Exploration Of The Molecular Mechanism Of Skp2 In Promoting The Malignant Progression And Drug Resistance In Osteosarcoma

Objective:S phase kinase associated protein 2(Skp2)plays an important role in ubiquitination-mediated modification and degradation of its substrates,and regulates tumor progression and progression via degrading cell cycle associated proteins.In recent years,Skp2 has been considered to play a key role in regulating in many kinds of tumors,while its role in osteosarcoma is still not clear.In this study,we will discuss the effect of Skp2 on the malignant biological behavior of osteosarcoma and its possible molecular mechanism.The OS cells will be transfected with Skp2 cDNA or Skp2 siRNA and cells will be detected for cell biological functions including cell growth,apoptosis,migration,and invasion.The role of Skp2 in MTX-resistant OS cells will be investigated.The molecular mechanisms of Skp2-mediated tumorigenesis and drug resistance will be dissected.Method:Part 1: Skp2 expression plasmids(Skp2 cDNA)were transfected into MG63 and U2 OS osteosarcoma cell lines,respectively.Control groups were identified as: control group(without any treatment),transfection control group(cells were transfected with empty vector).After transfections,a series of detections were performed as follows: 1)qRT-PCR and Western blotting detection of the mRNA and protein expression levels of Skp2;2)MTT analyses of cell proliferation;3)Annexin V/PI double staining and flow cytometry analyses of cell apoptosis;4)PI staining and flow cytometry analyses of cell cycle distributions;5)Transwell measurement of cell migration and invasion abilities;6)Western blotting detection of intracellular P21,P57,E-caderin andmm P protein level changes.Part 2: The expression of Skp2 in osteosarcoma cell lines,MG63 and SW1353 cells,was knocked out by RNA interference(RNAi).1)Interference sequences targeting human Skp2 were synthesis and denominated as siRNA1,siRNA2 and siRNA3,respectively.The siRNAs were transfer into cells,respectively.And control cells were transfected with non specific interference sequences of NC siRNA.qRT-PCR and Western blotting detection of Skp2 mRNA and protein expression were performed.According to the RNAi screening results,siRNA with the highest knockout efficiency of Skp2 was chosen for the subsequent experiment.Experimental groups including control group(without any treatment),NC siRNA group(transfected with non specific siRNA),siRNA group(transfected with Skp2 siRNA sequence),respectively;2)MTT analyses of cell proliferation between the above groups;3)Annexin V/PI double staining and flow cytometry analyses of cell apoptosis;4)PI staining and flow cytometry analyses of cell cycle distributions;5)Wound healing and transwell assays of cell migration and invasion abilities;6)Western blotting detection the protein level changes of Skp2 downstream targets,P27?p57?Foxo1?E-cadherin and pAkt.Part 3: Establishment of methotrexate resistant osteosarcoma cell lines using sustained induction with MTX in U2 OS and MG63 cell lines,respectively(MTX resistance cell lines,MR).1)MTT measurement of drug resistance in MR OS cells;2)Compare morphology changes of MR OS cells with the parental cells under a microscope;3)Detection of the epithelium mesenchymal cell transformation(epithelial-mesenchymal transition,EMT)markers changes in MR OS cells by qRT-PCR and Western blotting;4)Detection of Skp2 expression in MR OS cells by qRT-PCR and Western blotting;5)Test the invasion and migration of MR OS cells via wound healing and Transwell assay;6)Test the adhesive and shedding capacity of MR OS cells using cell attachment and detachment assay;7)Stable knockdown of Skp2 in MR OS cells by infection with Skp2 shRNA lentivirus particles.Verify its knockdown efficiency by Western blotting;8)Compare morphology changes of MR OS cells before and after Skp2 knockdown under a microscope;9)Measure the invasion and migration of MR OS cells after Skp2 stably knockdown through wound healing and Transwell assay;10)Test the adhesive and shedding capacity of MR OS cells after Skp2 stably knockdown using cell attachment and detachment assay;11)MTT measurement of drug sensitivity to methotrexate in MR OS cells after Skp2 stably knockdown;12)Detect the EMT markers changes in MR OS cells after Skp2 stably knockdown by Western blotting.Result:Part 1: 1)Our qRT-PCR and Western blotting results showed that the expression of Skp2 significantly up-regulated in both MG63 and SW1353 after Skp2 cDNA transfection,compared with the control group and empty vector transfected group(P<0.05);2)MTT results showed that the over-expression of Skp2 significantly enhanced cell growth(P<0.05);3)Annexin V/PI double staining and flow cytometry analyses showed that the up-regulation of Skp2 markedly weakened cell apoptosis in both OS cell lines;4)PI single staining experiments showed that after over-expression of Skp2,the number of S phase cells increased significantly,and cell cycle was arrested in S phase;5)Transwell assay showed that the ability of migration and invasion was enhanced in Skp2 overexpressing OS cells;6)Mechanically,our Western blotting data suggested that Skp2 decreased the expression of E-cadherin,Foxo1,p21,and p57,but increasedmmP-9 in OS cells.In conclusion,our study demonstrated that Skp2 exhibited an oncogenic function in OS cells,suggesting that inhibition of Skp2 may be a novel approach for the treatment of OS.Part 2: 1)In the present study,qRT-PCR and Western blotting experiments showed that Skp2 siRNA2 transfection resulted in Skp2 depletion efficiency in both MG63 and SW1353 cells.So we chose Skp2 siRNA2 transfection for the subsequent experiments;2)MTT results showed that depletion of Skp2 significantly inhibited cell proliferation in both MG63 and SW1353 cells,compared with control group and NC siRNA group(P<0.05);3)Annexin V/PI double staining and flow cytometry analyses showed that depletion of Skp2 triggered cell apoptosis in two OS cell lines;4)Downregulation of Skp2 induced cell cycle arrest in the G0/G1 phase in OS cells,compared with the other 2groups;5)Our wound healing assay demonstrated that inhibition of Skp2 significantly suppressed the migration ability of OS cells.In addition,transwell assay showed that Skp2 knockdown obviously inhibited OS cells invasion ability;6)Invariably,our western blot results demonstrated that depletion of Skp2 in OS cells inhibited activation of pAkt and increased expression of p27,p57,E-cadherin and Foxo1 in OS cells,suggesting that Skp2 exerted its oncogenic function partly through the regulation of pAkt and its substrates.Our findings revealed that targeting Skp2 could be a promising therapeutic strategy for the treatment of OS.Part 3: 1)MTT results showed that 20?Mand a higher concentration of MTX treatment led to significant growth inhibition in the parental osteosarcoma celsl,while MR OS cells displayed much higher resistant ability to MTX;2)MR cells were observed under a microscope.We found that the resistant cells appeared to become elongated and more spindle-like shapes,similar to fibroblast morphology,therefore possessed mesenchymal phenotype;3)We detected the mRNA and protein expression levels of EMT markers.Compared with the parental cells,our results showed that MR OS cells underwent EMT markers changes.The expression of E-caderin and ZO-1 were significantly decreased,while the other characterized proteins such as Slug,N-caderin,and Vimentin were observably increased in resistant cells.In combined with the morphological changes,the EMT markers changes further proved that MR OS cells went through EMT transformation;4)In order to further explore the role of Skp2 in MR cells,we detected the expression of Skp2 in MR cells and the parental OS cells.Our results showed that Skp2 was overexpressed in MR OS cells,both in mRNA level and protein level;5)Moreover,MR OS cells obtained enhanced invasion and migration abilities;6)The adhesive and shedding capacity of MR OS cells were enhanced compared with the parental cells;7)Western blotting results showed that stable knockdown of Skp2 was established in MR OS cells by infection with Skp2 shRNA lentivirus particles;8)After stable knockdown of Skp2,MR OS cells were observed under the microscope.We observed that MR cells became round-shaped,transformed from mesenchymal cells to epithelioid cells;9)Moreover,we found that the invasion and migration abilities of MR OS cells were significantly inhibited after Skp2 stably knockdown;10)Attachment and detachment assay revealed that the adhesive and shedding capacity of MR OS cells were suppressed after Skp2 stably knockdown;11)The use of targeted shRNA against Skp2 resulted in an enhancement of sensitivity to MTX in MR OS cell proliferation;12)Knockdown of Skp2 abrogated EMT characteristics in MR OS cells,including up-regulation of E-caderin and ZO-1,and down-regulation of Slug,N-caderin and Vimentin.Conclusion:Overexpression of Skp2 promotes the growth,invasion,migration and cell cycle progression of osteosarcoma cells.Skp2 overexpression enhances cell cycle progression at S phase.The expression of E-caderin?Foxo1?p21 and p57 were significantly downregulated after Skp2 overexpression.However,the expression of MMP-9 was up-regulated,which might contribute to the invasion and migration abilities of OS cells.After Skp2 knockdown by siRNA transfection,the growth,invasion,migration and cell cycle progression of osteosarcoma cells were markerdly inhibited.Moreover,Skp2 knockdown triggered apoptosis of OS cells.The expression of p27,p57,Foxo1,and E-cadherin was increased.The expression of pAkt was decreased upon Skp2 inhibition.These finding indicated that targeting Skp2 is a promising approach for the treatment of human OS.More importantly,we found that Skp2 signaling was activated in MTX resistant OS cells and closely involved in regulating the EMT features and drug resistance of osteosarcoma cells.Down regulation of Skp2 by shRNA infection partially reversed the EMT characterizations in MTX resistant cells,improves the sensitivity of resistant cells to MTX.Therefore,Skp2 could be used as a promising theraputic target for the treatment of patients with osteosarcoma.