Expression Of EMT In Osteosarcoma And The Effect Of IWR-1 On EMT Of Osteosarcoma Cells

Osteosarcoma is a bone cancer that occurs in children and adolescents.It mainly affects the metaphysis of the long bones and is most common in the distal femur and proximal tibia around the knee joint.The progress of osteosarcoma is rapid.Even at present,the 5-year survival rate of osteosarcoma patients remains at about 60% even though the treatment plan of“neoadjuvant chemotherapy + limb salvage surgery”is clinically improved.Therefore,looking for new methods of treatment of osteosarcoma or new targets become the main direction of clinical research.Epithelial cell interstitialisation(EMT)refers to the process of transformation from polar non-movable epithelial cells through cell phenotypic changes and changes in protein expression into non-polar and easily translocated interstitial cells.EMT is associated with tumor cells The transfer is closely related.Further study found that intracellular EMT and Wnt / β-catenin signaling pathway abnormal activation related,such as breast cancer,colorectal cancer and lymphoma.At present,the role of EMT in osteosarcoma is not clear.Therefore,this study mainly focused on the expression of EMT in osteosarcoma and the change of the growth and metastasis of osteosarcoma cells after inhibiting the Wnt / β-catenin signaling pathway,as well as the effect on the EMT process.Part I EMT related markers N-cadherin expression in osteosarcoma tissue and clinical significanceObjective: To investigate the expression of EMT marker N-cadherin in osteosarcoma and its relationship with the clinicopathological features and prognosis of osteosarcoma.Methods: The expression of N-cadherin in 37 patients with osteosarcoma was detected by immunohistochemical method,and the relationship between different levels of N-cadherin expression and clinicopathological features and prognosis of patients with osteosarcoma was investigatedResults: N-cadherin protein was positive in 29 of the 37 osteosarcoma tissues.Further analysis found: Ennecking surgical staging higher N-cadherin protein expression also increased.Of the 23 patients with metastasis,21 were positive for N-cadherin protein,with a positive rate of 91.30%.Of the 11 non-metastatic patients,6 were positive for N-cadherin with a positive rate of 54.54% The difference between the two groups was statistically significant(P <0.05).In patients with high expression of N-cadherin protein,the tumor is more malignant and more likely to metastasize.The N-cadherin protein and patient’s gender,age,tumor size,tumor type and other factors.Conclusion: The difference of expression of N-cadherin in osteosarcoma is closely related to the malignant degree and prognosis of osteosarcoma.Part II The effect of Wnt pathway inhibitor on the proliferation and metastasis in human osteosarcoma cells and the effect of EMT mechanismObjective: To investigate the effects of different concentrations of IWR-1 on the proliferation,migration and invasion of osteosarcoma and the effect of IWR-1 on the expression of EMT-related genes.Methods: Human osteosarcoma U2-OS cells were cultured and cultured in normal medium as a blank control group.U2-OS cells were stimulated with 0(DMSO group)and 5,10,15 umol / L IWR-1,0 umol/L group(DMSO group)served as the negative control group;CCK-8 method was used to detect the effect of IWR-1 and blank group on the cell proliferation;the wound healing test was used to detect the migration ability of the blank group and the different concentrations of IWR-1 cells Transwell invasion assay was used to detect the invasion ability of IWR-1 cells in different concentration groups.Western Blotting was used to detect the protein levels ofβ-catenin and MMP-2 in the blank group and IWR-1 group EMT marker E-cadherin,N-cadherin protein expression.Results: The results of CCK-8 assay showed that with the increase of IWR-1 concentration(0,5,10 and 15 μmol / L),the cell growth rate was significantly decreased and the cell proliferation inhibition rate was gradually increased(P<0.01).After the same concentration of IWR-1 was added to U2 OS cells,the increment rate increased with the extension of time(24,48,72,96h).The difference was statistically significant(P <0.05).The results of wound healing test and Transwell invasion assay showed that the change of IWR-1 concentration was negatively correlated with the migration and invasion of human osteosarcoma U2-OS cells,the difference was statistically significant(P <0.05).Western Blotting showed that the relative expression of β-catenin and MMP-2 decreased with the increase of IWR-1(0,5,10 and 15 μmol / L),and the difference was statistically significant(P <0.05);The relative expression of EMT-related protein E-cadherin gradually increased,while the relative expression of N-cadherin decreased gradually,the differences were statistically significant(P <0.05).Conclusion: Wnt/β-catenin signaling pathway inhibitor IWR-1 can inhibit the proliferation,migration and invasion of human osteosarcoma U2-OS cells by inhibiting the Wnt / β-catenin signaling pathway.At the same time,it has the effect of reversing the EMT process by decreasing the expression of N-cadherin and increasing the expression of E-cadherin.