High intensity focused ultrasound inhibits melanoma cell migration and metastasis through attenuating microRNA-21-mediated PTEN suppression.

High intensity focused ultrasound (HIFU) technology is becoming a potential noninvasive treatment for solid tumor. To explore whether HIFU can be applied to treat melanoma and its metastasis, we investigated the effect of HIFU on murine melanoma model. While there was little influence on cell survival, viability or apoptosis, HIFU exposure suppressed melanoma cell migration in vitro and metastasis in vivo. The expression of microRNA-21(miR-21) was down-regulated and PTEN expression was up-regulated in response to HIFU exposure, which was in concomitant with the reduction of AKT activity. Furthermore, ectopic miR-21 expression suppressed this effect of HIFU. These results demonstrate that HIFU exposure can inhibit AKT-mediated melanoma metastasis via miR-21 inhibition to restore PTEN expression. Therefore, targeting the miR-21/PTEN/AKT pathway might be a novel strategy of HIFU in treatment of melanoma.

[The effect of suppressive oligodeoxynucleotides on interferon-γ and phosphorylation of signal transducers and activators of transcription 4 expression of silica-induced pulmonary inflammation in mice].

OBJECTIVE: To investigate the protective effect of suppressive oligodeoxynucleotides (Sup ODN) on interferon-γ (IFN-γ) and signal transducers and activators of transcription (pSTAT4) expression of Silica-induced pulmonary inflammation in Mice. METHODS: Sixty Balb/c mice were randomly divided into 4 groups, normal control group, silicious group, suppressive oligodeoxynucleotides (Sup ODN) group, control oligodeoxynucleotides (Con ODN) group. Except the normal control group injected normal saline, the rest groups were induced by the intratracheal instillation of 0.1 ml (5 g/L) of sterilized silica suspension. Sup ODN group and Con ODN group were treated by i.p. injection of 0.3 ml (1mg/mL) of suppressive or control ODN 3 h before silica administration. After 7 days, the animals were killed and levels of IFN-γ were detected by ELISA. The pathologic changes in lung tissues of mice were observed with HE staining. Expressions of IFN-γ and pSTAT4 in lung tissue were detected with immunohistochemistry and quantified by Image-Pro Plus 7.0. RESULTS: HE staining showed that the lung tissue of silicious group were damaged seriously than Sup ODN group. Compared with the normal control group (serum: (280.1±41.3) pg/ml, lung tissue: (0.249±0.373), IFN-γ increased in silicious group (serum: (886.3±81.7) pg/ml, lung tissue: (0.270±0.300) (P < 0.05). Compared with the normal control group and Con ODN group [(894.5±91.6) pg/ml], IFN-γ in the serum of Sup ODN group decreased significantly (P < 0.01). Compared with the silicious group , IFN-γ in lung tissue decreased in Sup ODN group (0.241±0.250) (P < 0.05). Compared with the normal control group (0.279±0.353), pSTAT4 in lung tissue increased significantly in silicious group (0.313±0.231) (P < 0.01). Compared with the silicious group, pSTAT4 in lung tissue decreased significantly in Sup ODN group (0.269±0.523) (P < 0.01). CONCLUSION: Sup ODN attained protective effect on Silica treated mice by suppressing expression of IFN-γ and pSTAT4.

[Correlation analysis between the exposure levels and the serum protein fingerprints in population exposure to silica].

OBJECTIVE: To explore the correlation between the exposure levels and serum protein fingerprints in population exposed to silica. METHODS: Liquid chip time-of-flight mass spectrometry technology was used to investigate the serum profiles in control group (30 cases), group exposed to silica (30 cases), silicosis group (I stage, 25 cases) and suspected silicosis group (30 cases), and screen the differential expression proteins. The correlation between the levels of the differential expression proteins and the exposure levels was performed. RESULTS: Five differential expression proteins were found among 4 groups, the expression of 5081 and 5066 proteins was upregulated, and the expression of 3954, 2021 and 1777 proteins was downregulated. There was no the correlation between the exposure levels and the peak with M/Z among those proteins. CONCLUSION: the results of present investigation indicated there was no correlation between the exposure levels and protein/peptide peak.

Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells.

Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation of mesenchymal stem cells, the molecular mechanism involved remains to be fully elucidated. In this study, we showed that BMP9 simultaneously promotes the activation of Smad1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. Knockdown of Smad4 with RNA interference reduced nuclear translocation of Smad1/5/8, and disrupted BMP9-induced osteogenic differentiation. BMP9-induced osteogenic differentiation was blocked by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. SB203580 decreased BMP9-activated Smads singling, and yet PD98059 stimulated Smads singling in C3H10T1/2 cells. The effects of inhibitor were reproduced with adenovirus expressing siRNA targeted p38 and ERK1/2, respectively. Taken together, our findings revealed that Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation. Also, it is noteworthy that p38 and ERK1/2 may play opposing regulatory roles in mediating BMP9-induced osteogenic differentiation of C3H10T1/2 cells.

[Differential analysis of two-dimensional gel electrophoresis profiles in lung tissue of rats exposed to silica early].

OBJECTIVE: To analyze the differences of lung tissue proteins in rats exposed to silica early by using comparative proteomics method and investigate the related mechanism with the occurrence and development of silicosis. METHODS: Adult male Wistar rats were randomly divided into control group and silica-treated group. The animal model was established by intratracheal (IT) instillation with silica suspension. On the 14th day after establishment of animal model, rats were sacrificed and lung tissues were collected. The total proteins were separated by means of two-dimensional gel electrophoresis (2-DE) and the differentially expressed proteins were identified by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In addition, Western blotting was performed to verify the expression of certain candidate protein. RESULTS: Eleven differential expression protein spots were tested by MALDI-TOF-MS, and six proteins were identified. The levels of cathepsin D precursor, peroxiredoxin-1 (Prx-1), heat shock cognate 71 000 protein (HSP7C), heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3) and fatty acid-binding protein (epidermal, E-FABP) were up-regulated in silica-treated group with the optical density (A) values. These values were 116.50+/-12.56, 148.75+/-22.40; 40.00+/-1.63, 66.00+/-13.93; 51.25+/-7.37, 92.75+/-8.69; 83.00+/-6.48, 122.75+/-24.62; 50.75+/-6.50, 93.50+/-23.10 and 100.25+/-19.99, 142.50+/-21.21 respectively. The statistical difference was observed as compared with control group (t=-2.51, -3.71, -7.28, -3.12, -3.56 and -2.90, P<0.05). However, SEC14-like protein 3 with the A values 153.00+/-11.28, 109.75+/-18.32 was down-regulated (t=4.02, P<0.01). Western blotting showed that in the expression of Prx-1 was higher in silica-treated group (0.61+/-0.05) than that in the control (0.35+/-0.05) (t=-7.24, P<0.01) when calculating the semi-quantification of this protein using ratio of optical density. CONCLUSION: 2-DE pattern of lung tissue from rats exposed to silica has been established and six differentially expressed proteins have been identified. Our study is of help for further research of the mechanisms of silicosis.

[The proteomics research on relational expressed serum proteins among the recovered SARS patients complicating avascular necrosis of femoral head].

OBJECTIVE: To seek differentially expressed serum proteins in recovered SARS patients complicating avascular necrosis of femoral head (AVNFH). METHODS: 2-DE and MALDI-TOF MS were used to study the comparative serum proteomics among female SARS AVNFH group, female SARS non-AVNFH group and female healthy group. ELISA method was used to detect serum amyloid P component in individual serum; specificity and sensitivity of serum amyloid P component were analyzed. RESULTS: Average protein points on 2-DE of 3 groups were 632 +/- 28, 671 +/- 55, 688 +/- 42 respectively, and the matching rate of protein points was ranged from 85% to 95%; eighteen differentially expressed proteins were discovered including transthyretin, serpin peptidase inhibitor, alpha-1-antitrypsin precursor, serum amyloid P components, etc. Compared to healthy group and SARS non-AVNFH group, transthyretin, C4B3, fibrinogen gamma, apolipoprotein L, apolipoprotein A-IV precursor, albumin and prealbumin showed lower expression, inversely serpin peptidase inhibitor, alpha-1-antitrypsin precursor and serum amyloid P components showed higher expression in serum in the SARS AVNFH necrosis group. The serum amyloid P component in 3 groups were 0.54 +/- 0.30 ng/ml, 0.83 +/- 0.39 ng/ml, 1.21 +/- 0.29 ng/ml respectively. The areas under the ROC curve on serum amyloid P component was 0.854, the specificity was 77.8% and the sensitivity was 85.2%. CONCLUSION: There were differentially expressed serum proteins in three groups. Serum amyloid P components might be one of the potential biomarkers in serum of recovered SARS patients complicating avascular necrosis of femoral head.

[Expression of human spindle mitosis arrest deficiency gene in spontaneous abortion embryo tissues].

OBJECTIVE: To investigate the expression of human spindle mitosis arrest deficiency gene (hsMAD2) in spontaneous abortion embryos and the relationship between low expression of hsMAD2 and numerical chromosomal aberration. METHODS Spontaneous abortion embryo tissues were collected, including 23 cases of once spontaneous abortion tissue and 10 cases of twice or more spontaneous abortion tissue and induced abortion embryos (35 cases) from the Department of Gynaecology and Obstetrics of the Affiliated Hospitals of Chongqing University of Medical Science during the period of March 2006 to March 2007. FQ-PCR and western blot were used to evaluate the endogenous expression level of hsMAD2 mRNA and hsMAD2 protein; primary culturing of cells from the induced abortion embryos was conducted and 5 embryonic cells were selected by chromosomes karyotype analysis. Recombinant shRNA plasmids targeting hsMAD2 gene were constructed to inhibit the expression of endogenous hsMAD2 genes in embryonic cells which have normal karyotypes; the groups were defined as the first experimental group (transfected with pshRNA-hsMAD2-1) , the second experimental group (transfected with pshRNA-hsMAD2-2), the third experimental group (transfected with pshRNA-hsMAD2-3), the first control group (transfected with nothing), the second control group (transfected with pTZU6 + 1) and the independent group (transfected with pshRNA-N1). Interference efficiency was demonstrated by FQ-PCR and western blot; cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay; cell-cycle was assessed by flow cytometry (FCM); the chromosome numbers were calculated to analyze the variation of chromosomes. RESULTS: (1) The mRNA levels of hsMAD2 in the once spontaneous abortion tissue, twice or more spontaneous abortion tissue and induced abortion tissue were 0.00879 +/- 0.00035, 0.00901 +/- 0.00033 and 0.00941 +/- 0.00026 respectively, and there was no significant difference (P > 0.05) compared with each other; however, the protein levels of hsMAD2 in three groups were 0.2791 +/- 0.0311, 0.0431 +/- 0.0020 and 0.5790 +/- 0.0331 respectively, and there were significant differences (P < 0.05) compared with each other. (2) Recombinant shRNA plasmids could significantly and specifically inhibit hsMAD2 gene expression in embryonic cells. Compared with the first control group (4%) and the second control group (3%), the recombinant shRNA could inhibit embryonic cell proliferation to 54% at 48 h after transfection (P < 0.05); compared with the first control group (8.2%) and the second control group (8.0%), the ratios of G2/M phase cells in the experimental group (17.9%)was significantly increased (P < 0.05); compared with the first control group (4.8%), the ratios of abnormal chromosomes in the experimental group was increased to 30.0% (P < 0.05). CONCLUSIONS: Down-expression of hsMAD2 gene may be one of the mechanisms inducing numerical chromosome aberration, abnormal embryo development and the occurrence of spontaneous abortion.

[Identification of nm23-M2/NDPK B expression during the blastocyst adhesiveness in the mouse endomerium by two-dimentional electrophoresis and MALDI-TOF-MASS spectrometry].

The nm23 gene family was involved in cellular multiphysiopathological processes including differentiation, development, apoptosis and cancer promotion, progression or metastasis. Some data indicate that nm23 plays an important role in regulating reproductive processes. In the present study, we analyzed the proteome of the implantation sites and the peri-implantation sites in NIH. mice on Day 5 of gestation by using two-Dimensional gel electrophoresis (2-D PAGE), while the virgin mice as the control. A protein spot with pI 7.1, Mr 18 kDa showed up-regulated expression in endometrium during the blastocysts adhesiveness. Using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS), this protein was identified as nm23-M2/NDPK B. The nm23-M2 expression in mice endometrium was shown progressive increase on Day 5 of gestation by RT-PCR, which was consistent with the result obtained by immunohistochemistry. These findings suggest that nm23-M2/NDPK B was involved in the process of blastocyst implantation.

Expression of human soluble gp190 in yeast Pichia pastoris.

The cDNA of N-terminal extracellular domain of human leukemia inhibitory factor receptor a-subunit (gp190) ,which encoded soluble gp190 (sgp190) ,was cloned into the methylotrophic yeast Pichia pastoris secreted expression vector pPIC9K. The linearized plasmid sgp190-pPIC9K was then integrated with GS115 genome by spheroplasts transformation. MD medium and PCR method were used for screening positive transformants. The transformants (His+ Mut+) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418 and induced by 0.5% methanol in shaking flask to express sgp190. The sgp190 expressed level could reach up to 26% of total proteins in culture supernatant. SDS-PAGE and Western blotting analysis showed that the molecular weight of sgp190 is about 125 kD and could be specifically recognized by polyclonal antibodies against human sgp190,which indicated good antigenicity of this secreted expressed protein. Biological activity assay showed that purified sgp190 could inhibit the effect of LIF on hCG biosynthesis of cytotrophoblast in vitro.

[Expression of leukemia inhibitory factor gene in decidua from women with habitual abortion].

To evaluate the relationship between habitual abortion of unknown etiology and expression of leukemia inhibitory factor (LIF) gene in the decidua,the expression of LIF gene in the decidua were detected in 32 cases of habitual abortion of unknown etiology (abortion group) and 36 cases of normal pregnancy (control group) by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the level of LIF mRNA expression of the decidua in abortion group was 0.512+/-0.219, significantly lower than those in the control group (1.314+/-0.457, P<0.01). The findings indicated that the low expression of LIF gene in abortion group might be one of the causes of habitual abortion. Analysis on the LIF gene expression of decidua can be used as a index for diagnosis of the habitual abortion.