| Osteosarcoma(Osteosarcoma OS)is a type of bone cancer that develops from mesenchymal cells.It occurs mostly in China at 15-25 years old and is more common in the metaphysis of long bones,such as the distal end of the femur and the proximal end of the tibia.Osteosarcoma often undergoes distant metastasis through local infiltration,hematogenous metastasis,etc.,often resulting in lung metastasis,which leads to some patients with lung metastasis at the time of presentation,which is one of the main reasons for poor prognosis of osteosarcoma.Early in the development of science and technology,medical treatment of osteosarcoma with surgical amputation as the mainstream,the five-year survival rate is less than 20%,and after the amputation seriously affects the patient's appearance and limb function,also creates a pole for the patient's psychology Big damage.With the advancement of science and technology and the introduction of medical multi-modal treatment concepts,the current treatment of osteosarcoma is preoperative neoadjuvant chemotherapy-surgical treatment-postoperative neoadjuvant chemotherapy.Since the introduction of chemotherapy,the rate of limb salvage in patients with osteosarcoma Significantly improved,the 5-year survival rate has also increased to 70%,and also improved the quality of life of patients.However,there are more than ten subtypes of osteosarcoma,and the pathological mechanism for the development of osteosarcoma is still inconclusive.Therefore,it is difficult to achieve the flesh by even developing a new type of chemotherapy drug or a combination of multiple chemotherapy drugs.Accurate treatment of subtypes of tumors,and long-term chemotherapy increases the possibility of resistance to osteosarcoma cells.The treatment of osteosarcoma seems to enter a bottleneck period.With the deepening of the molecular mechanism of osteosarcoma,it is recognized that the molecular sites involved in the metastasis and recurrence of osteosarcoma can be explored and molecularly intervened to inhibit the proliferation,invasion and metastasis of osteosarcoma cells.Thereby opening up new treatment ideas for the treatment of osteosarcoma.Cathepsin B(CB)is a lysosomal cysteine protease similar in structure to papain.Recent studies have found that cathepsin B can play different roles in all stages of malignant tumors,and it is highly expressed in a variety of tumor cells,such as gastric cancer,lung cancer,oral squamous cell carcinoma,esophageal cancer,prostate cancer,colorectal Cancer,breast cancer,endometrial cancer,etc.However,reports on how cathepsin B affects the development of osteosarcoma in a molecular way are rare.Therefore,by constructing inhibitors that inhibit the expression of cathepsin B gene,we explored the effects of cathepsin B on cell function of osteosarcoma cell proliferation,invasion,migration,etc.,and further revealed the molecular mechanism of osteosarcoma metastasis and recurrence,for the treatment of osteosarcoma provide new targets for action.ObjectiveIn this study,the differences in the expression of cathepsin B protein in osteosarcoma and paracancerous tissues were studied by purchasing tumor tissue and paracancerous tissue samples from patients with osteosarcoma.We observed changes in proliferation,migration,and invasion of MG63 cells after silencing of cathepsin B to investigate the effects of cathepsin B on osteosarcoma cell function.Methods1.We used immunohistochemistry to observe differences in cathepsin B expression in osteosarcoma and adjacent tissues.2.We transfected small interfering RNA(siRNA)with specific silencing of cathepsin B gene into osteosarcoma MG63 cells by liposome 2000 transfection.The experimental groups were as follows:(1)Blank group: only transfected into liposome reagent;(2)siRNA-1 group: transfected CB-siRNA-1;(3)siRNA-2 group: transfected CB-siRNA-2;(4)siRNA-3 group: transfected CB-siRNA-3;(5)NC group: transfection of negative control-siRNA.3.We used Western blot to detect the difference of cathepsin B expression in each group after transfection,and selected the highest silencing efficiency from siRNA-1 group,siRNA-2 group and siRNA-3 group as the follow-up experimental group.4.We used CCK-8 assay and clone formation assay to detect the proliferation of MG63 cells in each group,and analyzed whether the proliferation of cells in each group was different.5.We used cell scratch assays to detect the migration of MG63 cells in each group and to analyze whether there was a difference in the migration of cells in each group.6.We used the Transwell experiment to detect the invasion of MG63 cells in each group and analyzed whether the invasion of each group was different.Result1.The expression of cathepsin B is significantly increased in osteosarcoma tissue compared with adjacent tissues.2.siRNA-1 group,siRNA-2 group and siRNA-3 group could significantly down-regulate the expression of cathepsin B in osteosarcoma MG63 cells compared with NC group and Blank group(P<0.001).The down-regulation effect was most obvious in the siRNA-2 group,which was compared with the siRNA-1 group and the siRNA-3 group(P<0.05).Therefore,the siRNA-2 group was used as a subsequent experimental group.3.In the CCK-8 experiment,the 450 nm wavelength absorbance(OD450 value)of the siRNA-2 group was significantly lower at 24 h,48h,72 h than that of the Black group,(P< 0.05),but there was no difference between 0h and 96 h.Significance(P>0.05).4.In the monoclonal formation experiment,the number of cell clones in the siRNA-2 group was significantly lower than that in the Blank group and the NC group(P<0.001).There was no significant difference between the Blank group and the NC group(P>0.05).5.The cell scratching experiment showed that the cell healing rate of siRNA-2 group was significantly lower than that of Blank group and NC group at 6h(P<0.01),and the cell healing rate was also significantly lower than that of Blank group and NC group at 12h(P<0.001).There was no significant difference in cell healing rate between the Blank group and the NC group at 6h and 12h(P>0.05).6.Transwell experiments showed that the cells in the siRNA-2 group that passed through the basement membrane of the ventricle were significantly reduced compared with the Blank group and the NC group,and the difference was statistically significant(P<0.001).Compared with the NC group,the number of cells passing through the basement membrane of the small group was not significantly different between the Black group and the NC group(P>0.05).Conclusion1.The expression of cathepsin B in osteosarcoma tissues was significantly increased.2.Cathepsin B has the ability to enhance the proliferation,invasion and migration of osteosarcoma cells. |
PDF Full Text Request