By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 micromol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.
Cartilage/*cytology ; Cell Differentiation/*drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Oligodeoxyribonucleotides/genetics ; Oligodeoxyribonucleotides/*pharmacology ; Rats, Sprague-Dawley ; Stathmin/*genetics ; Stathmin/pharmacology ; Stem Cells/*cytology

OBJECTIVE: To observe expression of stathmin gene in laryngeal squamous cell carcinoma (LSCC) and relation between expression of stathmin gene and occurrence and development of LSCC. METHOD: The expression of the stathmin gene was determined in 35 LSCC of specimens and 18 normal laryngeal tissues (NLT) of specimens by in situ hybridization with Digoxigenin labeled probe of stathmin mRNA. RESULT: Expression of stathmin gene was observed in 35 cases of laryngeal squamous cell carcinoma tissue (positive rate, 69%) and positive signal was observed in both cytoplasm and nuclear. Among 18 cases of normal tissue, only 6 showed weak positive signal. There was significant difference in expression of stathmin gene between laryngeal squamous cell carcinoma tissue and normal tissue. CONCLUSION Expression of stathmin gene may play a key role in the pathogenesis and development of laryngeal squamous cell carcinoma. It may be a very important biotherapy target in the treatment of laryngeal squamous cell carcinoma.
Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Gene Expression ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; Stathmin ; genetics ; metabolism

BACKGROUNDStathmin was identified as an endometriosis-related protein by comparative proteomics in our previous study. As a microtubule-destabilizing factor, stathmin was shown to participate in the relay and integration of diverse intracellular signaling pathways involved in cell proliferation, differentiation, and many other cellular activities. To investigate whether stathmin is involved in the pathogenesis of endometriosis, we examined the expression of stathmin in eutopic endometrium of women with or without endometriosis.

METHODSEutopic endometrium samples were collected from thirty-six patients who were diagnosed as endometriosis and the nineteen age-matched patients who were confirmed to be free of endometriosis surgically and histologically. The expression of stathmin mRNA was detected by real-time PCR, and its protein was detected by Western blotting and immunohistochemistry.

RESULTSStathmin was overexpressed in eutopic endometrium of women with endometriosis detected by real-time PCR in mRNA levels and by Western blotting in protein levels, without significant difference between proliferative and secretory phase. Immunohistochemistry showed that stathmin protein was localized in both endometrial glandular and stromal cells throughout the menstrual cycle.

CONCLUSIONSStathmin is overexpressed in endometrium of patients with endometriosis and may play a role in the pathogenesis of endometriosis.

Adult ; Blotting, Western ; Endometriosis ; metabolism ; Endometrium ; metabolism ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Polymerase Chain Reaction ; Stathmin ; genetics ; metabolism

To explore the biological function and possible underlying mechanism of stathmin gene during hepatocarcinogenesis. Three pairs of chemically synthesized small interfering RNA (siRNA) targeting on stathmin were transfected into HCCLM3 by LipofectamineTM 2000. After confirming the interfering effects of stathmin siRNAs through reverse transcription PCR and Western blotting, the HCCLM3 cells proliferation and apoptosis were detected by cell count kit-8 (CCK-8) and flow cytometry analysis, and the expressions of tumor-related genes (c-myc, c-fos, p53, etc) were observed by real-time PCR. Stathmin expression was effectively inhibited up to 90% by stathmin silencing in HCCLM3 cells (P is less than to 0.05) . By using CCK8 assay, it was shown that HCCLM3 cells proliferation were obviously depressed by 13.04%+/-0.10%, 28.10%+/-0.41% and 37.36%+/-2.15% at the time point of 24 h, 48 h and 72 h with the comparison to Mock group (F = 4.21, P is less than to 0.05). The results of flow cytometry demonstrated that the percentage of apoptotic cells was increased to 25.11%+/-1.62% in RNAi group, compared with 9.20 %+/-0.64 % in Mock group (F = 44.67, P is less than to 0.01). The results of real-time PCR showed that oncogenes c-myc and c-fos expressions were repressed, proliferation-associated gene ki-67 was down-regulated, and apoptosis-promoting gene caspase-3, bax and p53 were induced (P is less than to 0.05). Stathmin may promote cell proliferation, inhibit cell apoptosis and induce malignant transformation of hepatocytes by regulating some tumor-related genes expressions.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Stathmin

OBJECTIVETo investigate the expressions of stathmin gene and its coding protein in laryngeal squamous cell carcinoma, and to explore the relationship between stathmin gene and the biological behaviors of laryngeal squamous cell carcinoma for understanding the tumorigenicity and development of laryngeal squamous cell carcinoma.

METHODSLaryngeal carcinoma tissues (studying group) in the tumors center and laryngeal normal tissues (control group) parted from 1.0 cm of the safe borderline of the tumors were took from 38 patients with laryngeal squamous cell carcinoma while they were in operation. Semiquantitative method of reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression level of stathmin mRNA, and immunohistochemical staining (frozen section) was used to detect the expressions of stathmin protein, in laryngeal carcinoma tissues and laryngeal normal tissues of 38 cases, respectively.

RESULTSmRNA of stathmin gene was all positively expressed in laryngeal carcinoma tissues and in laryngeal normal tissues of 38 cases by RT-PCR. However, stathmin mRNA was obviously overexpressed in laryngeal carcinoma tissues than that in laryngeal normal tissues (t = 9.655, P < 0.05). Immunohistochemical staining showed stathmin protein was positively expressed in laryngeal carcinoma tissues of 26 cases (26/38, 68.4%), and mild-positively expressed in laryngeal normal tissues in 13 cases (13/38, 34.2%). There was significant difference between the expression rate of stathmin protein in laryngeal carcinoma tissues and in laryngeal normal tissues (chi2 = 8.901, P < 0.05). Meanwhile, the expression level of stathmin mRNA and the positive-expressed rate of stathmin protein in laryngeal carcinoma tissues of the advanced stage patients group (III stage and IV stage) were significantly higher than these in laryngeal carcinoma tissues of I and II stage patients group (t = 6.284, chi2 = 5.810, P < 0.05), and they were also significantly higher in laryngeal carcinoma tissues of the patients group with cervical lymph node metastasis than in laryngeal carcinoma tissues of the patients group without cervical lymph node metastasis (t = 9.350, chi2 = 6.923, P < 0.05).

CONCLUSIONSThe expression levels of stathmin gene and protein were significantly higher in laryngeal squamous cell carcinoma than these in laryngeal normal tissues, the levels are also significantly higher in advanced stage patients group (III stage and IV stage) than in the early stage patients group (I and II), and they are also related to the cervical lymph node metastasis of carcinoma. Stathmin gene may play an important role in the pathogenesis and development of laryngeal carcinoma and may be related to its prognosis.

Adult ; Aged ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Stathmin ; genetics ; metabolism

OBJECTIVETo explore the expression of stathmin gene in esophageal squamous cell carcinoma (ESCC) and its correlation to oncogenesis of ESCC.

METHODSThree ESCC cell lines, 75 ESCC samples, 25 tumor-adjacent samples and 30 normal esophageal mucosa samples were examined for the expression of stathmin mRNA and protein by in situ hybridization and immunohistochemistry, respectively. The correlations of stathmin expression to the clinicopathological features of the patients were analyzed.

RESULTSOverexpression of stathmin mRNA and protein was found in 3 ESCC cell lines EC9706, Eca109 and EC-1, with the positive expression rates exceeding 80%. The positive rates of stathmin mRNA and protein in ESCC samples were 82.7% and 81.3%, respectively. There were significant differences in the relative contents of stathmin mRNA and protein among normal mucosa tissue, tumor-adjacent tissue and cancer tissue (chi2=19.204 and 25.03, respectively, P<0.01). In addition, a positive correlation was noted between stathmin mRNA and protein expressions in ESCC (r=0.413, P=0.000). The relative contents of stathmin mRNA and protein were significantly correlated to the differentiation degree, lymph node metastasis, invasive depth and TNM stage of ESCC (P<0.05).

CONCLUSIONSThe expression of stathmin mRNA and protein is upregulated in ESCC with correlation to the differentiation degree, lymph node metastasis, invasive depth and TNM stage of ESCC, suggesting the possible involvement of stathmin in the oncogenesis of ESCC. Combined detection of stathmin mRNA and protein may prove valuable for early diagnosis and prognosis of ESCC, and stathmin may serve as a potential molecular target for biotherapy of the tumor.

Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis ; Stathmin ; genetics ; metabolism

OBJECTIVETo construct an eukaryotic expression vector of human stathmin gene and to assess its effect on esophageal cancer EC9706 cells.

METHODSStathmin cDNA coding sequence was amplified by RT-PCR from Eca109 cells and was cloned into pMD18-T vector. After identifying and sequencing, the correct inserting stathmin gene was sub-cloned into eukaryotic expression vector pEGFP-C2. EC9706 cells were transfected with this recombinant plasmid and control plasmid using Lipofectamine 2000, and the stable intergrant was selected with G418 medium. The expression of enhanced green fluorescent protein (EGFP) protein was detected by fluorescence microscopy and EGFP/stathmin fusion protein by Western blot assay in transfected EC9706 cells. The growth curve of the two stably transfected cells was protracted with cell counting. FACS was used to detect the cell cycle. The clone formation rate in plate and in nude mice was tested to investigate the tumorigenic characteristics of the two stably transfected cells in vitro and vivo.

RESULTSA 450 bp coding sequence of stathmin cDNA was amplified by RT-PCR, which was cloned into pMD18-T vector. After identified with restriction enzyme the recombinant plasmid pMD18-T-stathmin containing reverse inserting sequence was constructed successfully. Then, the sub-clone pEGFP-stathmin was sequenced, confirming that the recombinant vector was right. The recombinant plasmid pEGFP-stathmin and pEGFP-C2 vector were transfected separately into EC9706 cells. After selecting with G418, the cells were transfected steadily. EGFP in EC9706 cells was observed after transfection by fluorescence microscopy. The expressed product was proved to be 46,000 EGFP/stathmin fusion protein by Western blot. Compared with those transfected with pEGFP-C2, the growth of cells transfected with pEGFP-stathmin became slow, the cells were swelled, the cell cycle was blocked at G2/M phase, the average clone formation rate decreased in vitro, and the tumorigenicity of inoculated cells in nude mice was decreased.

CONCLUSIONThe recombinant eukaryotic expression vector pEGFP-stathmin has been constructed successfully. It expresses steadily in esophageal cancer cells and inhibits the proliferation and tumorigenicity of transfected cells.

Animals ; Cell Cycle ; Cell Proliferation ; Escherichia coli ; genetics ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Plasmids ; Recombinant Fusion Proteins ; genetics ; metabolism ; Stathmin ; genetics ; metabolism ; Transfection

OBJECTIVE: To determine the biological activity of ellagic acid extracted from gallnut against nasopharyngeal carcinoma and its molecular mechanism. METHODS: Nasopharyngeal carcinoma 5-8F cells were treated with 2, 4, 6 μg/mL ellagic acid for 48 h in vitro. The cell proliferation and cell apoptosis were analyzed by MTT and Hoechst33258 stain. The cell cycle and protein expression were measured by flow cytometry and Western blot. RESULTS: Ellagic acid inhibited the proliferation of 5-8F cells. The inhibition rates were (29.35±4.95)%, (53.32 ±4.44)% and ( 61.75 + 6.93)%, respectively, with significant difference from the control group (P<0.01). S phase cells in the experimental groups were (25.47±0.74)%, (28.08±1.41)% and (35.49±0.66)%, respectively, with significant difference (P<0.01) from the control group (21.26±0.70)%. Cells in the experimental groups showed nuclear pyknosis, karyorrhexis and poptotic cell morphology. The expression of COX-2 and stathmin in 5-8F cells was down-regulated with increased drug concentration. CONCLUSION Ellagic acid extracted from gallnut has activity against nasopharyngeal carcinoma cells, and its mechanism may be related to down-regulated expression of COX-2 and stathmin.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 ; genetics ; metabolism ; Down-Regulation ; drug effects ; Ellagic Acid ; pharmacology ; Humans ; Nasopharyngeal Neoplasms ; pathology ; Plant Extracts ; pharmacology ; Stathmin ; genetics ; metabolism

BACKGROUNDMultidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia. The resistance process is multifactorial. To realize the total factors involved in multidrug resistance, we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins (CRKL) using siRNA to determine its role in K562/ADM cells.

METHODSAltered protein expressions between K562/S (K562 ADM-sensitive cell line) and K562/ADM (K562 multidrug resistant cell line induced by adriamycin) were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile, we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis. Furthermore, we used RNA interference to silence the CRKL gene expression.

RESULTSAmong the 9 differentially expressed proteins, 3 were up-regulated in K562/ADM cells, while 6 were down-regulated in the K562/ADM cells compared with its parent cell line. The expression of CRKL was up-regulated significantly in K562/ADM cells, and it can be decreased by recombinant lentivirus. Moreover, the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression.

CONCLUSIONSThe data provided the differentially expressed proteins in K562 and its resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer. We found CRKL may be a new protein involved in the multidrug resistance of leukaemia cells.

Adaptor Proteins, Signal Transducing ; analysis ; antagonists & inhibitors ; genetics ; Amino Acid Sequence ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Humans ; K562 Cells ; chemistry ; drug effects ; Molecular Sequence Data ; Neoplasm Proteins ; analysis ; Nuclear Proteins ; analysis ; antagonists & inhibitors ; genetics ; Proteomics ; Stathmin ; analysis

To explore differentially expressed genes in leukemia gene expression profile and identify main related genes in acute leukemia, gene expression profiles were analyzed in bone marrow/leucopheresis peripheral blood stem cells samples from 9 acute leukemia patients and their sibling donors with the use of oligonucleotide microarrays. 163 reported leukemia-related genes were involved in the study. The oligonucleotide primers were designed, synthesized and spotted on the chemical-material-coated-glass plates in array. The total RNAs were isolated from nine patients' bone marrow or leucopheresis peripheral blood cells and from nine their sibling donors peripheral blood stem cells treated by G-CSF, then collected by CS-3000 cell selection machine, and were reversely transcribed to cDNAs with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the oligonucleotide microarray. The results showed that in four patient/donor pairs with B-ALL, 5 up-regulated (RIZ, STK-1, T-cell leukemia/lymphoma 1A, Cbp/p300, Op18) and 1 down-regulated genes (hematopoietic proteoglycan core protein) were identified; In five patient/donor pairs with AML-M(4) and AML-M(5), 6 up-regulated (STAT5B, ligand p62 for the Lck SH2, CST3, LTC4S, myeloid leukemia factor 2 and epb72) and 1 down-regulated genes (CCR5) were identified. In conclusion, on the basis of distinguishing study of specific genetic related recipient/sibling donor pairs, screening leukemia-related genes with oligonucleotide microarrays, a set of 13 up-regulated or down-regulated genes among 163 leukemia-related genes has been identified. The result has further confirmed that above genes play critical role in the molecular mechanism of acute leukemia.
Blood Donors ; DNA-Binding Proteins ; genetics ; Gene Expression Profiling ; Glutathione Transferase ; genetics ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Microtubule Proteins ; genetics ; Milk Proteins ; genetics ; Oligonucleotide Array Sequence Analysis ; Peripheral Blood Stem Cell Transplantation ; Phosphoproteins ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; STAT5 Transcription Factor ; Siblings ; Stathmin ; Trans-Activators ; genetics