Effect Of LIMK1on Leptin-Induced Human Osteosarcoma Cell In Proliferation And Migration

1. ObjectiveOsteosarcoma is the most common primary malignant bone tumors。And whichgenerates metaphysis of long bones between adolescents and young adults。Per one millionpopulation fall ill of four to five people。Poor prognosis。The main treatment is to take thesurgery combined with radiotherapy and chemotherapy。The5-year survival rate of thepatients with OS has significantly improved over the past decades to approximately60–70%since the introduction of combinatorial chemotherapy。But a considerable portion ofpatients are not sensitive for chemicotheraphy or have a emergence of drug resistance。Although these years，the understanding of the molecular mechanisms of osteosarcoma hasincreased，There are not more depth and comprehensive understanding of osteosarcomamolecular。Such as tumor formation、tumor development and metastasis、drug resistence。In recent years，multidrug resistance of osteosarcoma has become a hotspot。We focus ongene and protein level，and make gene and protein of the human osteosarcoma vincristineresistant cells as a target。We provide a model and theoretical foundation for furthertreatment of osteosarcoma multidrug resistance。And opens up ideas of reversal of the humanosteosarcoma multidrug resistance。Leptin,16kda the hormone of non-glycosylation, is the product of the obesity geneprotein. And locate in human chromosome. Adipose tissue complete synthesis and secretion,And play the hypothalamus-mediated regulation of energy balance, endocrine, immune andmetabolic function. In recent years some studies have shown that, Serum leptin andpostmenopausal women with osteoporosis are closely related. And leptin increase to a veryhigh risk of recurrence after liver cancer treatment. Some in vitro studies have shown thatbone marrow stromal cells are sensitive to leptin. This increases their proliferation anddifferentiation of the osteoblast lineage,and the number of mineralized nodules, But inhibitthey differentiate into fat cells. In recent years,the study found that leptin plays an importantrole in the development of many malignant tumors. Such as breast cancer,colorectalcancer,lung cancer. But yet to clarify the role and mechanism of leptin and human osteosarcoma and its drug-resistant cell growth at home and abroad.Our latest research finding show, LIMK1-expression was significantly higher indrug-resistant osteosarcoma cells than in human osteosarcoma cells. When using siRNAinterference to inhibit the LIMK1gene, significantly eased the resistance of the cells. Andinhibited the cell migration. From this, Overexpression of LIMK1plays an important role inregulating resistance and invasiveness. It may be caused by drug-resistance tumor cells andtransfer of one the key elements.In this study,LIMK1is the target, And provides a new theoretical foundation for furtherreseaech LIMK1in the leptin-mediated drug-resistant tumor cell proliferation and migrationmechanisms and drug-resistant tumor treatment studies.2. MethodsIn this experiment，we in vitro culture human osteosarcoma vincristine resistant cellMG63/VCR for the target. on the basis of established human osteosarcoma vincristineresistant cell model,suing small doses of sustained induction methods to maintain theirresistance. We detect the expression changes of MDR1and LIMK1before and afterleptin-induced by RT-PCR techonology. We detect the transfection efficiency by RT-PCR、Westernblot after LIMK1silenced.We use the CCK8,flow cytometry,the scratch test to detectthe proliferation,cycle,and migration of MG63/VCR cell after transfected with LIMK1induced by leptin.And then explore the role of the LIMK1on leptin-induced MG63/VCRcell.3. ResultsOur results show that the expression of MDR1gene and LIMK1gene was enhancedafter200ng/ml leptin-induced MG63/VCR cell by RT-PCR techonology detected.Afersilencing the LIMK1gene we test the transfection efficency by RT-PCR techonology andWesternblot techonology and we found the transfection efficiency up to70%.We test the proliferation of MG63/VCR cell that was induced by leptin aftertransfection and we found the proliferation of MG63/VCR significantly slow when theLIMK1gene was silenced. We found that when the silence LIMK1gene the cell cycle at G2phase and S phase cells was significantly fewer than the group plused leptinThe scratch test results showed that leptin-induced MG63/VCR cell migration distanceof24hours maximum.When the silence LIMK1,the migration of cells24hours slower thanleptin-induced MG63/VCR.After the silence LIMK1leptin-induced MG63/VCR cellmigration speed of24hours is still slower than the leptin group is not silent 4. Conclusion⑴We confirmed that leptin promote the proliferation and migration of MG63/VCR.⑵We found that leptin promote the expression of LIMK1.⑶We further confirmed that LIMK1reverse the proliferation and the migration ofMG63/VCR cell.