The Role And Mechanism Of Microtubule Affinity Regulated Kinase 2 On Cisplatin Resistance Of Osteosarcoma Stem Cells And Development Of Osteosarcoma Cells

Part 1The expression and clinical significance of microtubule affinity regulated kinase 2 in osteosarcomaBackgroundOsteosarcoma is the most common primary malignant tumor of bone in children and adolescents.Although the annual incidence rate is only 3/1 million,it seriously endangers the health of young patients.Due to its obvious heterogeneity,genomic instability,early metastasis,and insensitivity to chemotherapy effects,osteosarcoma has become the main cause of cancer-related deaths in adolescents and young people.With the advancement of chemotherapy and surgical techniques,the treatment plan for osteosarcoma has changed from the amputation treatment before the 1970s to the increasingly standardized neoadjuvant chemotherapy-surgery-adjuvant chemotherapy standard.The 5-year survival rate of patients with osteosarcoma without metastasis has increased from less than 20%to about 70%.Nevertheless,the survival rate of patients with osteosarcoma has not improved significantly in the past three decades,and the 5-year survival rate of patients with metastatic osteosarcoma is less than 25%.In recent years,research on targeted therapy of various malignant tumors has been in full swing,and many tumors have also made milestone progress.The NCCN guidelines recommend sorafenib(VEGFR inhibitor)as the second-line treatment for patients with osteosarcoma.Therefore,it is necessary to explore new molecular mechanisms of osteosarcoma,develop new effective therapeutic targets,and improve the prognosis of osteosarcomaMicrotubule affinity regulating kinase 2(MARK2).a serine/threonine protein kinase,is a key component of microtubule associated protein phosphorylation,and plays an important role in the phosphorylation of cell cycle regulatory proteins,type Ⅱ histones and other cytokines.Animal gene knockout experiments have shown that MARK2 plays an important role in nerve differentiation,neurodegeneration,cell polarization,intracellular transport and cell migration.However,recent studies have found that MARK2 is closely related to tumor malignant biological behavior and drug resistance.In non-small cell lung cancer,cervical cancer and other malignant tumors,MARK2 is highly expressed and is associated with poor clinical prognosis.However,the expression of MARK2 in osteosarcoma and its related research are lacking,so we first try to explore the expression of microtubule affinity-regulated kinase 2 in osteosarcoma and its clinical significancePurposeThis study aims to explore the expression of MARK2 in osteosarcoma and its relationship with clinicopathological characteristics and prognosis,and to provide a solid foundation for further research on the role and mechanism of MARK2 in the occurrence and development of osteosarcomaMethods1.We first analyze the relationship between the expression of MARK2 and clinical prognosis in bone tumor patients from public databases.2.Immunohistochemical detection of MARK2 protein expression in osteosarcoma tissue and osteoblastoma tissue,and analysis of the relationship between MARK2 expression in osteosarcoma tissue and clinicopathological characteristics and prognosis.Results1.The analysis of GEO database(GSE12865)found that compared with normal osteoblast tissue,the expression of MARK2 in osteosarcoma tissue was increased.Analysis of TARGET data shows that patients with osteosarcoma with high MARK2 expression have a low overall survival and disease-free survival and poor prognosis2.The results of immunohistochemistry experiments showed that the expression of MARK2 in osteosarcoma tissue was significantly higher than that in osteoblastoma tissue Moreover,the high expression of MARK2 was related to tumor size(P=0.0322),distant metastasis(P=0.0044)and response to chemotherapy(P<0.0001).Kaplan-Meier survival analysis showed that patients with higher MARK2 expression had shorter overall survival.ConclusionMARK2 is highly expressed in osteosarcoma tissues,and is related to tumor size,distant metastasis,response to chemotherapy,and poor prognosis of patientsPart 2The role and mechanism of MARK2 in cisplatin resistance of osteosarcoma stem cellsBackgroundThe standard treatment of neoadjuvant chemotherapy,surgery,and adjuvant chemotherapy has significantly improved the survival rate of patients with osteosarcoma.Standard chemotherapy drugs for osteosarcoma include cisplatin,doxorubicin,ifosfamide,and methotrexate.Despite recent advances in diagnosis and treatment,the survival rate of osteosarcoma patients has remained stagnant in recent years Previously,in fact,about 35%of osteosarcoma patients would still be resistant to the above four chemotherapeutics,especially cisplatin resistance.Chemoresistance often leads to treatment failure and leads to recurrence,metastasis or unresectable osteosarcoma,which limits the current therapeutic effects of osteosarcoma.Therefore,it is necessary to further explore the resistance mechanism of osteosarcoma and develop new effective therapeutic targets to reverse the chemotherapy resistance of osteosarcoma and improve the prognosis of osteosarcoma.Cancer stem cells(CSC)are a type of malignant tumor cell group with characteristics such as self-renewal,differentiation potential,high tumorigenicity,and high drug resistance.Although they account for a very small proportion in tumors,they are not sensitive to conventional radiotherapy and chemotherapy.They are considered to be the root cause of tumor development,chemotherapy resistance,and metastasis,and have important significance for tumor treatment and prognosis.Therefore,cancer stem cell theory is an important reason for chemotherapy resistance.Isolation of cancer stem cells for related research can find drug targets and related signal transduction pathways,which provides theoretical support for the development of new drugs and treatment methods.CD 133 antigen is considered as a stem cell marker for normal tissues and cancer tissues.Studies have identified that CD133+cells in the osteosarcoma cell line have many characteristics of cancer stem cells.The ability of tumor cells to repair DNA damage is also an important mechanism of chemotherapy resistance.Many studies have found that the activity and expression of DNA dependent protein kinase(DNA-PK)and DNA dependent protein kinase catalytic subunit(DNA-PKCs)are increased in glioma,cervical cancer,non-small cell lung cancer and B-cell chronic lymphocytic leukemia.Our previous research found that CD133+osteosarcoma cells have the characteristics of cancer stem cells.This group of cells highly express MDR1 and DNA-PK genes,which are prominent in chemotherapy resistance,tumor formation and tumor malignant biological behavior.The high expression of DNA-PKcs is associated with poor prognosis of osteosarcoma.The positive rate of DNA-PKcs in patients with recurrence and metastasis is significantly higher than that in patients without tumor.DNA-PKcs can be used as one of the prognostic factors affecting tumor-free survival.However,the upstream target of this mechanism is unclear.Microtubule affinity-regulating kinase 2(MARK2)is involved in the regulation of many cell functions.In recent years,studies have found that MARK2 is closely related to the chemotherapy resistance of tumors.MARK2 is overexpressed in cisplatin-resistant cervical cancer cell lines.Studies have also found that MARK2 is highly expressed in non-small cell lung cancer and is closely related to cisplatin resistance,and that MARK2 overexpression is closely related to DNA hypomethylation and replication.Both cell line and primary cell experiments have shown that MARK2 plays an important role in cell cycle activation and DNA damage repair.Down regulation of MARK2 expression increased DNA damage,S phase arrest and increased NF-κB activity in lung cancer cells.This study believes that MARK2 can increase cell DNA damage repair by regulating the cell cycle.The dysregulation of the PI3K/Akt/mTOR pathway is related to the chemoresistance of many tumors including osteosarcoma.The first part found that the high expression of MARK2 in osteosarcoma is related to the response to chemotherapy.However,whether MARK2 can regulate the expression of DNA-PKcs through the PI3K/Akt/mTOR signaling pathway to promote the resistance of osteosarcoma stem cells to cisplatin remains to be further studied.PurposesThis study aimed to explore the effect of MARK2 on cisplatin resistance in CD133+MG-63 and MNNG/HOS cells and its underlying mechanism.Methods1.CD133-and CD133+MG-63 and MNNG/HOS cells are obtained by MACS(magnetic activated cell sorting).The CCK8 cell viability test was used to detect their resistance to cisplatin,and the expression of MARK2 in CD 133-and CD 133+MG-63 and MNNG/HOS cells was detected by qRT-PCR and Western blotting.2.After downregulatinon of MARK2 in CD 133+MG-63 and MNNG/HOS cells by siRNA,the changes in cisplatin resistance were detected.3.Western blotting experiment and cellular immunofluorescence experiment were used to detect the expression of DNA damage and repair related proteins in CD133-and CD133+MG-63 and MNNG/HOS cells after cisplatin treatment.After knockdown of MARK2 in CD133+MG-63 and MNNG/HOS cells,the changes in DNA damage and repair were detected.4.Western blotting was used to compare the expression of PI3K/Akt/mTOR pathway related proteins and DNA-PKcs in CD133-and CD133+ MG-63 and MNNG/HOS cells,and after adding Akt inhibitor,the expression level of DNA-PKcs and cisplatin resistance were detected.After down-regulating the expression of MARK2 in CD133+MG-63 and MNNG/HOS cells,the changes in the expression of related proteins in the PI3K/Akt/mTOR pathway and DNA-PKcs were detected.Results1.Compared with CD133-MG-63 and MNNG/HOS cells,CD133+MG-63 and MNNG/HOS cells showed higher cisplatin resistance and MARK2 expression level.2.Down-regulating MARK2 reduced the cisplatin resistance of CD 133+MG-63 and MNNG/HOS cells,indicating that MARK2 promotes cisplatin resistance in CD 133+MG-63 and MNNG/HOS cells.3.Compared with CD133-MG-63 and MNNG/HOS cells,CD133+MG-63 and MNNG/HOS cells had fewer DNA double-strand breaks and increased DNA damage repair proteins after cisplatin treatment.After down-regulating the expression of MARK2 in CD133+MG-63 and MNNG/HOS cells,DNA double-strand breaks increased and DNA damage repair proteins decreased.The results indicated that MARK2 can promote the repair of DNA damage in CD133+MG-63 and MNNG/HOS cells.4.Compared with CD133-MG-63 and MNNG/HOS cells,the expression levels of key proteins in the PI3K/Akt/mTOR signaling pathway and DNA-PKcs in CD133+MG-63 and MNNG/HOS cells increased.After inhibiting the activity of PI3K/Akt/mTOR signaling pathway,the expression level of DNA-PKcs and cisplatin resistance in CD133+MG-63 and MNNG/HOS cells decreased.After down-regulating MARK2,the expression levels of key proteins in the PI3K/Akt/mTOR signaling pathway and DNA-PKcs in CD 133+MG-63 and MNNG/HOS cells decreased.The above results indicated that MARK2 can regulate the expression of DNA-PKcs through the PI3K/Akt/mTOR signaling pathway,thereby enhancing the cisplatin resistance of CD 133+MG-63 and MNNG/HOS cells.Conclusions1.MARK2 is highly expressed in CD133+MG-63 and MNNG/HOS cells and is associated with cisplatin resistance.2.MARK2 regulates the expression of DNA-PKCs through PI3K/Akt/mTOR signal,which promotes cisplatin resistance of CD 133+MG-63 and MNNG/HOS cells,which provides a new strategy and therapeutic target for reversing chemotherapy resistance of osteosarcoma.Part 3The role and mechanism of MARK2 in the development of osteosarcoma cellsBackgroundOsteosarcoma is characterized by high degree of malignancy,rapid progress and early metastasis.90%of the metastases occur in the lung.The main cause of death in patients with osteosarcoma is lung metastasis.About 10-20%of osteosarcoma patients have distant metastasis at the initial diagnosis,and more than 50%of patients will have metastasis at different stages of the disease.Although the survival rate of osteosarcoma under the standard treatment has been significantly improved,the 5-year survival rate of patients with metastatic osteosarcoma is about 25%.Therefore,distant metastasis is an important factor affecting the prognosis of patients with osteosarcoma.Sorafenib is a second-line drug recommended by NCCN for relapsed and metastatic refractory cases.Molecular targeted drugs are a new treatment for osteosarcoma,which will play an important role in improving the prognosis of osteosarcoma.Therefore,it is necessary to further study the mechanism of osteosarcoma development and find new biomarkers and therapeutic targets to improve the prognosis of osteosarcoma.Microtubule affinity modulating kinases(MARKs)are serine/threonine protein kinases that can phosphorylate microtubule-related proteins,such as Tau,and regulate cytoskeletal dynamics and cell cycle progression.There are four types of MARK(MARK1-4)in the human body.At present,more and more evidences show that MARKs have multiple functions,including cell polarity,neuronal differentiation,circulation,neurodegeneration,and intracellular Transport,cell growth and cell migration.Some previous studies have also shown that MARKs play a vital role in the occurrence and development of certain cancers.As a member of the MARKs family,it is reported that microtubule affinity-regulated kinase 2(MARK2)mediates cell proliferation,metastasis and chemical drug resistance through various signal pathways,so it plays a pivotal role in tumor progression and prognosis.The first part of the study found that MARK2 is highly expressed in osteosarcoma and is related to tumor size,distant metastasis and poor prognosis.However,the functional role and molecular mechanism of MARK2 in the pathogenesis and progression of osteosarcoma are still unknown.Studies have shown that osteosarcoma genes are highly unstable.The PI3K/AKT signaling pathway is activated in advanced osteosarcoma cases and participates in the occurrence and development of osteosarcoma cells,including the regulation of cell proliferation,migration and invasion,and chemotherapy resistance.In our previous experiments,we found that MARK2 enhances the cisplatin resistance of osteosarcoma cells through the PI3K/AKT/NF-κB signaling pathway.However,whether MARK2 promotes osteosarcoma cell proliferation and metastasis through PI3K/AKT/NF-κB signaling pathway remains to be determined.PurposeThe purpose of this study is to explore the role and potential mechanism of MARK2 in the progression and metastasis of osteosarcoma.Methods1.Use lentivirus to establishMG-63 and MNNG/HOS cells stably overexpressing or knocking down MARK2.CCK-8 method detects cell proliferation.Flow cytometry test cell cycle.2.After overexpression or knockdown of MARK2,wound-healing assay,Transwell cell migration and invasion assays were used to detect the migration and invasion ability of osteosarcoma cells,and the expression of matrix metalloprotein2(MMP2)was detected by Western blot.3.Western blot experiments to detect the expression levels of key proteins related to PI3K/AKT/NF-κB signaling pathway after MARK2 overexpression and knockdown.4.Use PI3K inhibitor LY294002 to treat MARK2 overexpressing cells for rescue experiment.CCK8,cell flow cytometry experiment,wound-healing assay,Transwell cell migration and invasion assay and western blot experiment to detect whether PI3K inhibitor can rescue the effect of MARK2 overexpression on the proliferation,migration and invasion of osteosarcoma cells.5.Establish a nude mouse xenograft osteosarcoma model,observe the effect of overexpression and knockdown of MARK2 on the volume and weight of subcutaneous tumors.Western blotting was performed to detect the expression levels of key proteins related to PI3K/AKT/NF-κB signaling pathway and MMP2 in nude mouse tumor specimens.The immunohistochemistry experiment detects Ki-67,an index related to proliferation ability.Results1.CCK8 experiment showed that overexpression of MARK2 promoted the proliferation of osteosarcoma cells.Flow cytometry showed that overexpression of MARK2 significantly decreased the percentage of G1 phase cells and increased the percentage of S phase cells.In contrast,the opposite effect was observed in osteosarcoma cells with MARK2 down-regulation.It is suggested that MARK2 may promote the growth of osteosarcoma cells by accelerating the G1-S phase transition.2.Overexpression of MARK2 promoted the migration and invasion of osteosarcoma cells,and increased the expression of MMP2.Knockdown of MARK2 inhibited the migration and invasion of osteosarcoma cells,and decreased the expression of MMP2.It is suggested that MARK2 can promote the migration and invasion of osteosarcoma cells by promoting the expression of MMP2.3.Overexpression of MARK2 increased the protein levels of p-PI3K p85,p-AKT,and p-NF-κB/p65 in the PI3K/AKT/NF-κB signaling pathway of osteosarcoma cells.Knockdown of MARK2 decreased the protein levels of p-PI3K p85,p-AKT,p-NF-κB/p65.The results showed that MARK2 can activate the PI3K/AKT/NF-κB pathway in osteosarcoma.4.The results of the rescue experiment showed that in the osteosarcoma cells overexpressing MARK2 treated with LY294002,the cell proliferation ability decreased,the cell cycle G1 phase ratio increased,the cell cycle S phase ratio increased,and the cell migration and invasion ability decreased.The expression levels of p-AKT,p-NF-κB/p65 and MMP2 were inhibited,while the expression of MARK2 was not affected.The above results suggested that PI3K inhibitors can rescue the effects of MARK2 overexpression on the proliferation,migration and invasion of osteosarcoma cells.MARK2 promoted the osteosarcoma cell proliferation,migration and invasion by regulating Gl-S phase transition and MMP2 expression through PI3K/AKT/NF-κB pathway5.The volume and weight of xenograft osteosarcoma tumors overexpressing MARK2 increased.Knockdown of MARK2 reduced the volume and weight of xenograft osteosarcoma tumors.The expression levels of p-AKT,p-NF-κB/p65,MMP2 and Ki-67 increased significantly in tumor specimens of mice overexpressing MARK2,while the expression levels of p-AKT,p-NF-KB/p65,MMP2 and Ki-67 decreased.These results indicated that MARK2 may promote tumor formation in osteosarcoma cells in vivo through PI3K/AKT/NF-κB pathway.ConclusionMARK2 regulates Gl-S phase transition and MMP2 expression through PI3K/AKT/NF-κB pathway,thereby promoting the proliferation,migration and invasion of osteosarcoma cells.The results suggest that MARK2 can be used as a new oncogene and a potential therapeutic target for patients with osteosarcoma.