The Relationship Between KDM5C And Osteosarcoma And Skeletal Muscle Regeneration

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Association of KDM5 C with SAC on mitosis of osteosarcomaObjectiveOsteosarcoma is the most prevalent primary bone tumor.Cell cycle-independent chemotherapy drugs are widely applied to chemotherapeutic regimens for osteosarcoma,such as doxorubicin,ifosfamide and cisplatin.However,their application are limited usually by the failed therapy due to side effects.The development of novel cell cycle-dependent chemotherapy drugs is a significant direction of osteosarcoma research.Spindle assembly checkpoint(SAC)as a significant regulation of mitosis can be inhibited by methylation of Lys4 of histone H3 near the centromeres,thus promoting mitosis from metaphase to anaphase,increasing genetic instability.Lysine demethylase 5C(KDM5C)has been shown to be able to mediate the demethylation of tri-and di-methylated H3K4(H3K4me3/me2),but not monomethylated H3K4(H3K4me1)and regulate H3K4me2/me3 levels on chromosomes.KDM5 C is associated with several malignancies.However,whether KDM5 C is associated with osteosarcoma had remained unclear,especially with SAC.This paper aims to investigate the alteration of KDM5 C protein level and the regulatory effect of KDM5 C on SAC,related mitotic progression of osteosarcoma cell lines.MethodsThe various osteosarcoma cells in prometaphase were collected by synchronization.The alteration of KDM5 C protein level and its potential posttranslational modification in mitosis were determined by Western blot.The effect of inhibitor CPI-455 on mitosis process was detected using Western blot.Results1.In mitosis of osteosarcoma,the alterations of SAC related markers,cell division cycle protein 27(cdc27)and Cyclin B1,were similar to previous literatures,but KDM5 C protein level had no obvious change.2.There were no differences on the alteration of KDM5 C protein level from prometaphase to G1 phase in He La.3.The constructed recombinant plasmids contained the sequence of KDM5 C genetic fragments that were identified by sequence analysis,and expressed truncate KDM5 C proteins in HEK293 T that were identified by Western blot.4.The shift and ubiquitination of KDM5 C truncations in U2 OS transfected recombinant plasmids were not observed by Western bolt.5.SAC related markers cdc27 and Cyclin B1 had no obvious change,compared to the DMSO control,whether increasing the level of exogenous KDM5 C protein or inhibiting demethylase activity of KDM5 C.6.There were no differences on SAC related phase from metaphase to anaphase and percentage of segregation error in mitotic phase,When demethylase activity of KDM5 C was inhibited by CPI-455.ConclusionMitotic process of osteosarcoma cell and the SAC is unaffected by KDM5 C protein level and its demethylase activity.Part ? The Effect of inhibiting KDM5 C activity by CPI-455 on proliferation and differentiation of C2C12ObjectiveSkeletal muscle injury is one of the common injuries in sports medicine,can lead to hematoma and the scar tissue after muscle injury,thus reducing the ability of skeletal muscles contraction,and increasing the opportunity for injury again.It is a potential threat to patients' athletic ability.Therefore,to investigate myogenic differentiation mechanism contribute to provide better treatments for skeletal muscle injury diseases.Methylation modification of histone H3 lysine 4(H3K4)can regulate many important biological processes by regulating target genes expression.Lysine methyltransferase 2D(KTM2D)is one of the KTM2 family members,and has been shown to be able to catalyze mono-and dimethylation of H3K4(H3K4me2/me1)specifically.The expression of Myogenin(Myo G)and Myosin in KTM2 D knockout mice are significantly reduced.KTM2 transmethylase family and KDM5 demethylase family play a critical role in the regulation of H3K4 methylation.KDM5 C is one of the KDM5 family members and has been shown to be able to mediate the demethylation of triand di-methylated H3K4(H3K4me3/me2).However,whether KDM5 C is associated with myogenic differentiation had remained unclear.The study aims to investigate the effect of inhibiting KDM5 C activity by CPI-455 on proliferation and differentiation of myoblast C2C12.MethodsThe alteration of KDM5 C protein level or m RNA relative level(RT-PCR)was detected during myoblast differentiation process(0h,48 h,72h,96h)by Western blot or Real-time quantitative PCR after inhibiting KDM5 C activity by CPI-455.The effect of inhibiting KDM5 C activity on mouse myoblast proliferation on was detected using CCK-8 and Hoechst 33342.Results1.The protein expression levels of myogenic differentiation markers of Myosin and Myo G were both lower after treatment by CPI-455 during myogenic differentiation process(48h,72 h,96h)than those by DMSO over the same period.However,there was no obvious difference in the comparison of the protein expression of myogenic differentiation markers of Actin and Alpha 1(ACTA1).Furthermore,the protein expression levels of H3K4me3 and KDM5 C were all higher in CPI-455 treatment group than those in DMSO treatment group on the same time.2.No obvious differences were found with respect to the relative expression of myogenic differentiation marker of Myosin,Myo G,ACTA1,Myo D,Myf5 and Pax7 m RNA following treatment by CPI-455 when compared to those by DMSO during myogenic differentiation process(48h,72 h,96h).3.There was no significant difference in relative KDM5 C m RNA level between CPI-455 treatment group and DMSO treatment group.4.CCK-8 assay and cell counting with Hoechst 33342 nuclei staining for live cells were used for the detection of the proliferation of C2C12 cells.Corresponding results indicated that there was no significant difference in relative cell activity or relative nuclei number between CPI-455 group and DMSO group.ConclusionInhibiting the demethylase activity of KDM5 C could reduce the myogenic differentiation of C2C12 cells by down-regulating the protein expression of myogenic differentiation markers,including Myosin and Myo G,without significant influence in the proliferation of C2C12 cells.