The Role And Mechanism Of MicroRNA-145 In Epithelial-Mesenchymal Transition,Invasion And Metastasis Of Osteosarcoma

BackgroundOsteosarcoma is the most common primary bone tumor in humans with high degree of malignancy.Invasion and distant metastasis often occur in patients with osteosarcoma.Recently,with the development of radiotherapy and chemotherapy,oncology and other medical technologies,neoadjuvant chemotherapy combined with limb salvage surgery and other comprehensive treatments are currently used to treat osteosarcoma.Surgery combined with adjuvant chemotherapy improve the cure rate,however,the survival rate of patients with metastasis are significantly reduced.Although the effect of adjuvant chemotherapy is more obvious,the patient is still prone to tumor recurrence,deterioration and distant metastasis.Therefore,we still need to find more targeted,more effective and safe treatment for osteosarcoma.MicroRNAs are a class of highly conserved,highly conserved endogenous non-coding single-stranded small RNAs with a length of approximately 18-25 bp.MicroRNA can degrade or inhibit the mRNA of its target gene during its translation through complementary binding to the 3’ untranslated region(3’UTR)of the target gene mRNA,which negatively regulate the expression of the target gene after the transcription,then participate in the regulation of a variety of cell activities.Studies have shown that the abnormal expression of miRNAs are closely linked to osteosarcoma proliferation,metastasis and prognosis and other biological behavior.For example,miR-199a-3p is significantly down-regulated in osteosarcoma cells.Upregulation of its expression in osteosarcoma cells inhibits proliferation and migration osteosarcoma cell.miR-21 is also involved in the occurrence and development of osteosarcoma,which is highly expressed in human osteosarcoma tissues.miR-21 regulates the migration and invasion of osteosarcoma cells by regulating RECK.miR-195 can inhibit the migration and invasion of osteosarcoma cell lines by negatively regulating the expression of fatty acid synthase.It is thought that miRNA is involved in epithelial-mesenchymal transition(EMT).EMT is the process that epithelial-derived tumor cells lose their epithelial phenotype to obtain mesenchymal phenotype,and the ability of invasion and metastasis.EMT is the early and crucial step of invasion and metastasis of tumor.EMT is mainly characterized by loss of tight junctions between epithelial cells and cytoskeletal rearrangements,accompanied by a decrease of E-cadherin,a marker of epithelial cells,an increase of N-cadherin and vimentin,the mesenchymal markers,and the acquirement of migration capacity.MiR-145 is widely expressed in many tissues and is considered as a tumor suppressor gene.MiR-145 is currently found low expressed in a variety of tumor tissue,such as breast cancer,prostate cancer,colon cancer,lung cancer.miR-145 can down-regulate the IRS-1 protein by binding to the 3’-UTR of IRS-1 gene to inhibit the growth of tumor cells.By directly acting on c-Myc,miR-145 can arrest tumor cells in G1 phase,promote apoptosis and inhibit cells growth,down-regulate RTKN,reduce the binding activity of Rho GTP,inhibition of cell proliferation.The latest study found that miR-145 not only can inhibit tumor growth,but also inhibit tumor cell invasion and metastasis.Objectives1.To reveal the expression of miR-145 in osteosarcoma tissue and cell line;2.To study the impact of miR-145 on the invasion and metastasis of osteosarcoma cell lines and the EMT;3.To elucidate the molecular mechanism of miR-145 involved in EMT in osteosarcoma.MethodsPart1 The expression of miR-145 in osteosarcoma tissue and cell lines1.Collected 15 cases of osteosarcoma tissue and corresponding normal adjacent tissue.2.Determined the mRNA level of miR-145 in 15 cases of osteosarcoma and its normal adjacent tissue by quantitative RT-PCR.3.Determined the mRNA level of miR-145 in osteosarcoma cell lines,U20S,MG63 and SAOS2,and normal human osteoblast cell line hFOB by quantitative RT-PCR.4.Predicted potential target genes of miR-145 using online target gene prediction software Targetscan7.0(http://www.targetscan.org/)and PicTar(http://pictar.mdc-berlin.de/).5.Determined the mRNA level of NEDD9 in 15 cases of osteosarcoma and its normal adjacent tissue by quantitative RT-PCR.6.Correlation analysis of the relative mRNA level of miR-145 and NEDD9 in osteosarcoma tissues by using Pearson method.Part 2 The role of miR-145 in the invasion,metastasis and EMT in osteosarcoma cells1.Transfected osteosarcoma cell line MG63 with miR-145 mimics by Lipofectamine 2000.MiRNA negative control and untransfected cells served as negative control and blank control,respectively.2.Detected the mRNA level of miR-145 in each group 48h after transfection by quantitative RT-PCR.3.Evaluated the proliferation ability of MG63 cells after transfection by using CCK8 experiment.4.Used Boyden chamber test to assess the invasion ability of MG63 cells after transfection.5.Examed the migration ability of MG63 cellls after transfection by using scratch test.6.Detected the mRNA level of E-cadherin,vimentin,N-cadherin and Snail 1 in each group after transfection by quantitative RT-PCR.7.Evaluated the protein expression of E-cadherin,vimentin,N-cadherin and Snail 1 in each group after transfection by Western blot.8.Inoculated MG63 cells transfected with miR-145 mimic into the axillary region of nude mice to observe the growth of transplanted tumor.MG63 cells transfected with miRNA negative control and untransfected cells served as negative control and blank control,respectively.9.Quantitative real time RT-PCR was used to detect the mRNA levels of Snail 1,E-cadherin,vimentin and N-cadherin in transplanted tumor of each group.10.Immunohistochemistry was used to detect the protein expression of E-cadherin,vimentin,N-cadherin in transplanted tumor of each group.Part 3 The regulation mechanism of miR-145 on the EMT in osteosarcoma1.Verified whether NEDD9 was the target gene of miR-145 through dual luciferase reporter gene.The wild-type(NEDD9-3’UTR-Wt)and mutant(NEDD9-3’UTR-Mut)sequences of NEDD9 3’-UTR were inserted into pmirGLO vector to construct luciferase reporter gene pmirGLO-NEDD9-3’-UTR-Wt/Mut,and then co-transfected with miR-145 inhibitor or miR-145 NC to MG63 cells.The luciferase activity in each group was detected.2.Transfected miR-145 mimics or miR-145 NC into osteosarcoma cell line MG63,then analyzed the mRNA levels and protein expression of NEDD9 by quantitative real time RT-PCR and Western blot.3.Transfected NEDD9 siRNA into MG63 cells.The cells transfected with siRNA negative and control and untransfected cells served as control.The mRNA levels and protein expression of NEDD9,E-cadherin,Snail 1,vimentin and N-cadherin were dected by using quantitative real time RT-PCR and Western blot respectively.The invasion ability of cells in each group was detected by Boyden Chamber assay.The scratch test was used to detect the migration ability of the cells in each group.4.Rescue experiments was used to further clarify whether miR-145 cells play a role in the EMT of osteosarcoma through NEDD9.The MG63 cells transfected with miR-145 inhibitor were transfected with NEDD9 siRNA or siRNA negative.The mRNA and protein levels of E-cadherin,Snail 1,vimentin and N-cadherin in each group were detected by real-time RT-PCR and Western blot.ResultsPart1 miR-145 expression was down-regulated in osteosarcoma tissue and osteosarcoma cell lines1.Quantitative real-time PCR showed that the mRNA level of miR-145 in osteosarcoma tissue was significantly lo-wer than that in normal tissue(p<0.05).2.The expression of miR-145 was significantly correlated with the distant metastasis of tumor(p<0.05).The expression of miR-145 mRNA in tumor tissue with distant metastasis was significantly higher than that without metastasis.3.Quantitative real-time PCR showed that the expression of miR-145 in 3 osteosarcoma cell lines(U30S,MG63and SAOS2)were significantly higher than that in normal human osteoblastic cell line hFOB(p<0.05).The expression of miR-145 in MG63 cell line with high ability of invasion and metastasis was significantly lower than that in U20S and SAOS2 cell lines with low invasion and metastasis ability.4.NEDD9 was predicted to be the one of the potential target genes of miR-145 through the online prediction using Targetscan7.0 and PicTar.5.The expression of NEDD9 in osteosarcoma tissue was significantly higher than that in normal tissue adjacent to cancer,and the expression of NEDD9 in osteosarcoma tissue was highly negatively correlated with miR-145.Part 2 The role of miR-145 on the invasion and metastasis of osteosarcoma cells and the EMT.1.Quantitative real-time PCR showed that the mRNA level of miR-145 in osteosarcoma cell line MG63 transfected with miR-145 mimics was significantly up-regulated compared with the miR-145 NC group and lipofectamine-treated group(p<0.05).2.The results of CCK8 test showed that compared with the miR-145 NC group and lipofectamine-treated group,the proliferation rate of miR-145 mimics transfection group was significantly inhibited 48h after transfection(p<0.05).3.The number of transmembrane cells in miR-145 mimics transfection group was was significantly lower than that in the miR-145 NC group and lipofectamine-treated group(p<0.05).4.Scratch assay showed that the scratch repair ability of miR-145 mimics transfection group was significantly lower than that of the miR-145 NC group and lipofectamine-treated group(p<0.05).5.Compared with the miR-145 NC group and lipofectamine-treated group,the mRNA level of E-cadherin in miR-145 mimics transfection group was significantly increased,while the mRNA levels of Snail 1,vimentin and N-cadherin were significantly down-regulated(p<0.05).6.Western blot showed that compared with the miR-145 NC group and lipofectamine-treated group,the protein expression of E-cadherin in miR-145 mimics transfection group was significantly increased,while the protein expressions of Snail 1,vimentin and N-cadherin were decreased.7.Nude mice tumorigenicity test showed that the tumor volume in mice inoculated with MG63 cells transfected with miR-145 mimics was significant smaller than the miR-145 NC group and lipofectamine-treated group,the difference was statistically(p<0.05),and the tumor growth rate was also significant lower than that in the two control groups.8.Quantitative real-time RT-PCR showed that the mRNA level of E-cadherin in inoculated the tumor cells transfected with miR-145 mimics group was significantly higher than that in the two control groups,while the mRNA levels of Snail 1,Vimentin and N-cadherin were statistically decreased(p<0.05).9.The results of immunohistochemistry showed that compared with the control group,the expression of E-cadherin protein was increased,and the expression of vimentin and N-cadherin protein in the tumor tissue of nude mice inoculated with miR-145 mimics transfection group were decreased.Part 3 The regulation mechanism of miR-145 in osteosarcoma EMT.1.The luciferase activity in the MG63 cells co-transfected with miR-145 mimics and pmirGLO-NEDD9-3’-UTR-Wt was significantly lower than that of the miR-145 NC group(p<0.05).The luciferase activity of cells co-transfected with miR-145 mimics and pmirGLO-NEDD9-3’-UTR-Mut showed no significant difference compared with the miR-145 NC group.2.The expression of NEDD9 in MG63 cells was significantly decreased after transfected with NEDD9 siRNA(p<0.05),indicating that the designed NEDD9 siRNA can effectively inhibit the expression of NEDD9.Meanwhile,the mRNA and protein levels of E-cadherin were significantly increased(p<0.05)and the mRNA and protein levels of Snail 1,vimentin and N-cadherin were significantly decreased(p<0.05).Boyden chamber test and scratch test showed that the invasion and migration ability of MG63 cells transfected with NEDD9 siRNA were significantly decreased compared with the siRNA NC control group(p<0.05).3.The protein expression of NEDD9 in MG63 cells transfected with miR-145 mimics was significantly lower than that in cells transfected with miR-145 NC(p<0.05).4.Rescue experimental showed that reintroduction of NEDD9 siRNA into MG63 cells transfected with miR-145 inhibitor partially rescued EMT caused by decreased miR-145.The mRNA and protein expression of E-cadherin in miR-145 inhibitor + NEDD9 siRNA group were significantly higher than those in miR-145 inhibitor transfected group,while the mRNA and protein expressions of Snail 1,vimentin and N-cadherin were significantly decreased(p<0.05).Conclusions1.The expression of miR-145 was decreased in osteosarcoma tissue.The expression level of miR-145 mRNA in tumor tissue was closely related to distant metastasis of osteosarcoma.2.The expression of miR-145 in human osteosarcoma cell lines U20S,MG63 and SAOS2 was down-regulated.The expression level of miR-145 in osteosarcoma cell lines was was closely related to the invasive ability of osteosarcoma cells.3.MiR-145 can inhibit EMT in osteosarcoma cell lines,thereby inhibited cell invasion and metastasis.4.NEDD9 was the target gene of miR-145,MiR-145 can regulate the expression of EMT in osteosarcoma cells by regulating the expression of NEDD9 3’-UTR region and regulate the invasion and metastasis of osteosarcoma cells.