BACKGROUND: Cluster of differentiation 8 positive (CD8 + ) T cells play a crucial role in the response against tumors, including hepatocellular carcinoma (HCC), where their dysfunction is commonly observed. While the association between elevated peroxisome proliferator-activated receptor alpha (PPARα) expression in HCC cells and exosomes and unfavorable prognosis in HCC patients is well-established, the underlying biological mechanisms by which PPARα induces CD8 + T cell exhaustion mediated by HCC exosomes remain poorly understood. METHODS: Bioinformatics analyses and dual-luciferase reporter assays were used to investigate the regulation of microRNA-27b-3p ( miR-27b-3p ) and thymocyte selection-associated high mobility group box ( Tox ) by Pparα . In vitro and in vivo experiments were conducted to validate the effects of HCC-derived exosomes, miR-27b-3p overexpression, and Pparα on T cell function. Exosome characterization was confirmed using transmission electron microscopy, Western blotting, and particle size analysis. Exosome tracing was performed using small animal in vivo imaging and confocal microscopy. The expression levels of miR-27b-3p , Pparα , and T cell exhaustion-related molecules ( Tox , Havcr2 , and Pdcd1 ) were detected using quantitative reverse transcription polymerase chain reaction analysis, Western blotting analysis, immunofluorescence staining, and flow cytometry analysis. RESULTS: Pparα expression was significantly increased in HCC and negatively correlated with prognosis. It showed a positive correlation with Tox and a negative correlation with miR-27b-3p . The overexpressed Pparα from HCC cells was delivered to CD8 + T cells via exosomes, which absorbed miR-27b-3p both in vitro and in vivo , acting as "miRNA sponges". Further experiments demonstrated that Pparα can inhibit the negative regulation of Tox mediated by miR-27b-3p through binding to its 3'untranslated regions. CONCLUSIONS HCC-derived exosomes deliver Pparα to T cells and promote CD8 + T cell exhaustion and malignant progression of HCC via the miR-27b-3p /TOX regulatory axis. The mechanisms underlying T-cell exhaustion in HCC can be utilized for the advancement of anticancer therapies.
MicroRNAs/metabolism* ; PPAR alpha/genetics* ; Carcinoma, Hepatocellular/genetics* ; Humans ; Liver Neoplasms/genetics* ; CD8-Positive T-Lymphocytes/immunology* ; Exosomes/metabolism* ; Animals ; Cell Line, Tumor ; Mice ; High Mobility Group Proteins/genetics* ; Male ; T-Cell Exhaustion

OBJECTIVEThe protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability.

METHODSThe yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry.

RESULTSWe identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1.

CONCLUSIONTgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth.

Apoptosis ; Blotting, Western ; DNA-Binding Proteins ; genetics ; metabolism ; Flow Cytometry ; HeLa Cells ; High Mobility Group Proteins ; genetics ; metabolism ; Humans ; Immunoprecipitation ; Phosphoprotein Phosphatases ; genetics ; metabolism ; Protein Phosphatase 2C ; Toxoplasma ; enzymology ; Transcriptional Elongation Factors ; genetics ; metabolism ; Two-Hybrid System Techniques

Gene expressions are sex-specific in the sex development of mammals. Different genes express in different phases and tend to change with the time. The functions of some genes, such as SRY, SOX9, SOX8, DAX1, and FGF9, have already been defined in male gonadal morphogenesis. This paper presents a review of the genes involved in the formation of the male gonad in mammals.
Animals ; DAX-1 Orphan Nuclear Receptor ; DNA-Binding Proteins ; genetics ; Gene Expression Regulation, Developmental ; Genitalia, Male ; embryology ; growth & development ; metabolism ; High Mobility Group Proteins ; genetics ; Male ; Mammals ; embryology ; genetics ; growth & development ; Morphogenesis ; genetics ; Receptors, Retinoic Acid ; genetics ; Repressor Proteins ; genetics ; SOX9 Transcription Factor ; Sex-Determining Region Y Protein ; genetics ; Transcription Factors ; genetics

OBJECTIVETo construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration.

METHODSBac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDuljgreen fluorescene protein (GFP)-CMV (pFGC)-hSox9. The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani (LB) agarose culture medium containing isopropyl-beta-D-thiogalactoside (IPTG), X-gal, gentamicin, kanamycin and tetracycline. The white colonies were selected and cultured for amplification, and the hSox9Bacmid DNA was extracted. After verification, recombinant baculovirus Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells. The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining.

RESULTSPolymerase chain reaction (PCR) confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein. Western blotting and immunohistochemical staining analysis indicated that exogenous hSox9 gene was expressed in the disc cells.

CONCLUSIONSThe successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.

Animals ; Baculoviridae ; genetics ; Cytomegalovirus ; genetics ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; High Mobility Group Proteins ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Intervertebral Disc ; cytology ; metabolism ; pathology ; Lumbar Vertebrae ; Plasmids ; Rabbits ; Recombinant Proteins ; SOX9 Transcription Factor ; Spinal Diseases ; therapy ; Transcription Factors ; genetics ; metabolism ; Transfection

OBJECTIVETo assess the regulating effects of interleukin-1 (IL-1) on gene expression of cartilage specificity gene Sox9 and type II collagen mRNA in the human intervertebral discs.

METHODSRT-PCR were used to investigate the effects of IL-1 on gene expression of Sox9 and type II collagen mRNA in intervertebral discs cells cultures of embryo.

RESULTSThe Sox9 and type II collagen mRNA in intervertebral discs were decreased progressively along with the addition concentrations of IL-1 than the controls. And the mRNA of Sox9 and type II collagen also markedly decreased with the time of culture.

CONCLUSIONSIL-1 could cause dose-dependent and time-dependent inhibition effects on Sox9 and type II collagen gene expression in human intervertebral discs.

Cells, Cultured ; Collagen Type II ; genetics ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; High Mobility Group Proteins ; genetics ; Humans ; Interleukin-1 ; pharmacology ; Intervertebral Disc ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; SOX9 Transcription Factor ; Time Factors ; Transcription Factors ; genetics

Mouse embryonic stem (mES) cells are capable of undergoing chondrogenesis in vitro. To enhance this process, the human SOX9 (hSOX9) cDNA was delivered into mES cells and the clones overexpressing hSOX9 (denoted as mES-hSOX9 cells) were verified by Western blot analysis. The transcripts of collagen IIA (a juvenile form), aggrecan and Pax1 were expressed in mES-hSOX9 cells grown on feeder layers, suggesting the immediate effect of exogenous SOX9 on chondrogenesis. However, SOX9 overexpression did not affect the cell cycle distribution in undifferentiated mES cells. Upon differentiation, collagen IIB (an adult form) was detected in day 3 immature embryoid bodies. In addition, the overexpression of exogenous SOX9 significantly induced transcriptional activity driven by SOX9 binding site. Taken together, we for the first time demonstrated that constitutive overexpression of exogenous SOX9 in undifferentiated mES cells might have dual potentials to induce both chondrogenic commitment and growth capacity in the undifferentiated status.
Animals ; Cell Differentiation/genetics ; Cell Line ; *Chondrogenesis ; Collagen Type II/genetics ; Embryo/*cytology ; Enhancer Elements (Genetics)/genetics ; Extracellular Matrix Proteins/genetics ; Genetic Markers/genetics ; High Mobility Group Proteins/genetics/*metabolism ; Humans ; Lectins, C-Type/genetics ; Mice ; Paired Box Transcription Factors/genetics ; Proteoglycans/genetics ; Research Support, Non-U.S. Gov't ; Stem Cells/*metabolism/physiology ; Trans-Activation (Genetics) ; Transcription Factors/genetics/*metabolism

Gondadal differentiation is genetically determined in humans. Sex is determined when the bipotential embryologic tissues differentiate into testes or ovary. SRY, a gene located on the Y chromosome, triggers a complex genetic cascade leading to testicular differentiation. However, only a minority of 46, XY sex reversal patients can be explained by SRY mutations, suggesting that other genes influencing sex determination are to be discovered. Recent studies show that testis differentiation requires insulin receptor family function in mice. SRY normally requires two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for gonadal differentiation.
Active Transport, Cell Nucleus ; Female ; Gene Expression Regulation ; Genes, sry ; physiology ; High Mobility Group Proteins ; genetics ; physiology ; Humans ; Male ; SOX9 Transcription Factor ; Sex Differentiation ; Transcription Factors ; genetics ; physiology

delta12-Prostaglandin (PG) J2 is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed delta12-PGJ2-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of delta12-PGJ2- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated delta12-PGJ2-induced caspase activation, loss of mitochondrial transmembrane potential (delta psi m), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the delta psi m was dissipated. One of the earliest events observed in delta12-PGJ2-induced apoptotic events was dissipation of delta psi m, the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of delta psi m depolarization in delta12-PGJ2- induced apoptosis. Up-regulation of Sox-4 protein by delta12-PGJ2 was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that delta12-PGJ2 induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that delta12-PGJ2-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by delta12-PGJ2.
Amino Acid Chloromethyl Ketones/pharmacology ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects/*physiology ; Caspases/physiology ; Cytochromes c/physiology ; Female ; Flavoproteins/metabolism/*physiology ; Hela Cells ; High Mobility Group Proteins/*physiology ; Humans ; Membrane Proteins/metabolism/*physiology ; Mitochondria/metabolism/physiology ; Prostaglandin D2/*pharmacology ; Protein Transport/physiology ; Proto-Oncogene Proteins c-bcl-2/biosynthesis/*physiology ; Research Support, Non-U.S. Gov't ; Trans-Activation (Genetics) ; Trans-Activators/*physiology

We reported earlier that expression of Sox-4 was found to be elevated during prostaglandin (PG) A2 and delta(12)-PGJ(12) induced apoptosis in human hepatocarcinoma Hep3B cells. In this study, the role of Sox-4 was examined using human Hep3B and HepG2 cell lines. Sox-4 induction by several apoptotic inducer such as A23187 (Ca(2+) ionophore) and etoposide (topoisomerase II inhibitor) and Sox-4 transfection into the cells were able to induce apoptosis as observed by the cellular DNA fragmentation. Antisense oligonucleotide of Sox-4 inhibited the induction of Sox-4 expression and blocked the formation of DNA fragmentation by PGA(2) and delta(12)-PGJ(12) in Hep3B and HepG2 cells. Sox-4-induced apoptosis was accompanied with caspase-1 activation indicating that caspase cascade was involved in this apoptotic pathway. These results indicate that Sox-4 is involved in Hep3B and HepG2 cells apoptosis as an important apoptotic mediator.
Apoptosis/*drug effects ; Blotting, Western ; Calcimycin/pharmacology ; Caspase 1/antagonists & inhibitors/metabolism ; Etoposide/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; High Mobility Group Proteins/genetics/*metabolism ; Human ; Liver Neoplasms/enzymology/metabolism/pathology ; Oligopeptides/pharmacology ; Prostaglandin D2/*analogs & derivatives/*pharmacology ; Prostaglandins A/*pharmacology ; Trans-Activators/genetics/*metabolism ; Transfection ; Tumor Cells, Cultured

OBJECTIVE: Thyroid nodule is frequent and occurs in about 5+ACU- of the general population. In contrast, thyroid cancer is much less frequent and occurs in about 5-10+ACU- of thyroid nodules. Distinguishing between benign and malignant lesions is an important task that is best accomplished by fine needle aspiration. Recently, Chiappetta et al. reported that the expression of the high mobility group (HMG) I(Y) proteins correlates with the malignant phenotype of human thyroid neoplasia, and suggested that the detection of the HMG I(Y) proteins might be a valid tool for an easy and sensitive discrimination assay between benign and malignant neoplastic thyroid disease. METHODS: We evaluated the expression of the HMG I(Y) mRNA in 39 frozen thyroid tissues from patients with thyroid nodule by semiquantitative RT-PCR. RESULTS: The expression of the HMG I(Y) mRNA was low in all of 10 normal thyroid tissues. In all of 3 adenomatous goiters, 6 follicular adenomas and 2 Hurthle cell adenomas, the HMG I(Y) mRNA expression level was low. In 11 of 13 papillary carcinomas and all of 5 follicular carcinomas, the HMG I(Y) mRNA expression level was high. CONCLUSION: These results indicate that there is a correlation between the expression of HMG I(Y) and the malignant phenotype of thyroid cancer, suggesting that these proteins may be useful as a marker in thyroid cancer.
Biopsy, Needle ; Comparative Study ; Diagnosis, Differential ; Female ; Gene Expression Regulation, Neoplastic ; High Mobility Group Proteins/analysis+ACo- ; Human ; Male ; Probability ; RNA, Messenger/analysis+ACo- ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity ; Thyroid Neoplasms/genetics+ACo- ; Thyroid Neoplasms/diagnosis+ACo- ; Thyroid Nodule/genetics+ACo- ; Thyroid Nodule/diagnosis+ACo- ; Tumor Markers, Biological/analysis+ACo-