| Osteosarcoma has become the most common malignant tumor in teenagers,its morbidity and mortality increase in all kinds of tumors because of its hidden symptoms and no obvious clinical symptoms.Patients are often in the advanced stage when diagnosed,because of lack of efficient of diagnosis method.The genetic pathogenesis of osteosarcoma includes gene mutations,single nucleotide polymorphisms and epigenetics,where gene mutations refer to genes that rely on DNA base sequence mutations to cause abnormal gene expression.The Epigenetics is independent of DNA base sequence changes,which gene promoter methylation abnormalities are the most common pathogenesis.The DKK3 gene is a member of DKK family.It can regulate the cell cycle and apoptosis by modulating Wnt signal transduction way.DKKs gene can modulate vascular endothelial growth factor receptor 2 to inhibit tumor angiogenesis.In addition,recent study showed that DKK3 could inhibit the Wnt/p-catenin signaling pathway,Wnt/matrix metalloproteinase protein 2 and Wnt/JNK pathways to inhibit cell proliferation and induce apoptosis.Reduced expression of DKK3 gene can lead to decrease the expression of mRNA and protein,regulating cell proliferation and apoptosis,which resulted in decreased ability to inducing a variety of malignant tumors.Studies showed that decreased DKK3 gene expressions might be pathogenesis for many malignant tumor,which could act as a biomarker in the disease condition and prognosis evaluation.Methylated oligonucleotide consists of 20bp,which is designed and synthesized artificialy.Methylated oligonucleotide could induce targetd gene siliencing with specificity and high efficiency,which has been proven by current study.Part 1:Methylation status of DKK3 gene promoter in Osteosarcoma and its clinical valueObjective To detect the methylation status of DKK3 gene promoter in Osteosarcoma and study its clinical value.Methods The patients with osteosarcoma(OST group)and benign bone disease(CON group)from Jan 2015 to Jan 2017 in Hubei Province People’s Hospital were enrolled for studied.The gender,age and body mass index between OST group of patients with osteosarcoma and CON group of patients with benign bone disease were comparable.The methylation status of DKK3 gene promoter in both groups was detected by methylation specific PCR in the tumor tissue of OST group and the tissue of CON group of benign bone disease.The difference of the methylation status of DKK3 gene promoter was detected and analyzed.The methylation rates of DKK3 gene promoter in different clinicopathological factors were analyzed.The mRNA and protein expressions in OST group and CON group were detected with RT-PCR and Western blot respectively.Results(1)The methylation rate of the DKK3 gene promoter in the tumor tissue of the OST group was 72.0%,while the methylation rate of the DKK3 gene promoter in CON group was 2.5%.The promoter methylation rate of DKK3 gene in tumor tissue of patients with osteosarcoma was significantly higher than that in the control group(P<0.05).(2)There were no significant differences in sex age,location,family story and pathological type(all P>0.05),but they were statistics significant in KPS score,pathological fracture,Enneking stage,maximum diameter of tumor,treatment method and distant metastasis.The methylation rate with KPS score<70,with pathological fracture,Enneking stage ⅡB+Ⅲ,maximum diameter of tumor ≥ 6cm,combined therapy method and with distant metastasis were significantly higher than in KPS score ≥70,without pathological fracture,Enneking stage Ⅰ + ⅡA,maximum diameter of tumor<6cm,surgical therapy method and without distant metastasis(All P<0.05);(3)The R0 radical operation rate,disease-free survival time and overall survival time in cases with DKK3 gene promoter methylation were significantly lower than in cases with unmethylation(P<0.05),while 3-year mortality rate was higher(P<0.05);(4)The mRNA and protein expressions in OST group were significantly lower than in CON group(P<0.05).Conclusion(1)The methylation rate of DKK3 gene promoter in osteosarcoma patients is significantly higher than that in patients with benign bone disease.The promoter of DKK3 gene in osteosarcoma patients is in hypermethylation state;(2)The methylation rates of DKK3 gene promoter are statistics significant in KPS score,pathological fracture,Enneking stage,maximum diameter of tumor,treatment method and distant metastasis.The methylation rate with KPS score<70,with pathological fracture,Enneking stage ⅡB+Ⅲ,maximum diameter of tumor ≥ 6cm,combined therapy method and with distant metastasis were significantly higher than in KPS score ≥70,without pathological fracture,Enneking stageⅠ + Ⅱ A,maximum diameter of tumor<6cm,surgical therapy method and without distant metastasis.(3)The mRNA and protein expressions in patient with osteosarcoma are in low status;(4)The hypermethylation of DKK3 gene promoter may be a triggering factor for the low expression of DKK3 gene.The pathogenesis of osteosarcoma may correlate with DKK3 gene promoter methylation,which could act as a biomarker for osteosarcoma’s disease condition and prognosis evaluation.Part 2:Effects of DKK3 gene promoter’s methylation induced by methylated oligonucleotide on Osteosarcoma’s proliferation and apoptosisObjective To study the effects of DKK3 gene promoter’s methylation induced by methylated oligonucleotide on Osteosarcoma’s proliferation and apoptosis.Methods Oligonucleotides(MON group,UMON group,CON1 group,CON2 group and CON3 group)were designed and transfected into SAOS-2 Osteosarcoma cell line.The DKK3 gene promoter’s methylation status was detected with bisulphite Sequencing.The growth curve were detected with CCK8 kit.The cell proliferation and apoptosis were detected by flow cytometry pre-and post-transfection.The DKK3 gene’s mRNA and protein expressions were detected with RT-PCR and Western blot respectively.Results(1)The DKK3 gene promoter in MON group were 1-5(100.0%)and 6-8 CG(0.0%),while they were all 0.0%in UMON group,CONI group,CON2 group and CON3 group;(2)The D1-D7 absorbance in MON group were significantly higher than in UMON group,CONI group,CON2 group and CON3 group;(3)The G0/G1 phase and apoptosis rate in MON group were lower than in UMON group,CONI group,CON2 group and CON3 group,while they were higher in S phase.G2/M phase and PI;(4)The DKK3 gene’s mRNA and protein expressions in MON group were significantly lower than in UMON group,CON1 group,CON2 group and CON3 group.Conclusion(1)The methylated oligonucleotide could successfully induce DKK3 gene promoter’s methylation and cause gene expression silencing;(2)Osteosarcoma cell’s proliferation increases after methylated oligonucleotide transfection but the apoptosis decreases;(3)DKK3 gene promoter’s methylation induced by methylated oligonucleotide could change osteosarcoma cell’s biological behavior,which may prove the correlation between DKK3 gene promoter’s methylation and pathogenesis of osteosarcoma. |
