabolism , Therapeutics , MicroRNAs , Genetics , Metabolism , Proto-Oncogene Proteins c-bcl-6 , GeneticsABSTRACT
MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression. The deregulated expression of miRNAs is associated with a variety of diseases, including breast cancer. In the present study, we found that miR-495 was markedly up-regulated in clinical breast cancer samples by quantitative real time-PCR (qRT-PCR). Junctional adhesion molecule A (JAM-A) was predicted to be a potential target of miR-495 by bioinformatics analysis and was subsequently verified by luciferase assay and Western blotting. JAM-A was found to be negatively correlated with the migration of breast cancer cells through loss-of-function and gain-of-function assays, and the inhibition of JAM-A by miR-495 promoted the migration of MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of JAM-A could restore miR-495-induced breast cancer cell migration. Taken together, our findings suggest that miR-495 could facilitate breast cancer progression through the repression of JAM-A, making this miRNA a potential therapeutic target.
Subject(s)
Adult , Aged , Female , Humans , Middle Aged , 3' Untranslated Regions , Genetics , Blotting, Western , Breast Neoplasms , Genetics , Metabolism , Pathology , Cell Adhesion Molecules , Genetics , Metabolism , Cell Line, Tumor , Cell Movement , Genetics , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , MCF-7 Cells , MicroRNAs , Genetics , RNA Interference , Receptors, Cell Surface , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The mechanism underlying T cell-mediated fulminant hepatitis is not fully understood. In this study, we investigated whether myeloid derived suppressor cells (MDSCs) could prevent the concanavalin A (ConA)-induced hepatitis through suppressing T cell proliferation. We observed an increase in the frequencies of MDSCs in mouse spleen and liver at early stage of ConA treatment, implicating that the MDSCs might be involved in the initial resistance of mice against ConA-mediated inflammation. Subpopulation analysis showed that the MDSCs in liver of ConA-induced mice were mainly granulocytic MDSCs. Adoptive transfer of the bone marrow-derived MDSCs into ConA-treated mice showed that the MDSCs migrated into the liver and spleen where they suppressed T cell proliferation through ROS pathway. In addition, the frequencies of MDSCs in mice were also significantly increased by the treatment with immune suppressor glucocorticoids. Transfer of MDSCs into the regulatory T cell (Treg)-depleted mice showed that the protective effect of MDSCs on ConA-induced hepatitis is Treg-independent. In conclusion, our results demonstrate that MDSCs possess a direct protective role in T cell-mediated hepatitis, and increasing the frequency of MDSCs by either adoptive transfer or glucocorticoid treatment represents a potential cell-based therapeutic strategy for the acute inflammatory disease.
Subject(s)
Animals , Male , Adoptive Transfer , Blotting, Western , Bone Marrow Cells , Allergy and Immunology , CD11b Antigen , Allergy and Immunology , Metabolism , Cell Movement , Allergy and Immunology , Cell Proliferation , Chemical and Drug Induced Liver Injury , Allergy and Immunology , Concanavalin A , Toxicity , Dexamethasone , Pharmacology , Flow Cytometry , Glucocorticoids , Pharmacology , Liver , Allergy and Immunology , Pathology , Mice, Inbred C57BL , Mitogens , Toxicity , Myeloid Cells , Allergy and Immunology , Metabolism , Transplantation , Receptors, Chemokine , Allergy and Immunology , Metabolism , Spleen , Allergy and Immunology , Pathology , T-Lymphocytes , Allergy and Immunology , T-Lymphocytes, Regulatory , Allergy and ImmunologyABSTRACT
The intestinal absorption of berberine (Ber) and its structural modified compound 8-hydroxy dihydroberberine (Hdber) was compared, and their effects on the intestinal absorption of sugar by perfusion experiment were investigated in order to reveal the mechanism of low dose and high activity of Hdber in the treatment of hyperglycemia. The absorption of Hdber and Ber in rat small intestine was measured by in situ perfusion. High performance liquid chromatography (HPLC) was used to determine the concentrations of Hdber and Ber. In situ perfusion method was also used to study the effects of Hdber and Ber on sugar intestinal absorption. Glucose oxidase method and UV spectrophotometry were applied to examine the concentrations of glucose and sucrose in the perfusion fluid. The results showed that the absorption rate of Ber in the small intestine was lower than 10%, but that of Hdber was larger than 70%. Both Hdber and Ber inhibited the absorption of glucose and sucrose at the doses of 10 and 20 μg/mL. However, Hdber presented stronger activity than Ber (P<0.01). It is suggested that Hdber is absorbed easily in rat small intestine and that its inhibitory effect on the absorption of sugar is better than Ber.
ABSTRACT
The intestinal absorption of berberine (Ber) and its structural modified compound 8-hydroxy dihydroberberine (Hdber) was compared, and their effects on the intestinal absorption of sugar by perfusion experiment were investigated in order to reveal the mechanism of low dose and high activity of Hdber in the treatment of hyperglycemia. The absorption of Hdber and Ber in rat small intestine was measured by in situ perfusion. High performance liquid chromatography (HPLC) was used to determine the concentrations of Hdber and Ber. In situ perfusion method was also used to study the effects of Hdber and Ber on sugar intestinal absorption. Glucose oxidase method and UV spectrophotometry were applied to examine the concentrations of glucose and sucrose in the perfusion fluid. The results showed that the absorption rate of Ber in the small intestine was lower than 10%, but that of Hdber was larger than 70%. Both Hdber and Ber inhibited the absorption of glucose and sucrose at the doses of 10 and 20 μg/mL. However, Hdber presented stronger activity than Ber (P<0.01). It is suggested that Hdber is absorbed easily in rat small intestine and that its inhibitory effect on the absorption of sugar is better than Ber.
Subject(s)
Animals , Rats , Absorption , Berberine , Carbohydrate Metabolism , Carbohydrates , Chemistry , Chromatography, High Pressure Liquid , Glucose , Metabolism , Intestinal AbsorptionABSTRACT
This paper studied the effects of different mulching modes on the soil moisture in a semi-arid rainfed area of Loess Plateau, Northwest China. Seven treatments were installed, i. e., mulching plastic film in summer (T1), mulching plastic film in autumn (T2), mulching 5 cm long wheat straw in summer (T3), mulching whole wheat straw in summer (T4), mulching plastic film in summer plus wheat straw (T5), mulching used plastic film after harvest (T6), and un-mulching (CK). In T6, the soil moisture in different layers at different crop growth stages was all higher than that in CK. In the other five mulching treatments, the soil moisture in 0-90 cm layer before flowering stage was obviously higher, but that in 0-90 cm layer after flowering stage and in 90-200 cm layer during the whole growth season was lower than that of CK. The soil moisture in 0-200 cm layer in T6 during the whole growth period was significantly higher than that in CK, with a difference of 0.9%, but the soil moisture in 0-200 cm layer in other mulching treatments was lower. As compared with plastic film mulching, straw mulching increased the soil moisture in 0-200 cm layer. The soil moisture under mulching with used plastic film after harvest was higher than that under mulching with new plastic film. As compared to CK, the grain yield of winter wheat with plastic film mulching was increased by 20.3%-29.0%, and that With straw mulching was increased by 5.0%-16.7%. There was a significant positive correlation between the crop productivity and the soil water consumption during the growth period (r = 0.77*).
Subject(s)
Agriculture/methods , Biomass , Soil/chemistry , Triticum/growth & development , Water/analysis , Altitude , China , EcosystemABSTRACT
Tumor-associated macrophages (TAMs) mostly exhibit M2-like (alternatively activated) properties and play positive roles in angiogenesis and tumorigenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic factor. During tumor development, TAMs secrete VEGF and other factors to promote angiogenesis; thus, anti-treatment against TAMs and VEGF can repress cancer development, which has been demonstrated in clinical trials and on an experimental level. In the present work, we show that miR-150 is an oncomir because of its promotional effect on VEGF. MiR-150 targets TAMs to up-regulate their secretion of VEGF in vitro. With the utilization of cell-derived vesicles, named microvesicles (MVs), we transferred antisense RNA targeted to miR-150 into mice and found that the neutralization of miR-150 down-regulates miR-150 and VEGF levels in vivo and attenuates angiogenesis. Therefore, we proposed the therapeutic potential of neutralizing miR-150 to treat cancer and demonstrated a novel, natural, microvesicle-based method for the transfer of nucleic acids.
Subject(s)
Animals , Humans , Male , Mice , Carcinogenesis , Genetics , Metabolism , Pathology , Cell Line, Tumor , Exosomes , HEK293 Cells , Heterografts , Macrophages , Metabolism , Mice, Inbred C57BL , MicroRNAs , Genetics , Metabolism , Neoplasm Transplantation , RNA, Antisense , Genetics , Up-Regulation , Vascular Endothelial Growth Factor A , Genetics , MetabolismABSTRACT
MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that are involved in post-transcriptional gene regulation. It has long been assumed that miRNAs exert their roles only in the cytoplasm, where they recognize their target protein-coding messenger RNAs (mRNAs), and result in translational repression or target mRNA degradation. Recent studies, however, have revealed that mature miRNAs can also be transported from the cytoplasm to the nucleus and that these nuclear miRNAs can function in an unconventional manner to regulate the biogenesis and functions of ncRNAs (including miRNAs and long ncRNAs), adding a new layer of complexity to our understanding of gene regulation. In this review, we summarize recent literature on the working model of these unconventional miRNAs and speculate on their biological significance. We have every reason to believe that these novel models of miRNA function will become a major research topic in gene regulation in eukaryotes.
Subject(s)
Humans , Cell Nucleus , Genetics , Cytoplasm , Genetics , Eukaryota , Genetics , Gene Expression Regulation , MicroRNAs , Genetics , RNA Stability , Genetics , RNA, Long Noncoding , Genetics , RNA, Messenger , Genetics , MetabolismABSTRACT
Colostrum provides essential nutrients and immunologically active factors that are beneficial to newborns. Our previous work demonstrated that milk contains large amounts of miRNA that is largely stored in milk-derived microvesicles (MVs). In the present study, we found that the MVs from colostrum contain significantly higher levels of several immune-related miRNAs. We hypothesized that the colostrum MVs may transfer the immune-related miRNAs into cells, which contribute to its immune modulatory feature. We isolated colostrum MVs by ultracentrifugation and demonstrated several immune modulation features associated with miRNAs. We also provide evidence that the physical structure of milk-derived MVs is essential for transfer miRNAs and following immune modulation effect. Moreover, we found that colostrum powder-derived MVs also contains higher levels of immune-related miRNAs that display similar immune modulation effects. Taken together, these results show that MV-containing immunerelated miRNAs may be a novel mechanism by which colostrum modulates body immune response.
Subject(s)
Animals , Cattle , Female , Mice , Pregnancy , Cell Movement , Cell Proliferation , Colostrum , Metabolism , Cytokines , Metabolism , Liposomes , Chemistry , Metabolism , Macrophages , Allergy and Immunology , Metabolism , MicroRNAs , Allergy and Immunology , Metabolism , Milk , Allergy and Immunology , Metabolism , Phagocytosis , UltracentrifugationABSTRACT
A new class of RNA regulatory genes known as microRNAs (miRNAs) has been found to introduce a whole new layer of gene regulation in eukaryotes. The intensive studies of the past several years have demonstrated that miRNAs are not only found intracellularly, but are also detectable outside cells, including in various body fluids (e.g. serum, plasma, saliva, urine and milk). This phenomenon raises questions about the biological function of such extracellular miRNAs. Substantial amounts of extracellular miRNAs are enclosed in small membranous vesicles (e.g. exosomes, shedding vesicles and apoptotic bodies) or packaged with RNA-binding proteins (e.g. high-density lipoprotein, Argonaute 2 and nucleophosmin 1). These miRNAs may function as secreted signaling molecules to influence the recipient cell phenotypes. Furthermore, secreted extracellular miRNAs may reflect molecular changes in the cells from which they are derived and can therefore potentially serve as diagnostic indicators of disease. Several studies also point to the potential application of siRNA/miRNA delivery as a new therapeutic strategy for treating diseases. In this review, we summarize what is known about the mechanism of miRNA secretion. In addition, we describe the pathophysiological roles of secreted miRNAs and their clinical potential as diagnostic biomarkers and therapeutic drugs. We believe that miRNA transfer between cells will have a significant impact on biological research in the coming years.
Subject(s)
Animals , Humans , Diagnosis , Extracellular Space , Genetics , Gene Transfer, Horizontal , MicroRNAs , Genetics , Metabolism , TherapeuticsABSTRACT
<p><b>OBJECTIVE</b>To improve the diagnostic criteria of cervicogenic headache by summary the clinical features of cervicogenic headache in patients.</p><p><b>METHODS</b>Among 448 patients with the chief complaint of headache for the first visit from October 2008 to June 2009, 399 cases meet the diagnostic criteria, 330 cases were diagnosed as cervicogenic headache. There were 97 males and 233 females, ranging in age from 14 to 76 years, with an average of 46.1 years. The duration of the disease ranged from 0.33 to 50 years, with an average of 13.4 years. The gender, age, occupation, headache characteristics of the patients were recorded, and the tenderness point position and the range of diffuse pain were summarized for analysis.</p><p><b>RESULTS</b>There were 89.1% headache patients were cervicogenic headache, the male to female ratio was 1:2.4, 78.8% (260 cases) patients aged 21 to 60 years old, especially those 41 to 50 years old was the majority. There were 97.3% (321 cases) patients with paroxysmal headache, and only 2.7% (9 cases) patients had persistent headache. Majority patients had swelling pain (181 cases, accounting for 54.8%) or pulsatile pain (135 cases, accounting for 40.9%). Above two types pain may coexist, and other types were few. The position of headache concentrated at occiput, corona capitis and tempus, and could spread to the forehead, fossa orbitalis, or eye (about 1/3 patiens), very few spread to the ears, nasal wing or bridge of the nose, etc. There were 76.4% (252 cases) patients with full or bilateral headache, and only 12.1% (40 cases) with unilateral headache. Almost all the patients had symptoms of occipital nerve entrapment, among which about 73.3% (242 cases) patients had greater and lesser occipital large involved. There were 82.7% (273 cases) patients showing radiating pain by pressing the special point of occiput.</p><p><b>CONCLUSION</b>(1) Cervicogenic headache patients accounted for a higher proportion; (2) The existing diagnostic criteria is not perfect, and need to be improved; (3) Radiating pain occurred when pressing the specific location is an important diagnosis criteria.</p>
Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Post-Traumatic Headache , Diagnosis , Retrospective StudiesABSTRACT
<p><b>OBJECTIVE</b>To identify HCV core protein binding proteins.</p><p><b>METHODS</b>The library was amplified, purified, and then were transformed into yeast strain Y187. The reconstructed plasmid pGBKT7-core was transformed into yeast strain AH109 and screened on the nutrient deficiency medium SD/-Trp. The transformed AH109 mated with Y187 containing the library plasmid. The diploid yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade containing X-alpha-gal for selecting. The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5alpha. The plasmids in DH5alpha were extracted and sequenced.</p><p><b>RESULTS</b>Eleven proteins, including chymotrypsinogen B1 precursor, carboxypeptidase A1, trypsinogen 2, chymotryptic peptide C, trypsin 1, carboxypeptidase B1, kinesin superfamily proteins 3B, trypsin 2, mitochondria protein gene, elastase 3A and colipase were found to be able to bind to HCV core protein.</p><p><b>CONCLUSIONS</b>Proteins related with metabolism of glucose and lipid may bind to HCV core protein.</p>
Subject(s)
Gene Library , Hepacivirus , Genetics , Plasmids , Genetics , Polymerase Chain Reaction , Transformation, Genetic , Two-Hybrid System Techniques , Viral Core Proteins , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the effect of PGC-1alpha in human liver carcinogenesis, and explore the regulatory role of PGC-1alpha in the development of liver cancer.</p><p><b>METHODS</b>The changes of PGC-1alpha mRNA level in normal human liver tissues and human liver tumors was examined by quantitative RT-PCR. PGC-1alpha mRNA level was interfered by siRNA in human liver cell line L02 in vitro, and their morphological changes were observed by pathology with HE staining. The ultrastructure of cells was observed by electron microscopy. In addition, the gene expression pattern of decreasing PGC-1alpha in L02 cells and liver tumor tissue was compared by human genome 70-mer oligonucleotide microarray analysis.</p><p><b>RESULTS</b>PGC-1alpha expression was weaker in the malignant liver tumors compared with that in normal liver tissues. When PGC-1alpha expression was suppressed in human liver L02 cells, the cells became smaller with enlarged nuclei, and myelin figures were observed in mitochondria by electron microscopy, similar with the ultrastructure of liver cancer cells. Microarray analysis showed that the decrease of PGC-1alpha in L02 cells induced up-regulation of some oncogenes and adhesive genes, and down-regulation of a number of tumor suppressor genes and cell proliferation suppressor genes. The changes of decreasing expression pattern of PGC-1alpha gene in L02 cells were similar to those in human liver cancer tissue.</p><p><b>CONCLUSION</b>The results of the present study show that PGC-1alpha is down-regulated in liver cancers and is involved in the malignant transformation in human normal liver cells in vitro, suggesting an important regulatory role of PGC-1alpha in the development of liver cancer.</p>