| Part 1 The expression of DNA-PKcs in osteosarcoma cells and tissues and clinical significanceBackgroudOsteosarcoma(OS)defines neoplasm that shares the histological finding of osteoid production in association with malignant mesenchymal cells.OS is the most common primary solid tumor of bone in children and adolescence among various types of bone tumors,with an overall incidence of 4-5/106.With the introduction of chemotherapy in treatment of OS,the 5-year survival rate after surgery has increased to 50%～700%in patients without metastasis.However,there has been no further improvement during the last three decades in term of survival rate which even remains at 20%-30%for patients with detectable metastasis.In order to improve the treatment and survival rate,it is meaningful and valuable to investigate the mechanisms of-occurrence and progression of OS.DNA-dependent protein kinase catalytic subunit(DNA-PKcs)is a member of the large phosphatidylinositol 3-kinase(PI3K)-related kinase(PIKK)family.DNA-PKcs,along with accessory heterodimeric complexes,Ku70 and Ku80,constitute DNA-dependent protein kinase complex and are involved in DNA damage repair via non-homologous end joining(NHEJ).DNA-PKcs plays a significant role in the stability of genome.Moreover,DNA-PKcs is high expressed or possesses evaluated activity in malignances,and was widely involved in many functional activities in tumor cells.To date there was few studies about the expression and clinical significance of DNA-PKcs in OS.Therefore,the study aims to investigate the expression and clinical significance of DNA-PKcs in osteosarcoma cells and tissue.PurposesTo investigate the expression of DNA-PKcs in osteosarcoma cells and tissues and clinical significance.Methods1.The expressions of DNA-PKcs in mRNA and protein were examined in hFOB1.19 and osteosarcoma cells(MNNG/HOS,MG-63,U2-OS,Saos-2)by quantitative real-time polymerase chain reaction(qRT-PCR)and Western Blot.2.The location and expression of DNA-PKcs protein were examined in hFOB1.19 cells and osteosarcoma cells(MNNG/HOS,MG-63.U2-OS.Saos-2)by immunofluorescence.3.The expression of DNA-PKcs protein was examined in osteochondroma and osteosarcoma tissues by immunohistochemistry,respectively.The relationship between the expression of DNA-PKcs protein and clinicpathologic features(gender,age,tumor site,Enneking surgical staging)in osteosarconma was analyzed.Results1.DNA-PKcs was expressed in osteosarcoma cell lines(MNNG/HOS,MG-63,U2-OS,Saos-2),and the expressions of DNA-PKcs in mRNA and protein in osteosarcoma cells(MNNG/HOS,MG-63,U2-OS,Saos-2)were higher compared with hFOB 1.19 cells(P<0.05),2.The results of immunofluorescence showed that DNA-PKcs protein was located in nucleus of osteosarcoma cells(MNNG/HOS,MG-63,U2-OS,Saos-2)and was evaluated expressed compared with hFOB1.19 cells.3.The positive rate of DNA-PKcs protein was higher in osteosarcoma tissue compared with osteochondroma tissue(70.83%vs 5%,P<0.05).There was a positively relationship between the expression of DNA-PKcs and Enneking surgical staging rather than gender,age and tumor site.Conclusions1.The expressions of DNA-PKcs in mRNA and protein in osteosarcoma cells were higher compared with hFOB1.19 cells,and DNA-PKcs protein was expressed in cell nucleus in osteosarcoma.2.The positive rate of DNA-PKcs protein in osteosarcoma tissue was higher than osteochondroma tissue The expression of DNA-PKcs was positively correlated with Enneking surgical staging in osteosarcoma.Part 2 The effect of DNA-PKcs on cisplatin resistance and the mechanisms in CD133-positive MG-63 cellsBackgroundWith the introduction of chemotherapy in treatment of osteosarcoma(OS),the 5-year survival rate after surgery has increased to 50%～700/%in patients without metastasis.However,the treatment result of OS has reached a plateau during the last three decades.The development of chemo-resistance in OS is an important cause for the plateau of survival rate.It is necessary to further investigate the mechanisms of drug-resistance in OS.The cancer stem cell model is one merging model for the development of drug-resistance in malignancies.Cancer stem cells(CSCs)markedly promote drug-resistance in various cancers.It has been demonstrated that CD 133-positive cells in OS exhibit cancer stem cells characteristics,such as self-renewal,multiple differentiation,tumorigenicity and so on,and resistance to chemotherapeutic agents.However,the mechanisms of drug resistance in CD133-positive OS cells need to be further elucidated.DNA-dependent protein kinase catalytic subunit(DNA-PKcs),and accessory heterodimeric complexes,Ku70 and Ku80,are involved in DNA damage repair via non-homologous end joining(NHEJ).DNA-PKcs plays a significant role in chemo-resistance via DNA damage repair in OS.Moreover,DNA-PKcs was over-expressed in osteosarcoma CSCs.It is meaningful to research whether DNA-PKcs can cause chemo-resistance in cancer stem cells of OS via DNA damage repair.In addition,P-glycoprotein(P-gp),a member of ATP-binding cassette(ABC)transporter,plays an important role in chemo-resistance in tumors.It has been demonstrated that P-gp expression is at higher level in osteosarcoma CSCs compared with non-CSCs.Although DNA-PKcs and P-gp are both involved in chemo-resistance and over-expressed in osteosarcoma CSCs,there has been no study about the effect of DNA-PKcs on the regulation of P-gp in osteosarcoma CSCs chemo-resistance to date.Previous studies demonstrated that PI3K/Akt pathway could promote the NF-κB translocation into nucleus,and NF-κB regulated gene transcriptiont of P-gp.Hence,this prompts us to investigate whether DNA-PKcs regulates P-gp expression via Akt/NF-KB pathway to enhance cisplatin resistance in osteosarcoma CSCs.In this part,we investigated the effct of DNA-PKcs on cisplatin resistance and underlying mechanisms in CD133-positive MG-63 cells.Purposes1.To investigate the effect of DNA-PKcs on cisplatin resistance of CSCs in OS.2.To investigate whether DNA-PKcs could promote cisplatin resistance of osteosarcoma CSCs through DNA damage repair and regulation of P-gp expression.Methods1.Magnetic activated cell sorting(MACS)was performed and the magnetically labeled CD 133-positive cells and unlabeled CD 133-negative cells were collected,respectively.Then the levels of mRNA and protein of DNA-PKcs were compared between the two kinds of cells and the cells were treated with cisplatin to test cell survival rate.The expression of DNA-PKcs was down-regulated by small interference RNA(siRNA)in CD 133-positive cells,and the cells were treated with cisplatin to test cell survival rate.2.DNA damages were tested after cisplatin treatment in CD 133-positive cells and CD 133-negative cells,respectively.The change of DNA damages in CD133-positive cells treated with cisplatin was tested after downregulation of DNA-PKcs by siRNA.3.The expressions levels of P-gp in mRNA and protein were examined in CD 133-positive and CD133-negative MG-63 cells,respectively.Then the survival rate of CD133-positive MG-63 cells was examined after P-gp inhibitor treatment.4.The expressions of P-gp in mRNA and protein were examined after downregulation of DNA-PKcs by siRNA in CD 133-positive MG-63 cells.5.The activity of the Akt/NF-KB pathway was examined in CD133-positive and CD 133-negative MG-63 cells,respectively.Then the pathway was inhibited by Akt inhibitor or down-regulation of NF-κB,and the expressions of P-gp in gene and protein were examined.6.The activity of the Akt/NF-KB pathway was examined in CD 133-positive cells after downregulation of DNA-PKcs.Results1.Compared with CD133-negative MG-63 cells,CD 133-positive MG-63 cells were more resistant to cisplatin and expressed high levels of DNA-PKcs in mRNA and protein.However,downregulation of DNA-PKcs sensitized CD 133-positive MG-63 cells to cisplatin.2.Compared with CD 133-negative MG-63 cells,CD 133-positive MG-63 cells yielded less DNA double-strand breaks after cisplatin treatment.Downregulation of DNA-PKcs increased DNA double-srand breaks after cisplatin treatment.3.The expressions of P-gp in mRNA and protein were markedly elevated in CD133-positive MG-63 compared with CD133-negative MG-63 cells.However,inhibition of P-gp sensitized CD133-positive MG-63 cells to cisplatin.4.The expressions of P-gp in mRNA and protein were significantly decreased after downregulation of DNA-PKcs in CD 133-positive MG-63 cells.5.The Akt/NF-KB pathway was hyperactivated in CD133-positive MG-63 cells.After inhibition of the Akt/NF-KB pathway,the expressions of P-gp in mRNA and protein were significantly decreased in CD 133-positive MG-63 cells.Downregulation of DNA-PKcs suppressed the activation of the Akt-NF-κB pathway in CD 133-positive MG-63 cells.Conclusions1.DNA-PKcs was high expressed and promoted resistance to cisplatin in CD133-positive MG-63 cells.2.DNA-PKcs promoted cisplatin resistance of CD133-positive MG-63 cells through DNA damage repair and regulation of P-gp expression via Akt/NF-κB pathway.Part 3 The effect of DNA-PKcs on osteosracoma metastasis and the molecular mechanismsBackgroudOsteosarcoma(OS)has a high incidence of metastasis and the most common site of metastasis is lung.Metastasis is the main cause of death for osteosarcoma patients with poor prognosis.The 5-year survival rate is 20%-30%for patients with metastasis.It is necessary to investigate the mechanisms of the occurrence and progress of metastasis in OS in order to decrease the incidence of metastasis and improve survival rate.DNA-dependent protein kinase catalytic subunit(DNA-PKcs)is a member of phosphatidylinositol 3-kinase-related kinase(PIKK)family,and is involved in cell survival,inflammatory responses,metabolic regulation,cell apoptosis and cell cycle control through regulation of the target gene transcription.It has been demonstrated that DNA-PKcs is overexpressed in many malignances and is correlated with prognosis.Our study in part 1 also finds that the expression of DNA-PKcs is at a high level and correlated with Enneking staging.However,the studies about the effect of DNA-PKcs on tumor metastasis are few,and there is no report about the relationship between DNA-PKcs and osteosarcoma metastasis.It is reported that matrix metallopeptidase 2(MMP2)and phospho-myosin light chain 2(p-MLC2)were involved in cell motility via extracelluar matrix degradation and actin-myosin contractility respectively,and are both regulated by Rho/ROCK pathway.Hence,we hypothesize that DNA-PKcs may be involved in osteosarcoma metastasis through the regulation of MMP2 and p-MILC2 by Rho/ROCK pathway.In this study,the osteosarcoma cells were taken as object to investigate the effect of DNA-PKcs on osteosarcoma imetastasis and underlying mechanisms.It may provide theoretical evidence for osteosarcoma treatment targeting DNA-PKcs in future.Purposes1.To investigate the effect of DNA-PKcs on osteosarcoma cells migration and invasion in vitro,and formation and development of metastatis in vivo.2.To investigate whether DNA-PKcs could regulated expressions of MMP2 and p-MLC2 via Rho/ROCK pathway,and thus promoted osteosarcoma metastasisMethods1.Osteosarcoma cells were treated with NU7026.a specific inhibitor of DNA-PKcs,then migration ability of cells was tested using wound-healing assay and Transwell migration assay,and invasion ability of cells was tested using Transwell invasion assay.The methods above were used to prove the effect of DNA-PKcs on osteosarcoma cells migration and invasion in vitro.2.After osteosarcoma cells were infected with lentiviral vector of DNA-PKcs siRNA,the viral infection rate was observed by fluorescence microscope.The changes of the levels of DNA-PKcs in mRNA and protein were examined by qRT-PCR and Western Blot after infection of lentiviral vector of DNA-PKcs siRNA,to evaluate the effect of-siRNA interference.The changes of MMP2 and p-MLC2 protein levels in cells and MMP2 secretion in supernatants were examined after downregulation of DNA-PKcs by infection of lentiviral vector of DNA-PKcs siRNA in osteosarcoma cells,to demonstrate the effect of DNA-PKcs on the regulation of MMP2 and p-MLC2.3.After downregulation of DNA-PKcs.the level of active RhoA-GTPase was tested,and the changes of the levels of ROCK 1 and ROCK2 in mRNA and protein were examined by qRT-PCR and Western Blot.This was to investigate the ef-fect of DNA-PKcs on RhoA/ROCK pathway regulation.4.The osteosarcoma cells were treated with SLx-2119,a specific ROCK2 inhibitor,then the migration and invasion of cells were tested using wound-healing assay,Transwell migration assay and Transwell invasion assay,to research the effect of RhoA/ROCK2 pathway on migration and invasion of osteosarcoma cells.5.After downregulation of ROCK2 by siRNA transfected with liposome,the changes of MMP2 and p-MLC2 protein levels were examined by Western Blot,and the change of MMP2 secretion in supernatants was test by ELISA.All this methods above were used to investigate the effect of RhoA/ROCK2 pathway on regulation of’ MMP2 and p-MLC2.6.The MNNG/HOS cells infected with viral vector of negative control siRNA or DNA-PKcs siRNA were injected into NOD/SCID mice via caudal vein to establish the osteosarcoma metastasis model.The growth condition of the mice were observed and recorded.The mice were killed after 10 weeks and the metastatic lesions in NOD/SCID mice were examined and measured,to evaluate the effect of DNA-PKcs on osteosarcoma metastasis in vivo.Results1.Compared with negative control group,the migration and invasion ability of osteosarcoma cells were decreased significantly after treatment with NU7026(P<0.05).DNA-PKcs played positive role in osteosarcoa cells migration and invasion.2.After infection of viral vector of DNA-PKcs siRNA in osteosarcoma cells,the viral infection rate reached more than 80%,and the levels of DNA-PKcs in mRNA and protein were significantly lower than negative control group(P<0.05).The interference result was satisfactory.3.The MMP2 and p-MLC2 protein and MMP2 secretion were both markedly decreased after downregulation of DNA-PKcs in osteosarcoma cells(P<0.05).These results above demonstrated that DNA-PKcs was involved in cells migration and invasion via regulation MMP2 and p-MLC2 in osteosarcoma.4.The active RhoA-GTPase expression and the levels of ROCK2 in mRNA and protein were significantly decreased after downregulation of DNA-PKcs(P<0.05).This demonstrated that DNA-PKcs played role in the regulation of RhoA/ROCK2 pathway.5.The migration and invasion of osteosarcoma cells were significantly decreased after treatment with SLx-2119(P<0.05).The expression of MMP2 and p-MLC2 protein and MMP2 secretion were also markedly decreased after downregulation of ROCK2(P<0.05).The results showed that RhoA/ROCK2 pathway promoted the migration and invasion of osteosarcoma cells and regulated MMP2 and p-MLC2 expression.6.Compared with negative control group,the metastatic lesions in NOD/SCID mice injected with MNNG/HOS cells infected with viral vector of DNA-PKcs siRNA were much less and the average tumor volume was much smaller.There was a statistically significant difference between the two groups(P<0.05).This demonstrated that DNA-PKcs played role in formation and development of osteosarcoma metastasis in vivo.Conclusions1.DNA-PKcs promoted osteosarcoa cells migration and invasion in vitro,and formation and development of’ osteosarcoma metastasis in vivo.2.DNA-PKcs regulated the expression of MMP2 and p-MLC2 via RhoA/ROCK2 pathway,and therefore enhanced metastasis of osteosarcoma. |
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