The Role Of Histone Deacetylase SIRT2 In Promoting The Invasion And Metastasis Of Osteosarcoma Cells And The Molecular Mechanism

BackgroundOsteosarcoma（OS）is the most common primary malignant bone tumor and is composed of sarcomatous osteoblasts and the resulting bone-like tissues and new bone.The pathogenesis is still unclear.The incidence is slightly higher in men.It can occur at all ages,but it is most common in 15～19 years old people.In adolescents,it is the second leading cause of cancer-related deaths.Osteosarcoma usually occurs in the distal femur,proximal tibia,and proximal humerus,that is,it mainly occurs in the lower limbs,especially around the knee joint.Osteosarcoma mostly occurs in the vigorous period of bone growth and development.It is highly malignant,easy to relapse and metastasize.Therefore,it is an important tumor that seriously affects labor productivity.Early diagnosis and early treatment are important.At present,the increase of alkaline phosphatase and lactate dehydrogenase in the laboratory examination of osteosarcoma can indicate the occurrence of osteosarcoma,but imaging examination and tumor biopsy pathology are still needed.The current standard treatments for osteosarcoma include a combination of surgery,chemotherapy（such as methotrexate、adriamycin、cisplatin）,and radiation therapy.The proneness of osteosarcoma cells to invasion and metastasis is one of the main reasons of the poor prognosis.Therefore,targeting the metastasis of osteosarcoma may be a good strategy for the treatment of osteosarcoma.Sirtuins are NAD+（nicotinamide adenine dinucleotide）-dependent protein deacetylases,including a family of proteins（SIRT1-SIRT7）with homology to the silent information regulator 2（Sir2）in Saccharomyces cerevisiae.The seven sirtuins family members show diversity in subcellular localization and function.SIRT1,SIRT6 and SIRT7 are mainly located in the nucleus,and SIRT3,SIRT4 and SIRT5 are located in the mitochondria,while SIRT2 is mainly located in the cytoplasm.Current research shows that SIRT2 plays an important role in many biological processes and diseases,and is a potential therapeutic target for many diseases.In different tumor types,SIRT2 may act as an activator or inhibitor.There are many studies on SIRT2 in breast cancer,liver cancer,lung cancer,leukemia and other malignant tumors,but no relevant research on its role in the tumorigenesis and development of osteosarcoma has been reported.Epithelial-Mesenchymal Transition（EMT）is the first step for tumors to gain invasion and metastasis potentials.During the EMT process,epithelial cells lose their cell polarity,change their morphology and structure,and lose the characteristics of epithelial cells.Epithelial cells loosen contact with their neighbors,and they turned into invasive and migratory mesenchymal cells,then break through the basement membrane to the surrounding tissues.EMT transforms early tumors into aggressive malignant tumors.EMT is the key step to metastasize malignant tumors of epithelial origin.However,its role in tumors of mesenchymal origin such as osteosarcoma is still unclear.This study aims to reveal the expression and function of SIRT2 in osteosarcoma,and explores the regulation and molecular mechanism of SIRT2 on the invasion and metastasis of osteosarcoma cells through in vivo and in vitro experiments,which will help to develop new targets for the invasion and metastasis of osteosarcoma.Methods and Results1.Using real-time quantitative polymerase chain reaction（qRT-PCR）and Western blot to detect the mRNA and protein expression levels of SIRT2 in human osteoblasts hFOB1.19 and osteosarcoma cells MG63,HOS,U20S and Saos-2.We showed that SIRT2 mRNA and protein were highly expressed in osteosarcoma cells MG63 and Saos-2.Immunohistochemical staining was used to detect the expression of SIRT2 in tumor tissues of 38 osteosarcoma patients and 9 cases of normal bone and bone marrow tissues.It was shown that SIRT2 expression in osteosarcoma tissues was significantly up-regulated.2.The small interfering RNA（siRNA）of SIRT2 was used to transfect MG63 and Saos-2 cells respectively to inhibit the expression of SIRT2 in these cells.The effect of SIRT2 on the proliferation of osteosarcoma cells was explored through CCK-8 assay,and the Transwell migration and invasion assays were used to detect migration and invasion ability of osteosarcoma cells,and the wound-healing assay was used to compare the migration ability of osteosarcoma cells.The results showed that interference with SIRT2 significantly inhibited the proliferation,migration and invasion of osteosarcoma cells.We transfected the pENTER-SIRT2-C-Flag-His expression plasmid into U2OS cells（low SIRT2 expression）to up-regulate the expression of SIRT2 in U2OS osteosarcoma cells.CCK-8 experiment,Transwell migration and invasion assay and wound-healing assay were used.Our results showed that overexpression of SIRT2 promoted the proliferation,migration and invasion of osteosarcoma cells.3.We knocked down the expression of SIRT2 in MG63 and Saos-2 cells,and overexpressed SIRT2 in U2OS cells,then used Western blot to detect the expression of EMT-related proteins which were related to cancer invasion and metastasis.The results showed that after interfering with SIRT2,the expression of mesenchymal markers N-cadherin and Vimentin were decreased;matrix metalloproteinase 2（MMP-2）and MMP-9 which were used to degrade and remodel the extracellular matrix（ECM）were decreased.After overexpression of SIRT2,the expression of E-cadherin,an epithelial marker of EMT,was decreased,and the expression of mesenchymal markers N-cadherin,Vimentin,MMP2,and MMP9 were increased.The expression of N-cadherin and Vimentin was further detected by immunofluorescence.It was shown that after knocking down SIRT2,the expression of N-cadherin and Vimentin was decreased,and after overexpression of SIRT2,the expression of N-cadherin and Vimentin was increased.4.SIRT2 shRNA lentivirus was used to infect MG63 cells.After 48 hours of infection,the efficiency of lentivirus infection was observed by fluorescence microscope,and Western blot was used to detect the knockdown of SIRT2.The results showed that SIRT2 shRNA down-regulated the expression of SIRT2 in MG63 cells,confirming that an osteosarcoma cell line stably interferes with SIRT2 was established.The subcutaneous tumor formation experiment in nude mice was used to detect the tumor formation under the skin of nude mice transplanted with cells that stably interfere with SIRT2.Subcutaneous injection of MG63 cells that stably interfere with SIRT2 and control cells were performed into the left forelimb of nude mice.The tumor volume was measured to draw the growth curve of the tumor.The tumor size of the two groups of nude mice was observed and compared.It was shown that knockdown of SIRT2 significantly inhibited tumor growth.The mice were sacrificed 12 days later,and the tumors were isolated and weighed.The results showed that the volume and weight of the tumors in the SIRT2 knockdown group were significantly lower than those in the control group,indicating that the interference with SIRT2 inhibited the proliferation of osteosarcoma cells in nude mice.HE staining of tumor tissues showed that the SIRT2 knockdown group had a complete envelope,indicating that SIRT2 knockdown inhibited tumor invasion.In order to explore the role of SIRT2 in the metastasis of osteosarcoma cells in vivo,we injected osteosarcoma cells with stable knockdown of SIRT2 and control cells into the tail vein to establish an in vivo tumor metastasis model.Our results showed that the fluorescence intensity of nude mice in the control group was significantly stronger than that of the SIRT2 knockdown group,and the fluorescence was mainly distributed in the chest and abdomen.Importantly,the fluorescence intensity of the SIRT2 knockdown group was weakened,and the metastatic nodules were obviously reduced.HE staining further confirmed that in the SIRT2 knockdown group,metastatic nodules in the lung and liver were significantly reduced.5.In order to explore the mechanism by which SIRT2 promotes the invasion and metastasis of osteosarcoma cells,we used the BioGRID database and HitPredict database to search the molecules that interact with SIRT2.The results showed that SIRT2 may bind to Snail.We overexpressed SIRT2（penter-SIRT2-C-Flag-His plasmid）and Snail（pcDNA3.1-Snail-c-HA plasmid）in MG63 cells,and used SIRT2 and Snail antibodies for immunoprecipitation.The results of co-immunoprecipitation（Co-IP）proved that the SIRT2 bound to Snail.Western blot was used to detect the expression of Snail in osteosarcoma cells with high SIRT2 expression.It was shown that Snail was highly expressed in MG63 and Saos-2 cells,indicating that the expression of SIRT2 was positively correlated with the expression of Snail.Interfering with the expression of SIRT2 in MG63 and Saos-2 cells reduced the expression of Snail;while overexpressing SIRT2 in U2OS cells increased the expression of Snail,further suggesting that SIRT2 positively regulates the expression of Snail.Histochemical staining of SIRT2 and Snail of the tumor tissues was performed,and it was found that the staining of SIRT2 and Snail was weakened in the SIRT2 knockdown mice group.Next,we applied immunofluorescence experiments,and the results showed that after interference with SIRT2 expression in MG63 and Saos-2 cells,the fluorescence intensity of Snail was decreased,while overexpression of SIRT2 in U2OS cells increased the fluorescence intensity of Snail,further proved the positive regulation of SIRT2 on Snail.Immunofluorescence staining was performed on U2OS cells with SIRT2 overexpression,and it was shown that SIRT2 was mainly located in the nuclear and co-localized with Snail.6.The molecular mechanism of SIRT2 regulating Snail was further explored.The small molecule inhibitor AGK2 of SIRT2 was used to treat MG63 cells,and it was shown that the expression of Snail was reduced,indicating that SIRT2 up-regulated Snail through its deacetylase activity.The protein synthesis inhibitor cycloheximide（CHX）was used to treat MG63 cells to inhibit protein synthesis,and then treated the cells with the proteasome inhibitor MG132.We showed that the expression of Snail was significantly increased,indicating that Snail was degraded through the proteasome pathway.Cells were treated with CHX to inhibit protein synthesis,and cells were treated with AGK2,we showed the expression of Snail was decreased,further indicating that SIRT2 up-regulates Snail through its enzyme activity;combined treatment of cells with MG 132 and AGK2 inhibited the degradation of Snail,and the expression of Snail increased,indicating that SIRT2 inhibited the proteasome degradation of Snail by its enzyme activity to upregulate Snail.Conclusions and Significance1.Our research showed that histone deacetylase SIRT2 was highly expressed in osteosarcoma cells and tissues.SIRT2 was mainly located in the cytoplasm of osteosarcoma tissues.2.SIRT2 promoted the proliferation,migration and invasion of osteosarcoma cells,and up-regulated the expression of mesenchyme-related proteins N-cadherin,Vimentin and matrix metalloproteinases MMP2 and MMP9.3.Animal experiments confirmed that knockdown of SIRT2 in ostrosarcoma cells inhibited tumor growth and metastasis in nude mice.4.Mechanism studies showed that SIRT2 bound to transcription factor Snail,and their expressions were positively correlated,and SIRT2 up-regulated Snail by inhibiting Snail’s proteasome degradation.Osteosarcoma is highly aggressive,and the mechanism of its invasion and metastasis has not been fully elucidated.Mounting evidences have confirmed that Snail can induce EMT by regulating E-cadherin and mesenchymal markers,therefore,our study suggests that SIRT2 promotes the EMT process by up-regulating the transcription factor Snail,which is beneficial to the invasion and metastasis of osteosarcoma.This study clarified the role and part of the mechanism by which SIRT2 promotes the invasion and metastasis of osteosarcoma,and provides new targets and ideas for the treatment of osteosarcoma.