The Expression And Effects Of Slingshot-1L Phosphatase In Osteosarcoma Multidrug Resistance Cell

1. ObjectiveOsteosarcoma is the most common malignant bone tumor in clinical, accountingfor primary malignant bone tumor20%~30%, multiple children and adolescents. Andclinical features which is the rapid progress of the high degree of malignancy andpoor prognosis, which affect the quality of life in the patients seriously. Recently themain treatment is to take the surgery combined with radiotherapy and chemotherapy.The effect of clinical treatment is greatly improved of patients with osteosarcoma, theobstacle of treament is not sensitive for chemicotheraphy or have a emergence of drugresistance. In order to understanding molecular and mechanisms of osteosarcoma, theresearch object is the multi-drug resistant cell line of human osteosarcomaMG63/VCR in our previous work. We focus on gene and protein level, in order tofind new targets for the treatment of osteosarcoma, we also provide the oreticalfoundation for further treatment of osteosarcoma multidrug resistance.Slingshot(SSH) was a new gene identified in the genetic research of drosophilaby Niwa. It is highly expressed in the thyroid gland, heart, spleen, kidney, smallintestine and so on. The function of SSH including directional migration, mitosis,neurite formation and extension, the growth cone mobility/morphology. The previousstudies focus on the activity and mechanism in proliferation and migration of cells. Itshowed that several proteins have been mediated by SSH, including cofilin, threonineprotein kinase(PAK4), calcineurin, phosphoinositide3-kinase (PI3K)and LIM kinase(LIMK). But the correlation between the expression of SSH1L and multidrugresistance in human osteosarcoma multidrug resistance cells has not been reported.We investigated that the expression of SSH1L and multidrug resistance in humanosteosarcoma multidrug resistance cells, suppression of SSH1L expression by RNAinterference，we provided the role of SSH1L in the expression of drug resistance andrelated genes, cell migration and resistance, to find new targets for the treatment of osteosarcoma.2. MethodsIn this experiment，we in vitro culture human osteosarcoma vincristine resistantcell MG63/VCR for the object. using small doses of sustained induction methods tomaintain their resistance. We detect the expression changes of MDR1and SSH1Lbetween MG63cell and MG63/VCR cell by RT-PCR techonology, we observe thelevel of cofilin phosphorylation that is mediated by SSH1L;And the application ofgene transfection technology, we detect the transfection efficiency after SSH1Lsilenced and increased, using RT-PCR、the scratch test、Transwell migration assay、CCK8to detect the expression of drug resistance and related genes, cell migration andresistance in human osteosarcoma vincristine resistant MG63/VCR cell.3. ResultsWe detect the expression changes of MDR1and SSH1L between MG63cell andMG63/VCR cell by RT-PCR techonology, we found that the expression of MDR1andSSH1L in MG63/VCR were more increased than MG63, there was the same trend ofincrease. The cofilin phosphorylation in MG63/VCR was lower than MG63, it wasshowed that SSH1L activity was increase.Osteosarcoma multidrug resistant MG63/VCR cells transfected with highexpression plasmid pEYFP-SSH1L, siRNA interference plasmid pSUPER-SSH1L tocompared without transfected. We found that the transfection efficiency up to70%~80%when the quality volume ratio is1:2.5between plasmid and lipofectamine.we detected the expression of drug resistance MDR1and related genes MRP、BCL-2were significantly reduced；The scratch test results showed that cell migrationdistance was no change after expression increased and silence of SSH1L,themigration distance significantly increase after200ng/ml leptin-induced MG63/VCRcell, When the increased and silence of SSH1L, the migration of cells24hours nodifference to compared with control of leptin-induced MG63/VCR cell.We detected SSH1L expression increased and decreased MG63/VCR cellsensitivity to vincristine by CCK-8, The results showed that, reducing the expressionof SSH1L, multidrug resistant MG63/VCR cell sensitivity has increased, but increased SSH1L expression reduces the sensitivity of cells to vincristine.4. Conclusion(1) We confirmed that SSH1L expression increased in osteosarcoma multidrug resis-tant cell line MG63/VCR, and plays a positive regulatory role for the expression ofdrug resistance MDR1and related genes MRP、BCL-2.(2)SSH1L has no associated with migration of MG63/VCR, but leptin promote themigration of it.(3)Between SSH1L expression and drug resistance have significant correlation,SSH1L may be regulated multidrug resistance in osteosarcoma.