The role of EXT1 gene mutation and its high expression of calcitonin gene-related peptide in the development of multiple exostosis.

OBJECTIVE: Screening and identifying the gene mutation of EXT1, EXT2 and EXT3 associated with multiple exostosis (ME) and the expression in tumor tissues. METHODS: Nine patients with multiple exostosis were collected and genomic DNA was extracted. Polymerase chain reaction (PCR) amplification and direct sequencing techniques were used to screen all exons, 5' and 3' ends of the EXT1, EXT2 and EXT3 related causative genes. EXT1, EXT2 and EXT3 gene were screened and quantified by RNA-SEQ and RT-qPCR. The concentration of calcitonin gene-related peptide (CGRP) in peripheral blood of tumor patients and normal controls was detected by ELISA. RESULTS: Between the two patients with ME, the EXT1 gene was found in one patient to have c.79 T>A mutation, which caused the change of p.M27T, the non polar methionine was replaced by the high frequency mutation of polar threonine, and the rest of patients was found the splicing mutation c.1284 + 8 delAT of the heterozygosity of the EXT1 gene. The serum CGRP concentration of ME patients (623 + 49 pg/ml) was significantly higher than that of normal controls (196 + 68 pg/ml), and EXT1 mutation patients were also higher than non mutation patients.

The clinical significance of the Ezrin gene and circulating tumor cells in osteosarcoma.

PURPOSE: The aim of this study was to investigate the clinical significance of circulating tumor cells (CTCs) in the peripheral blood of an osteosarcoma and the Ezrin gene expressed in CTCs. PATIENTS AND METHODS: CTC enrichment was done with CanPatrol™ CTC enrichment technique in 41 patients with osteosarcoma. The characterization of CTCs was performed using a multiple messenger RNA in situ analysis (MRIA). The expression of the Ezrin gene in CTCs was detected by RNA probe technology. The correlations of CTC counts, cell type and the expression level of the Ezrin gene with clinical stage and metastasis of osteosarcoma were analyzed using SPSS 16.0 software. RESULTS: The CTC counts correlated significantly with Enneking stage (P<0.001). The ratio of mesenchymal CTCs correlated with the distant metastases (P<0.001). Ezrin gene expression in CTCs correlated significantly with distant metastases (χ2=152.51, P=0.000). CONCLUSION: The ratio of mesenchymal CTCs in the peripheral blood of osteosarcoma correlates with distant metastases. High expression of Ezrin gene in CTCs correlates with distant metastases.

Electrochemical immunosensor for detection of topoisomerase based on graphene-gold nanocomposites.

A facile electrochemical immunosensor based on graphene-three dimensional nanostructure gold nanocomposites (G-3D Au) using simple and rapid one-step electrochemical co-reduction technique was developed for sensitive detection of topoisomerase. The resultant G-3D Au nanocomposites were characterized by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy, and then were used as a substrate for construction of the "sandwich-type" immunosensor. Amperometric current-time curve was employed to monitor the immunoreaction on the protein modified electrode. The proposed method could respond to topoisomerase with a linear calibration range from 0.5 ng mL(-1) to 50 ng mL(-1) with a detection limit of 10 pg mL(-1). This new biosensor exhibited a fast amperometric response, high sensitivity and selectivity, and was successfully used in determining the topoisomerase which was added in human serum with a relative standard deviation (n=5)<5%. The immunosensor served as a significant step toward the practical application of the immunosensor in clinical diagnosis and prognosis monitor.

FIM-A, a phosphorus-containing sirolimus, inhibits the angiogenesis and proliferation of osteosarcomas.

The mTOR pathway is a central control of cell growth, proliferation, metabolism, and survival, and is deregulated in most cancers. Cancer cells are addicted to increased activity of mTOR kinase-mediated signaling pathways, leading to numerous inhibitors of mTOR signaling in preclinic and clinical trials for cancer therapy. Phosphorus-containing sirolimus (FIM-A), which targets mTOR signaling, inhibits cancer cell growth in vitro. Here we report that FIM-A reduces the angiogenesis and proliferation of osteosarcoma both in vitro and in vivo. In cultured osteosarcoma cell lines, FIM-A inhibited cell proliferation and arrested cells in the G1 phase of the cell cycle, accompanied with reduction of VEGF and HIF-1alpha. With in vivo mouse osteosarcoma xenografts, FIM-A treatment resulted in the inhibition of mTORC1 signaling as demonstrated by the decreased phosphorylation of p70S6K1 and 4E-BP1. Consistent with this finding, FIM-A significantly decreased the average tumor volume, nuclei staining of PCNA, and the number of intratumoral microvessels. Our data demonstrated that targeting mTORC1 by FIM-A inhibited the growth of osteosarcoma in vitro and in vivo, providing the basis for further development of FIM-A as a therapy for osteosarcoma patients.

Pilose antler polypeptides promote chondrocyte proliferation via the tyrosine kinase signaling pathway.

BACKGROUND: Pilose antler polypeptides (PAP) have been reported to promote chondrocyte proliferation. However, the underlying mechanism remains unclear. The present study was to investigate the effects of PAP on the proliferation of chondrocytes and its underlying mechanism. METHODS: Chondrocytes isolated from the knee of Zealand white rabbits were cultured. The second generation chondrocytes were collected and identified using safranin-O staining. The chondrocytes were divided into the following 4 groups including serum-free, PAP, genistein (an inhibitor of tyrosine kinases), and PAP plus genistein group. Cell viability was analyzed using the MTT assay. The cell cycle distribution of the chondrocytes was analyzed by flow cytometry. The expression levels of cyclin A was detected using immunocytochemical staining. RESULTS: No significant difference was observed between serum-free and genistein group. Treatment of the cultures with PAP produced a significant dose-dependent increase in cell viability, the percentage proportion of chondrocytes in the S phase and Cyclin A expression as well. However, the promoting effect of PAP on chondrocyte proliferation were dose-dependently inhibited by genistein, whereas genistein alone had no effect on proliferation of isolated chondrocytes. CONCLUSIONS: The data demonstrate that PAP promotes chondrocyte proliferation with the increased cell number, percentage proportion of chondrocytes in S phase and expression of protein cyclin A via the TK signaling pathway.

KISS1 expression in osteosarcoma: high in chinese clinical cases, but lower in cell lines.

PURPOSE: Osteosarcoma is the most common primary bone malignancy with a notorious feature of high metastasis. KISS1 has been identified as a putative human metastasis suppressor gene in numerous types of cancer. This study was aimed to evaluate the relationship between expression of KISS1 and invasion ability in osteosarcoma cell lines, and the relationships between KISS1 expression levels and prognosis of clinical cases. METHODS: Expression levels of KISS1 in 3 types of osteosarcoma cell lines (MG-63, Saos-2 and U-2 OS) and a normal osteoblast cell line (hF-OB 1.19) were examined using semi-quantitative RT-PCR and immunochemistry staining. Transwell assays were used to detect the cell invasion ability. The osteosarcoma cell lines and specimen sections of osteosarcoma together with control were immuno-stained with KISS1 antibody. The relationship between the clinical data and the expression of KISS1 was evaluated. RESULTS: KISS1 mRNA expression was moderate in U-2 OS, weak in Saos-2 and lost in MG-63. Transwell assays displayed a gradually increased aggressive phenomenon in osteosarcoma cell lines U-2 OS, Saos-2 and MG-63. However, a contrary conclusion was obtained with clinical specimen, a higher positive rate of KISS1 expression being displayed in osteosarcoma patients, especially in metastastic cases, compared to the benign osteochondroma patients. Furthermore, significant earlier distant metastasis was observed in KISS1 positive than negative cases. CONCLUSION: KISS1 expression levels were found to be decreased with the increasing aggressive ability in osteosarcoma cell lines. However, expression of KISS1 positively correlated with metastasis in osteosarcoma patients.

[Experimental study on the role of VEGF autocrine loop in K562 leukemia cells].

OBJECTIVE: lo investigate the effects of anti-VEGF antibody and anti-VEGF hairpin ribozyme gene on the proliferation, apoptosis and related gene expression of the leukemia K562 cells and the possible molecular mechanisms. METHODS: K562 cells were cultured in different concentrations of anti-VEGF antibody. The recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the vector-alone were introduced into K562 cells by lipofectamine mediation. Cell proliferative capacity was determined by MTT, colony formation assay and cells cycles analysis. Cell apoptosis was assayed by DNA ladder and Annexin V -FITC/PI flow cytometry. RESULTS: The anti-VEGF antibody was able to inhibit growth and induce apoptosis of K562 cells in a dose-dependent manner. Exposure to anti-VEGF antibody at 0. 165 microg/ml for 72 hours, the cells exhibited typical DNA ladders. The apoptosis rate peaked at antibody concentration of 0. 825 microg/ml. RT-PCR showed a decrease of MRP and TOPO II expression but a relative constant expression of GST. The introduction of exogenous anti-VEGF ribozyme gene resulted in a decrease of the proliferative capacity and colony forming efficiency from (30.5 +/- 3.3) % in control group to (16.3 +/- 2.8) % in K562/RZ group, higher G1 and lower S ratio in cell cycle distribution in comparison with the control groups. Typical DNA fragmentation and higher Annexin V + ratio occurred in K562/RZ cells after treated with 0.5 micromot/L of As2O3 for 3 days, the apoptosis rate increased from 13.4% in control group to 31. 5% in As2O3 treated group. CONCLUSION: Anti-VEGF antibody can inhibit growth, induce apoptosis and down-regulate the expression of MRP, TOPO II genes of K562 cells in vitro. Transfection with anti-VEGF ribozyme gene can inhibit the proliferation of the cells by delaying the progression G1 into S phase in cell cycles and induce cell apoptosis by down-regulating VEGF gene expression.

Vector-based RNAi approach to isoform-specific downregulation of vascular endothelial growth factor (VEGF)165 expression in human leukemia cells.

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. K562 human leukemia cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. In the present study, three 19 bp reverse repeated motifs targeting exons 5 and 7 boundary of VEGF165 gene sequence with 9 bp spacer were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-VR1, pGenesil-VR2, pGenesil-VR3 and pGenesil-con (plasmid containing random DNA fragment) were transfected into K562 cells, respectively, through lipofectamine reagent. A vector-based small interfering RNA(SiRNA) inhibited VEGF165 mRNA expression by 72% and protein production by 67% in K562 cells. Human microvascular endothelial cell migration induced by conditioned medium from VEGFsi-transfected K562 cells was significantly less than that induced by conditioned medium from K562 cells and control vector-transfected K562 cells. Furthermore, the VEGF shRNA dramatically suppressed tumor angiogenesis and tumor growth in a K562 s.c. xenograft model. Vessel density as assessed by vWF immunohistochemical analysis was also decreased. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF-specific treatment in cancer.

[The down regulation of VEGF gene expression may link to change the expression profile of genes in leukemia cell line K562].

OBJECTIVE: To explore the potential effects of anti-VEGF hairpin ribozyme gene to gene expression profiles in leukemia cell line K562. METHODS: The lipofectamine mediation was used to transfect the recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the non-recombinant vector as control into K562 cells. And the positive clones were screened by G418. Ribozyme gene in K562 cells was confirmed by PCR. Fluorescent real time reverse transcription-PCR(RT-PCR) and Western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. cDNA microarray was used to explore the alteration of gene expression profiles when decreasing VEGF gene expression in leukemia cells. Expression of PCNA and GSN genes were verified by semi-quantitative RT-PCR. RESULTS: The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418. Stable expression of the ribozyme gene in K562 cells was confirmed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC (K562 cell transfected with empty vector) cells. The gene expression profiles were changed by transfection of anti-VEGF hairpin ribozyme gene into K562 cells. Among 4096 gene clones on the microarray, 191 (4.86%) genes were detected to have the marked changes with 104 down-regulated and 87 up-regulated, that were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, and oncogenes etc. An increased expression of GSN gene and a decreased expression of PCNA gene in K562/RZ cells have been detected by RT-PCR. CONCLUSION: Down-regulation of VEGF gene by introducing anti-VEGF hairpin ribozyme gene can alter the gene expression profiles in K562 cells, leading to change of cell growth, differentiation and apoptosis in K562/RZ cells.

[Inhibition of vascular endothelial growth factor gene expression and proliferation of leukemia cells by RNA interference].

OBJECTIVE: To explore the feasibility of selective inhibiting VEGF expression using VEGF short hairpin RNA (shRNA) interference, and observe the effects of VEGF gene silencing on NB4 cells growth. METHODS: Three 19 bp reverse repeated motifs targeting exons 3, 4, 5 respectively of VEGF gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-VR1, pGenesil-VR2, pGenesil-VR3 and pGenesil-con (plasmid containing random DNA fragment) were transfected into NB4 cells respectively through lipofectamine reagent. The alteration of VEGF expression was examined by fluorescent real time RT-PCR and Western blot. The proliferation capacity of leukemia cells was measured by trypan blue exclusion, MTT assay, colony formation assay and cell cycles analysis. RESULTS: Recombinant plasmids containing three shRNAs and random fragment were successfully constructed and transfected into NB4 cells respectively by liposome-mediated gene transfer method. shRNA in pGenesil-VR3 cells knocked down the expression of VEGF mRNA and protein dramatically in a sequence-specific manner when compared with that of pGenesil-VR1, Genesil-VR2 and pGenesil-con. The NB4 cells transfected with pGenesil-VR3 (NB4-VR3) had a more significant decrease in proliferation ability than NB4 and that transfected with pGenesil-con (NB4-con). The colony forming efficiencies of NB4-VR3, NB4-con and NB4 cell were (13.3 +/- 3.8)%, (21.3 +/- 6.4)% and (24.5 +/- 5.2)%, respectively (P < 0.05). Higher G(1) and lower S proportion were found in cell cycle distribution in comparison with the control groups by FCM. CONCLUSIONS: The shRNA can efficiently suppress VEGF expression in NB4 cells. Selective VEGF gene silence can inhibit the malignant proliferation of leukemia cells.

[Repair of articular cartilage defect with homogeneous chondrocytes combined with pluronic].

OBJECTIVE: To investigate the possibility of repairing the cartilage cartilage defect with homogeneous chondrocytes combined with Pluronic. METHODS: Homogeneous cartilage chondrocytes of adult New Zealand rabbits were harvested and cultured in vitro, which were marked by 3H-TdR and mixed with Pluronic. The medial or lateral condyle defects were made (phi 4 mm, extending down to the calcified zone) in 20 rabbits. In the experimental group, the right defects were repaired by homogeneous chondrocytes combined with Pluronic; in the control group, the left defects were repaired by Pluronic only or were left un-repaired. The animals were sacrificed in the 4th, 8th and 16th weeks after operation respectively. The repair results were observed and the cell source of repair tissue was distinguished. RESULTS: In the experimental group, the cartilage defects were repaired by the cartilage-like tissue after 8 weeks of operation; the defects were completely filled with mature cartilage tissue, which integrated smoothly with articular cartilage 16 weeks later. In the control group, only a small amount fibrous tissues were seen on the surface of defects. Autoradiographic assessment showed that the repair cells came from the implants, but not from self-chondrocytes. CONCLUSION: It is a good way to repair articular cartilage defects with homograft of tissue engineering cartilage. It is a convenient method to mark with 3H-TdR to discriminate the resource of the repair cells.