| Objectives1 To understand the role of TREX1(Three Prime Repair Exonuclease 1)level in distant metastasis and prognosis of OS(Osteosarcoma)patients,we used immunohistochemical assay to detect the expression level of TREX1 in tumor tissues from OS patients with or without metastasis within 3 years.2 We compared the expression level of TREX1 in CD133~+ and CD133-subpopulations of human OS cell line to determine the relationship between TREX1 and cells biological characteristics.3 We compared the changes of biological characteristic of OS cells before and after the TREX1 expression level was regulated,to check its influence on the biological characteristics of OS cells.4 We investigated the expression levels of downstream genes by TREX1 regulation to search for TREX1-related signaling pathways,which will provide experimental evidence to understand the functions of TREX1.Methods1 Clinical specimen collection: the specimens were divided into two groups according to OS patients with or without metastasis within 3 years,immunohistochemical assay was used to detect the expression level of TREX1 protein in OS tissues.2 CD133~+ and CD133-subpopulations were isolated by FACS from the human OS cells stained with PE-conjugated anti-human CD133 antibody.The TREX1 expression levels of them were detected by qPCR and Western Blot.Sphere-forming assay,clonogenic assay,growth(MTT assay),drugs sensitivity assessment,osteogenic and adipogenic assay,and tumorigenic assay were performed to compare the biological differences between CD133~+ and CD133-subpopulations of MNNG/HOS cell line.3 TREX1 of MNNG/HOS CD133-subpopulation was down-regulated to observe the changes of growth,the sensitivity to chemotherapeutic drugs,osteogenic and adipogenic differentiation potency,migration,invasion and tumorigenic ability.4 After TREX1 of MNNG/HOS CD133-subpopulation was down-regulated,q PCR was adopted to detect the expression changes for stemness related-genes Nanog and Oct4.Results1 Immunohistochemistry results indicated that: TREX1 protein was highly expressed in non-metastatic OS tissues,while lower expression was found in metastatic OS tissues with significant difference(P <0.05).Follow-up results showed that(final follow-up as the deadline)OS patients with high expression level of TREX1 had an average survival time for 52.6 months,while the value in the low expression level group was only 24.4 months;a significant difference was found between these two groups(P <0.05).2 Stemness-related genes Nanog and Oct4 were highly expressed,and TREX1 was lowly expressed in CD133~+ subpopulation of human OS cell line MNNG / HOS;this subpopulation possessed high ability to form sphere clusters and colonies,and had strong proliferation ability,low sensitivity to cisplatin,osteogenicity and adipogenicity differentiation potential and high tumorigenicity in vivo.In contrast,CD133-subpopulation showed low expression levels of Nanog and Oct4,but a higher level of TREX1;they were unable to form sphere clusters,had poor clonogenic ability,slow proliferation,low sensitivity to cisplatin,and lacked osteogenicity and adipogenicity differentiation potential with weak tumorigenicity.3 Down-regulation of TREX1 in OS MNNG/HOS CD133-subpopulation demonstrated that its proliferation ability was not altered significantly.However,this regulation reduced the sensitivity to cisplatin,increased osteogenicity and adipogenicity related-gene expression(mRNA levels),and enhanced the migration and invasion capacity and tumorigenicity in nude mice.4 Down-regulation of TREX1 in OS MNNG/HOS CD133-subpopulation showed that the expression levels of Oct4 and CD133 were much higher than that without interference(3-fold and 2-fold respectively,P <0.05);Nanog was slightly increased without significance(P > 0.05).Conclusions1 The expression level of TREX1 protein was low in 21 of 25 OS tissues for patients with metastasis;and 17 of 20 OS patients without metastasis showed TREX1 overexpression.That indicated the expression level of TREX1 protein was closely related to metastasis in OS patients,which could be used as a prognostic indicator for OS patients.2 Human OS MNNG/HOS CD133 ~+ subpopulation possessed low expression of TREX1,and the potential of self-renewing,multipotent differentiation,chemotherapy resistance,tumorigenicity and other cancer stem cell properties.While CD133-subpopulation showed high expression for TREX1 and did not have cancer stem cell characteristics.Hence,TREX1 is lowly expressed in OS stem cells.3 After down-regulation of TREX1 in human OS MNNG/HOS CD133-subpopulation,a number of stemness properties for this subpopulation were enhanced,such as osteogenicity and adipogenicity differentiation,chemotherapy resistance,migration,invasion and tumorigenicity in vivo etc.,but no the proliferation ability.4 After down-regulation of TREX1 in human OS MNNG/HOS CD133-subpopulation,stemness-related gene Oct4 was highly expressed,suggesting that TREX1 mediated signaling pathways probably regulated the expression of Oct4 to affect the cellular characteristics. |
