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

OBJECTIVETo screen the differently expressed proteins related to regulating the depolymerization of microtubules in the spinal cord of hens exposed to tri-o-cresyl phosphate (TOCP) and to provide target protein evidence for exploring the mechanisms of the delayed neurotoxicology (OPIDN) induced by organophosphorus compounds (OPs).

METHODSForty two Roman hens were randomly divided into three groups, i.e. TOCP group treated with 1000 mg/kg TOCP; intervention group treated with 40 mg/kg phenylmethanesulfonyl fluoride (PMSF) before 1000 mg/kg TOCP treatment and control group treated with tap water. Four hens in each group were sacrificed on the 5th and 20th days after exposure, respectively. Spinal cords were separated and homogenates at low temperature, and the total proteins were extracted. The OPIDN symptoms observed and recorded in the remaining 6 hens in each group. The differently expressed proteins related to regulating the depolymerization of microtubules were screen by two-dimensional electrophoresis and mass spectroscopy (MS).

RESULTSThe OPIDN symptoms appeared on the 5th day after exposure in TOCP group, which were gradually serious with time. The results by two-dimensional electrophoresis and MS showed that the Stathmin expression was downregulated 3.4 times and 2.8 times in TOCP group, respectively, as compared with the control and PMSF intervention groups. However, there was no significant difference of the Stathmin expression between control group and PMSF intervention group.

CONCLUSIONThe Stathmin expression in the spinal cord tissues of hens exposed to TOCP significantly downregulated. Moreover, the downregulated Stathmin expression may be related to excess polymerization of microtubules and the mechanism of OPIDN.

Animals ; Chickens ; Environmental Exposure ; Female ; Spinal Cord ; metabolism ; Stathmin ; metabolism ; Tritolyl Phosphates ; toxicity

The purpose of this study was to compare the differences of the protein expression profiles between human myeloid leukemia K562 cells and adriamycin-resistant K562/A02 cells, as well as to select novel resistance-related proteins in myeloid leukemia by means of proteomics. The total cellular proteins were separated from K562 and adriamycin-resistant K562/A02 cells by using technique of two dimensional difference in gel electrophoresis (2D-DIGE). Differentially expressed proteins were analyzed by matrix-assisted laser desorption ionization/time of flight-mass spectrometry (MALDI-TOF/MS), and by protein database searching. Moreover, the differentially expressed proteins were verified at protein and mRNA levels by Western blot assay and quantitative real time PCR. The results showed that 8 proteins differentially expressed in adriamycin-resistant K562/A02 cells, among them 2 proteins were identified to be down-regulated and 6 to be up-regulated. These identified proteins involved in the cell energy metabolism, cell proliferation, cell apoptosis, signal transduction, gene transcription and translation respectively. The results assayed by Western blot were similar to those detected by 2D-PAGE. Two up-regulated proteins Stathmin and CrkL were selected for verification in K562 and K562/A02 cells. As a result, the results detected by Western blot were identical with results from 2D-DIGE; real time quantitative PCR assay showed that the changes of CrkL at mRNA level were identical with changes at protein level, but no complete identity of Stathmin changes at mRNA level and protein level was observed. It is concluded that the difference of protein expression profile exists in K562 and K562/A02 cells. Stathmin and CrkL proteins may be involved in the drug resistance and suggest a novel clue for the resistant mechanisms in myeloid leukemia, which is worth further to explore.
Adaptor Proteins, Signal Transducing ; metabolism ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; K562 Cells ; Leukemia ; metabolism ; Nuclear Proteins ; metabolism ; Stathmin ; 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

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

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

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

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.
Animals ; Cryptorchidism ; metabolism ; Male ; Membrane Proteins ; analysis ; Mice ; Phosphatidylethanolamine Binding Protein ; analysis ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stathmin ; analysis ; Testis ; chemistry

This study was aimed to investigate the expression of stathmin1 mRNA and stathmin1 protein in de novo patients with acute leukemia (AL), relapsed patients with AL and complete remission patients with AL, and its clinical significance. The expression of stathmin1 mRNA and stathmin1 protein in peripheral blood samples from 76 cases of AL and 25 healthy persons were examined by fluorescent quantitative PCR (FQ-PCR) and Western blot, respectively. The results showed that the stathmin1 protein expression could not be detected in healthy persons, only the low level of its mRNA could be observed in them. The stathmin1 mRNA expression level in de novo AL patients was higher than that in healthy persons (P < 0.05), the stathmin1 mRNA expression level in relapsed patients with AL was higher than that in de novo patients (P < 0.05), and there was no significant difference of stathmin1 mRNA expression between patients with AML and patients with ALL. The positive rate of stathmin1 protein expression in de novo patients with AL was 89%, while it obviously decreased or did not express in complete remission patients with AL. The stathmin1 protein expression in relapsed patients with AL did not display significant difference as compared with that in de novo patients (P > 0.05). There was no significant difference in stathmin1 protein expression between patients with AML and patients with ALL (P > 0.05). It is concluded that stathmin1 protein and mRNA are overexpressed in de novo patients and relapsed patients, and lowly expressed in complete remission patients. Therefore, the stathmin1 may be a new biological marker for evaluation of minimal residual disease.
Acute Disease ; Adolescent ; Adult ; Case-Control Studies ; Humans ; Leukemia ; blood ; pathology ; Middle Aged ; Neoplasm, Residual ; blood ; diagnosis ; Stathmin ; blood ; Young Adult

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