OBJECTIVETo study the effect of the inhibition of CD44 gene expression on the growth of human nasopharyngeal carcinoma cell CNE-2L2 in vitro.

METHODSCD44 gene expression in cells was suppressed by siRNA which was introduced into cells through retrovirus infection. Integration of siRNA into genomic DNA was examined by genomic PCR. CD44 gene expression in cells was detected by Western blot analysis. Cell growth in vitro was assayed using Cell Titer 96 AQueous One Solution Cell Proliferation Assay kit Promega. Cells were stained with propidium iodium and cell DNA content was detected upon a flow cytometer.

RESULTSsiRNA was integrated into genomic DNA of host cells. The 4 cell pools integrated with one of the 4 siCD44s showed a significant inhibition of CD44 gene expression comparing to the controls, the wild type cell and the cell pool integrated with siegfp. The cell pools integrated with siCD44-1 or siCD44-2 showed the most profound inhibition. Growth of these 2 cells in vitro was compared to that of the controls and was found to be significantly inhibited. Cell DNA content analysis indicated 44.4%, 45.5%, 53.9%, and 53.3% in G0/G1 phase; 39.3%, 40.0%, 27.1%, and 28.2% in S phase; and 16.3%, 14.5%, 19.0%, and 18.5% in G2/M phase for the wild type cell, the cell pool integrated with siegfp, the cell pools integrated with siCD44-1, and the cell pools integrated with siCD44-2, respectively.

CONCLUSIONReduction in CD44 expression inhibit the growth of CNE-2L2 cell and affects the development of cells from G0/G1 into S phase, but may somehow promote cells to develop from S into G2/M phase.

Cell Line, Tumor ; Cell Proliferation ; DNA ; genetics ; Humans ; Hyaluronan Receptors ; genetics ; Nasopharyngeal Neoplasms ; pathology ; RNA, Small Interfering ; genetics

Carcinoma, Hepatocellular ; metabolism ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Hyaluronan Receptors ; biosynthesis ; genetics ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; Osteopontin ; biosynthesis ; genetics

OBJECTIVETo investigate the expression of CD44 (intron 9) and CD44v6 in brain neoplasm.

METHODSCD44 (intron 9) and CD44v6 were detected in malignant meningoma, encephalic glioma, and normal encephalic tissues around the brain noeplasm respectively by using RT-PCR.

RESULTSThe positive expression rate of CD44v6 was 92.86% (26/28) in malignant meningoma and 88.0% (22/25) in encephalic glioma. They were significantly higher than 5.0% (1/20) in normal encephalic tissues (P < 0.01). Mass values of malignant meningoma and encephalic glioma were higher than that of normal encephalic tissues (P < 0.01). The expression of CD44 (intron 9) was higher in malignant meningoma and encephalic glioma than in normal encephalic tissues (P < 0.01).

CONCLUSIONSDetection of CD44 (intron 9) and CD44v6 might be helpful to the diagnosis of malignant cerebroma.

Adolescent ; Adult ; Aged ; Brain Neoplasms ; genetics ; metabolism ; Child ; Child, Preschool ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Hyaluronan Receptors ; biosynthesis ; genetics ; Introns ; genetics ; Middle Aged ; RNA, Messenger ; biosynthesis

This study was aimed to investigate the effect of CD44 gene silence on the drug resistance and biologic activity of human multidrug resistant leukemia cell line K562/A02. The oligonucleotides of CD44 gene were designed according to related data of GenBank, double-stranded DNA was produced by annealing, and was inserted into pGCsilencerU6/Neo/GFP vector. The resultant recombinant plasmid pGCsiRNA-CD44 was transfected into K562/A02 cell line. Expressions of CD44, mdr-1 and blc-2 mRNA were assayed by real time RT-PCR. The 50% inhibitory concentration (IC50) of doxorubicin (ADM) for K562/A02 cell line was determined by MTT method. Cell cycle was determined by flow cytometry. The morphology of apoptotic cells was examined by Hochst 33258 staining. The results indicated that the siRNA eukaryotic plasmid directing at CD44 gene could effectively silence the CD44 gene of K562/A02 cells; as compared with control group, the CD44 expression in K562/A02 cells transfected with 4 pGCsiRNA-CD44 plasmids was obviously inhibited, while the inhibition of CD44 expression in cells transfected with siCD44-1 was strongest. After being transfected with pGCsiRNA-CD44, the expression of CD44 mRNA in K562/A02 cells reduced by 64.1% (p<0.05), at the same time the expression of mdr-1 and bcl-2 mRNA in pGCsiRNA-CD44-transfected K562/A02 cells reduced by 25.6% and 50.8% respectively. IC50 of K562/A02 cells after transfection decreased to (8.77+/-1.63) microg/ml and was obviously lower than that of control (17.97+/-1.61) microg/ml (p<0.01). After transfection for 48 hours, the ratio of K562/A02 cells in G0/G1 increased by 10.7%, and the cells displayed karyopyknosis, nuclear margination and apoptotic bodies. It is concluded that the siRNA plasmid specifically targeting CD44 gene can remarkably down-regulate the expression of CD44 gene, inhibit K562/A02 cell proliferation, induce its apoptosis and effectively reverse the multidrug resistance of K562/A02 cells.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Gene Silencing ; Genetic Vectors ; Humans ; Hyaluronan Receptors ; genetics ; K562 Cells ; RNA, Small Interfering

OBJECTIVETo explore the possibility of treating solid tumor with siCD44.

METHODSHuman nasopharyngeal carcinoma cell CNE-2L2 with high expression of CD44 was used in this study. The malignant activities of cells were examined by colony formation test, tumorigenesis, and lung metastasis of the tumor in nude mice. Ad5-siCD44 was constructed and adenoviruses were produced in 293 cells. CNE-2L2 cells were subcutaneously inoculated into nude mice. When tumors grew to 50-100 mm3, Ad5-siCD44 was injected into tumors, and Ad5-egfp and PBS were also injected as controls. The size and weight of tumors were compared after 2 weeks.

RESULTSSuppression of CD44 expression profoundly inhibited the malignant activities of CNE-2L2 cell. The average sizes of the tumors were (3.139 +/- 0.850), (3.612 +/- 0.888), and (1.512 +/- 0.742) cm3 after the intra-tumor injection of PBS, Ad5-egfp, and Ad5-siCD44, respectively, after two weeks. Significant difference was found between Ad5-siCD44 group and control groups (P < 0.05). The average weights were (2.28 +/- 0.73), (1.83 +/- 0.26), and (1.20 +/- 0.64) g, respectively, and significant difference was also found between Ad5-siCD44 group and control groups (P < 0.05).

CONCLUSIONIntra-tumor injection of Ad5-siCD44 can exhibit the therapeutic effect on the tumor inoculated with CNE-2L2 cells with high expression of CD44 in nude mice.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Genetic Therapy ; Humans ; Hyaluronan Receptors ; biosynthesis ; genetics ; Injections, Intralesional ; Mice ; Mice, Nude ; Nasopharyngeal Neoplasms ; genetics ; pathology ; therapy ; Neoplasm Transplantation ; Transplantation, Heterologous

OBJECTIVETo investigate the impact of S518 phosphorylation in Merlin on the interaction with CD44 in vestibular schwannoma and the tumor growth.

METHODSThirty-five samples of vestibular schwannoma were identified by pathology. Immunohistopathology and western blot were employed to analyze the expression and localization of S518 phosphorylated Merlin in the tumor tissues. Nerve tissues that were collected during other surgical operation were used as control. The expression level of S518 phosphorylated Merlin was compared with clinical stages, tumor size, clinical course and cystic degeneration. Immunoprecipitation was used to evaluate the impact of S518 phosphorylation in Merlin on the interaction with CD44.

RESULTSIn vestibular schwannoma, Merlin was phosphorylated at S518 and demonstrated perinuclear localization. The S518 phosphorylation level was much lower in the normal control nerve tissues than that in vestibular schwannoma tissues. There was no correlation between the phosphorylation level on Merlin and clinical stages, tumor size, clinical course and cystic degeneration. The S518 phosphorylated Merlin bound CD44 was higher than wild-type Merlin bound CD44 in vestibular schwannoma tissues.

CONCLUSIONSThe affinity of Merlin to CD44 was increased after phosphorylation at S518. Different cellular biological results might be triggered through binding to wild type Merlin and S518 phosphorylated Merlin.

Adult ; Aged ; Female ; Genes, Neurofibromatosis 2 ; Humans ; Hyaluronan Receptors ; genetics ; metabolism ; Male ; Middle Aged ; Neoplasm Staging ; Neurofibromin 2 ; genetics ; metabolism ; Neuroma, Acoustic ; genetics ; metabolism ; pathology ; Phosphorylation

OBJECTIVETo study the influence of CD44 a cell-matrix adhesion molecule on the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines, in order to investigate the growth and invasion mechanism of osteosarcoma.

METHODSThree osteosarcoma cell lines MG-63, HOS and U2-OS were routinely cultured. Flow cytometry and Western blot analysis were used for detecting the positive rates and relative amount of CD44 protein in the three cell lines. RT-PCR method was also used to compare the differences in the expression of CD44 mRNA among the 3 cell lines. Then, MTT method, adhesion detection, and Microcon-migration assay were used to detect the changes of the cells' proliferation rate, adhesive and invasive abilities after blocking the role of CD44 by using a special neutralizing antibody.

RESULTSThe results of flow cytometry showed that the percentage of CD44 positive cells were both over 99% in HOS and U2-OS, while that in MG-63 was only (2.10 +/- 0.46)%. The average fluorescence density of CD44 in HOS was significantly higher than in U2-OS. Western blot also showed that the relative content of CD44 protein in HOS was notably higher than that in U2-OS, while CD44 was negatively expressed in MG-63. The expression of CD44 mRNA was significantly lower in MG-63 than in both HOS and U2-OS, which were consistent with the expression of CD44 protein. The proliferation rates and adhesive abilities of MG-63 and HOS have no significant difference, but both were significantly higher than that of U2-OS. The invasive abilities of HOS was dramatically higher than MG-63 and U2-OS. After the role of CD44 was blocked by anti-CD44 neutralizing antibody, the proliferation rates of the 3 cell lines did not change significantly. While the HOS and MG-63 adhesion indices decreased dramatically (P < 0.05), the invasive abilities of HOS and U2-OS also decreased notably (P < 0.01).

CONCLUSIONSCD44 could promote the adhesiveness and invasiveness of osteosarcoma cell line HOS. CD44 may take part in promoting the process of U2-OS invasion and the adhesion of MG-63. On the other hand, CD44 could not affect the osteosarcoma cell proliferation rates.

Bone Neoplasms ; metabolism ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Hyaluronan Receptors ; biosynthesis ; genetics ; physiology ; Neoplasm Invasiveness ; Osteosarcoma ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics

As an important part of tumor microenvironment, neutrophils are poorly understood due to their spatiotemporal heterogeneity in tumorigenesis. Here we defined, at single-cell resolution, CD44-CXCR2- neutrophils as tumor-specific neutrophils (tsNeus) in both mouse and human gastric cancer (GC). We uncovered a Hippo regulon in neutrophils with unique YAP signature genes (e.g., ICAM1, CD14, EGR1) distinct from those identified in epithelial and/or cancer cells. Importantly, knockout of YAP/TAZ in neutrophils impaired their differentiation into CD54+ tsNeus and reduced their antitumor activity, leading to accelerated GC progression. Moreover, the relative amounts of CD54+ tsNeus were found to be negatively associated with GC progression and positively associated with patient survival. Interestingly, GC patients receiving neoadjuvant chemotherapy had increased numbers of CD54+ tsNeus. Furthermore, pharmacologically enhancing YAP activity selectively activated neutrophils to suppress refractory GC, with no significant inflammation-related side effects. Thus, our work characterized tumor-specific neutrophils in GC and revealed an essential role of YAP/TAZ-CD54 axis in tsNeus, opening a new possibility to develop neutrophil-based antitumor therapeutics.
Humans ; Animals ; Mice ; Adaptor Proteins, Signal Transducing/metabolism* ; Transcription Factors/metabolism* ; Stomach Neoplasms/pathology* ; Neutrophils/pathology* ; Signal Transduction/genetics* ; YAP-Signaling Proteins ; Tumor Microenvironment ; Hyaluronan Receptors/genetics*

OBJECTIVE: To study the correlation of CD44 with epithelial-mesenchymal transition(EMT) and metastasis in nasopharyngeal cancer cells, and explore the possible mechanism of CD44 regulates EMT and metastasis in nasopharyngeal cancer cells. METHOD: The CD44 and EMT-associated proteins in 5-8F and 6-10B nasopharyngeal cancer cell lines were assayed by Western blotting. The erasion trace test was performed to observe the migratory ability of 5-8F and 6-10B nasopharyngeal cancer cells. Using lipid-mediated DNA transfection technique, the low metastatic nasopharyngeal cancer cells 6-10B were transfected in vitro with plasmid which contained CD44 gene, and then new nasopharyngeal cancer cells were obtained. The CD44 and EMT-associated proteins in 6-10B, empty vector transfected and CD44-transfected cells were assayed by Western blotting. The erasion trace test was performed to observe the alteration of migratory ability of nasopharyngeal cancer cells before and after CD44 transfection. RESULT: The expression of CD44 and EMT-associated protein MMP-9 in 5-8F was higher than that in 6-10B, but EMT-associated protein E-Cadherin in 5-8F was lower than that in 6-10B. The migratory ability of 5-8F was higher than that of 6-10B. The expression of CD44 and MMP-9 were significantly higher in the CD44-transfected nasopharyngeal cancer cells than in the control groups. Compared with control groups, the migratory ability of CD44-transfected nasopharyngeal cancer cells was significantly increased. CONCLUSION CD44 positively regulates the metastatic ability of nasopharyngeal cancer cells, which is relevant to the process of EMT.
Carcinoma ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition ; Humans ; Hyaluronan Receptors ; genetics ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neoplasm Metastasis ; Transfection

The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD44-specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44 might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.
Cell Adhesion ; Cells, Cultured ; Glaucoma, Open-Angle ; metabolism ; pathology ; Humans ; Hyaluronan Receptors ; biosynthesis ; genetics ; Hyaluronic Acid ; metabolism ; Oligonucleotides, Antisense ; pharmacology ; Trabecular Meshwork ; cytology