Effect Of PTEN Gene On Adhesion, Invasion And Migration Of Osteosarcoma Cells

Objective Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene located at chromosome 10q23.31, which the full-length gene fragment is 200kb. Many studies found that the deletion or mutation of PTEN gene, encoding the protein with double special activities of lipid and protein phosphatase, was observed in a variety of human malignant tumor tissues and cell lines. On other hand, PTEN was also verified as one tumor suppressor primarily.PTEN gene encodes the particular proteins for the regulation of cell biological processes, including cell growth, proliferation and migration, which causes to inhibition of tumor cell cycle progression and promotes cell apoptosis. Moreover, abnormal expression of PTEN gene seem to be bound up relative with occurrence, development and prognosis of a wide range of human cancers, including breast, prostate, leukaemia and gastroenteric tumor,as well as gliomas. Accordingly, downregulation and dysfunction of PTEN gene may play an important role in progress of many malignant tumors. Though the downregulation of PTEN expression is also found in osteosarcoma, it is ambiguous whether change of PTEN gene results in occurrence and progress of osteosarcoma or not. Meanwhile, its molecular mechanism remains unclear either. In this study, the immunohistochemical method was used to evaluate the expressions of PTEN in osteosarcoma and osteochondroma tissues,and compared the difference of the expressions.Then,wild-type PTEN gene from lympholeukocyte was amplified to construct the eukaryotic vector p-EGFP-N1-PTEN, followed by transfecting into osteosarcoma U2-OS cell. The effect of PTEN gene on the capability of adhesion, invasion and metastasis of U2-OS cells was further observed. The levels of FAK, MMP-2 and MMP-9 and the phosphorylation of FAK were detected as well. The relationship between malignant phenotypes and PTEN gene expression was analyzed to provide the experiment basis for seeking target of osteosarcoma therapy.Methods 1. The S-P immunohistochemical method was used to evaluate the expressions of PTEN in 25 cases of osteosarcoma and 36 cases of osteochondroma tissues.2. After human peripheral blood lymphocytes were isolated and purified, total RNA of human lymphocytes were extracted under the condition without RNA enzyme contamination.3. The full-length of PTEN was amplified by reverse transcription polymerase chain reaction (RT-PCR). cDNA fragment of PTEN isolated and extracted from gel electrophoresis and the blank plasmid were digested by EcoKI and BamH I digestion enzymes simultaneously. After they were purified, the digesting products via ligation were transformed into DH-5a, one competent Escherichia coli. The positive clones were selected and amplified, followed by being verified through DNA sequencing. The homology analysis of results of sequencing was performed by BLAST and Genebank.4. Recombinant vector GFP-PTEN after purification was transfected into U2-OS cells with LipofectamineTM-2000. Transfection efficiency was detected by observing the expression of GFP green fluorescent protein under inverted fluorescence microscope.5. U2-OS cells with transfecting GFP-PTEN or with transfecting GFP were used to process subsequent experiments. Meanwhile, parent U2-OS cells were used as control. Cell scratch test, cell adhesion assay and Transwell detection were utilized to evaluate the difference of cells’ capability in various time phases after those cells were cultured continuously.6. The cells, after transfection at 48h, were used to detect the expression of PTEN mRNA and protein through RT-PCR and Western blot.7. The expression of FAK, MMP-2 and MMP-9 were measured by RT-PCR and Western blot, subsequently the differences analyzed according to statistics.Results 1. The positive rate of PTEN were44% in osteosarcoma,83.3% in osteochondroma, The positive rates of PTEN were significantly lower in osteosarcoma than those in osteochondroma tissues, P< 0.001.2.Total RNA was obtained successfully from human peripheral blood lymphocytes.3. The fullth frame of PTEN cDNA, about 1210 bps, was amplified with RT-PCR;and then eukaryotic expression vector GFP-PTEN was successfully constructed. One uniform size of the inserted fragment was separated by double enzyme digestion and PCR. It was certificated that the recombinant plasmid was successfully constructed according to sequencing result and Blast analysis.4. The eukaryotic expression vector GFP-PTEN was transfected into U2-OS cell and the expression of intracellular green fluorescent protein could be observed after 24h.5. U2-OS cells transfected after 48h, the results from adhesion experiment of the cells at different phases in 10min,30min and 60min showed that the adhesion rate GFP-PTEN/U2-OS cells was significantly decreased compared with those of other groups, namely U2-OS cells and GFP/U2-OS cells (P<0.05). But there was not one significant difference between the adhesion rates of U2-OS cells and GFP/U2-OS cells (P> 0.05). The results of cell scratch healing experiment displayed that all cells migrated in scratch area at each time point after scratch. U2-OS and GFP/U2-OS cells could heal the scratch area at 12h, however, GFP-PTEN/U2-OS cells failed to full the scratch. The data from invaion experiment showed that the numbers of cells permeating through transwell chamber micropore were 141.33±17.09,152.67±24.66 and 84.33±7.09 respectively. It was indicated that the invasion capacity of GFP-PTEN/U2-OS cells was obviously reduced compared with GFP/U2-OS and U2-OS cells (P<0.01).6. All cells were collected and split after transfection at 48h. Subsequently, intracellular total RNA and protein were extracted to observe the expression of recombinant PTEN gene by RT-PCR and Western blot. It was clear that the high expression of PTEN mRNA and protein was found in GFP-PTEN/U2-OS cells. There was one bright band from electrophoresis experiment consistent with the size of PTEN gene. Meanwhile, molecular weight of PTEN protein was 83KD through Western blot assay.7. The expression level of mRNA and protein of FAK, MMP-2 and MMP-9 gene was decreased significantly in the high level expression of the cells with PTEN gene, notably, the expression level of MMP-9 gene was more lower (P<0.01).Conclusion 1. Downregulation of PTEN expressions was found in osteosarcoma.2.The integrity fragment of PTEN gene was obtained from human peripheral blood lymphocytes.3. The eukaryotic recombinant vector with high expression of PTEN gene has been constructed and was certificated through double enzyme double enzyme digestion, PCR, sequencing assay and Blast analysis.4. Eukaryotic expression vector GFP-PTEN was successfully transfected into U2-OS cells and could be stably expressed in U2-OS cells.5. The adhesion capability of U2-OS cells with PTEN transfection was down-regulated; the potentialities of scratch healing and invasion of these cells were attenuated as well. It is implied that PTEN gene could produce the inhibitory effect on the potentialities of adhesion, invasion and migration of osteosarcoma U2-OS cells.6. GFP-PTEN/U2-OS cells could stably express mRNA and protein of PTEN gene.7. Up-regulation of PTEN gene could enormously decrease the levels of expression and phosphorylation of intracellular FAK, MMP-2 and MMP-9. Taken together, present study primarily reveals that absence of PTEN gene might be associated with some biological behaviors of osteosarcoma cells, containing adhesion, migration and invasion.