| [Backgrounds]Osteosarcoma is the primary malignant bone tumor that most commonly affects children,and young adults.It is a tumor of mesenchymal origin and mainly affects long bones,but may also affect other bones in the body.In China,The prevalence of OS patients accounts for about 0.2%of all malignant tumor patients,and the proportion is about three parts per million.Despite current medical methods have increased the 5-year survival rate of OS patients,the therapeutic effect is still unsatisfied.In addition,there are still many problems to be resolved in the etiology,cell biological behavior,treatment and evaluation of OS.Osteosarcoma occurs in rapid growth of bone tissue.Puberty is at a high stage of OS,which may be related to the rapid growth of bone tissue in puberty.Besides,the disease is appeared more often in males than in females and the peak of onset in female is earlier than males.The typical presentation includes onset of pain and swelling in the affected bone that it is sufficiently intense to wake the patient from sleep.The pain in OS patients is similar to the growth pain,but the former is more malignant.OS is not only easy to relapse,but also has a higher probability of metastasis in the early stage.In most OS patients,chemotherapy or radiotherapy is usually performed preoperatively or postoperatively to prevent the tumor from metastasizing to the body.However,the current standard therapies for the advancement of treatment to improve prognosis has been something of a maze.Hence,it is necessary to develop comprehensive and multidimensional therapies for OS.Currently,viral vectors,immunotherapy,anti-angiogenesis therapy and apoptosis therapy have been used in OS patients.Long noncoding RNAs(lncRNAs)are more than 200 nucleotides in length and cannot encode proteins.In addition,lncRNAs have a variety of types and they participate in various biological regulation processes.There are many quantities and effect models,including sense lncRNA,antisense lncRNA,intronic lncRNA,intergenic lncRNA and bidirectional IncRNA.Previous studies showed that IncRNAs participate in cell growth,differentiation,proliferation,cell cycle regulation and programmed cell death as well as tumor progression and metastasis,such as colon cancer,breast cancer,liver cancer and OS.LncSox4 acts as an important regulator in liver tumor-initiating cells and it can promote the self-renewal of cells via activating Sox4.However,its roles in malignant bone tumor remains largely underexplored.[Objects]This study aimed to study the role of lncSox4 in OS.Firstly,we checked the lncSox4 expression level in cells and tissues.Then,we investigated the effect of abnormal expression of lncSox4 on Mg63 cell proliferation and migration.Finally,we explored the mechanism of lncSox4 in OS by exploring the effect of lncSox4 on Wnt/β-catenin signaling pathway[Methods]Part IIn order to study the expression level of lncSox4 in OS,U-20S cell,Mg63 cell and OS tissues Tumor sample#1,Tumor sample#2 were required as research objects.Then,quantitative real-time PCR(qRT-PCR)was used to detect the expression of lncSox4 in OS cells and tissues.Furthermore,Northern blot was utilized to test the expression of lncSox4 in OS cells.Part ⅡTo study the effect of abnormal expression of lncSox4 on tumor cell proliferation and migration,silncSox4 1#,silncSox4 2#,oelncSox4 and their controls were transfected into Mg63 cell,respectively.Then,the expression of lncSox4 wasexamined by qRT-PCR.Then,the number of Ki-67+ positive in Mg63 cells was detected by flow cytometer.Finally,we identified the proliferation and migration capacity of Mg63 cells which were detected by flow cytometer and methyl thiazolyl tetrazolium(MTT)assay after silencing or overexpression of lncSox4 level.Part ⅢTo investigate the mechanism of lncSox4 in OS,firstly,we checked whether Sox4 is the down-regulator of lncSox4 in Mg63 cells by qRT-PCR.Next,we checked the roles of lncSox4 in the classic tumor-related factors(GLI1,GLI3,TCF1,CCND1,MYC1,HES1 and HEY1)using qRT-PCR and western blot.Then,after silncSox4 transfection,the expression levels of p-catenin under 5μM cycloheximide treatment for 0 h,4 h,8 h and 24h were tested by Western blot,and after oelncSox4 transfection,the expression levels of β-catenin under 5μM cycloheximide treatment for 0 h,12 h,24 h and 36h were tested by the same method.Further,the interactions between lncSox4 and β-catenin were proved by RNA pull-down experiment.Finally,the cell that the expression of lncSox4 were up-regulated/down-regulated were treated with 500 nM Wnt agonist CID11210285/1μM Wnt inhibitor IWP3,and cell viabilities were checked by MTT assay.[Results]Part ⅠqRT-PCR detection showed that the mRNA level of lncSox4 was significantly higher in U-20S and Mg63 cell lines compared with 293T cells(P<0.001).Similar results were achieved by Northern blot assay.The expressions of lncSox4 were up-regulated in U-20S and Mg63 cells.We also found that lncSox4 mRNA expression levels were strongly up-regulated in human osteosarcoma tissues compared with normal human tissues(P<0.001).Part ⅡLncSox4 expression was evaluated using qRT-PCR analysis,and the results showed that mRNA expression levels of lncSox4 were significantly down-regulated by silncSox4 1#and silncSox42#(P<0.001),while significantly up-regulated by oelncSox4 compared with negative control transfection(P<0.01).Then,the number of Ki-67+ positive Mg63 cells were significantly decreased after silncSox4 1#or silncSox4 2#transfection(P<0.001).However,the number of Ki-67+ positive Mg63 cells were significantly increased after oelncSox4 transfection(P<0.001).Further,Mg63 cells were cultured with silncSox4 1#or silncSox42#,the viability of Mg63 cells were inhibited compared with negative control(P<0.001),while was enhanced by overexpression of lncSox4(P<0.001).Finally,the effect of lncSox4 on migration of Mg63 cells was analyzed by Transwell assay.Results showed that lncSox4 silencing by silncSox4 1#or silncSox4 2#significantly attenuated the migration of Mg63 cells(P<0.01),while overexpression of lncSox4 resulted in a reverse impact(P<0.01).Part ⅢqRT-PCR detection showed that there were no influences on the Sox4 mRNA level either under lncSox4 silencing or overexpression conditions.Besides,silencing of lncSox4 decreased the mRNA levels of TCF1 and CCND1(P<0.001 or P<0.01),while overexpression of lncSox4 enhanced their mRNA levels(P<0.01).The protein levels of TCF1 and CCND1 were promoted by oelncSox4 but reduced by silncSox4.Further,the expression levels of β-catenin under CHX treatment for 24h were degraded by silncSox4 treatment.However,the expression level of β-catenin treated by CHX for 36 h was stable despite overexpression of lncSox4.RNA pull-down experiment showed that the direct combination of lncSox4 and β-catenin protected β-catenin from degradation.Finally,MTT assay results showed that the decrease of Mg63 cells viability induced by lncSox4 silencing was completely abolished by the Wnt agonist CID11210285 when the cells were cultured for 3 and 4 days(P<0.01).Moreover,the enhanced Mg63 cell viability induced by overexpression of lncSox4 was abolished by the Wnt inhibitor IWP3 after treatment for 2,3 and 4 days(P<0.01 or P<0.001).[Conclusions]In this study,we concluded that lncSox4 was highly expressed during osteosarcoma.The silencing of lncSox4 decreased the number of Ki-67+ positive Mg63 cell,inhibited the cell viability and migration,while overexpression of lncSox4 resulted in a reverse impact.LncSox4 could interact with β-catenin,and activate Wnt/β-catenin signaling pathway.Above all,lncSox4 enhanced proliferation through promoting Wnt/β-catenin pathway in osteosarcoma.The findings may provide a new perspective for for the diagnosis,treatment and prognosis of OS. |
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