ROCK2 Promotes Osteosarcoma Growth And Metastasis By Stabilizes PFKFB3 Expression

Background and Objective:Osteosarcoma is a common malignant tumor derived from bone tissue,and it is also the main type of tumor in children.It has the characteristics of high degree of malignancy,rapid growth,prone to invasion and migration,the overall prognosis of patients is poor,and the postoperative survival rate is low.Therefore,effective molecular targets are extremely important for the treatment of osteosarcoma.ROCK2 plays an important role in tumor progression.Studies have shown that it can regulate cell contraction and adhesion,invasion and migration,proliferation and apoptosis.However,its expression and mechanism in osteosarcoma are unclear.The level of glycolysis has increased significantly in the malignant progression of tumors.As a regulator of glycolysis and energy metabolism,PFKFB3 has been shown to play a key role in the development of tumors.However,little is known about the role and mechanism of PFKFB3 in osteosarcoma.The purpose of this study was to explore the expression and correlation of ROCK2 and PFKFB3 in osteosarcoma,and further to explore the mechanism of its impact on the growth and metastasis of osteosarcoma.Method:1.Immunohistochemistry,qRT-PCR and immunoblotting analysis the expression level of ROCK2 in osteosarcoma tissue.2.Select the shROCK2 plasmid with the best down-regulation effect and downregulate the expression of ROCK2 in osteosarcoma cells.Observe the effect of cell growth ability through CCK8 and EdU assays;then use flow cytometry to detect changes in cell cycle and apoptosis ratio;The effects of cell invasion and migration were detected through Wound healing and Transwell assays.Finally,we used stable transfected shNC / U2-OS,shROCK2 / U2-OS osteosarcoma cell lines to establish a human osteosarcoma subcutaneous tumor model and observe the tumor formation of nude mice.And transfected shNC / U2-OS,shROCK2 / U2-OS osteosarcoma cells through tail vein injection to observe the occurrence of metastasis of osteosarcoma cells in both lungs.3.The expression of PFKFB3 in osteosarcoma was analyzed After changing the expression of ROCK2.And then detect the expression of PFKFB3 in osteosarcoma tissue and the corresponding non-cancer tissue,and further analyze the correlation between the expression of PFKFB3 and ROCK2 in tumor tissue.Finally,PFKFB3 was up-regulated in cells with stable low expression of ROCK2 and PFKFB3 was decreased in cells over-expressing ROCK2.EdU was used to observe the growth of tumor cells,and their metastatic ability was analyzed by Transwell assay.4.Co-IP assays detected whether ROCK2 and PFKFB3 can directly combine with each other in U2-OS and MG-63 cells.On the basis of blocking the degradation and synthesis of cellular proteins,the effect of overexpression and silencing ROCK2 on the expression of PFKFB3 was detected by immunoblotting.Co-IP was used to detect the effect of overexpression and silencing of ROCK2 on the ubiquitination level of PFKFB3 in tumor cells.Result:1.Immunohistochemical results showed that the expression of ROCK2 in tumor tissues increased by 72.55%(37/51),while the corresponding in normal bone tissue was increased only 13.73%(7/51)(p <0.01).The results of qRT-PCR and immunoblotting confirmed that the expression level of ROCK2 in osteosarcoma tissue was significantly increased(p <0.01).2.After down-regulating the expression of ROCK2 in U2-OS and MG-63 cells,CCK8 and EdU experiments showed that their growth ability was reduced(p <0.01);the cell cycle was blocked in G1 phase;the percentage of apoptosis was significantly increased(p <0.05);Wound healing and Transwell assays showed that its invasion and migration ability was also significantly inhibited(p <0.01);in vivo experiments also confirmed that the number of tumor formation and lung metastasis in nude mice after reducing ROCK2 levels was less than that of the corresponding control group(p <0.05).3.After down-regulating the expression of ROCK2 in tumor cells,the expression level of PFKFB3 also decreased.On the contrary,after overexpressing ROCK2,PFKFB3 increased accordingly.The expression level of PFKFB3 in Osteosarcoma was significantly increased,and it was positively correlated with the expression level of ROCK2.In addition,we found that up-regulating the expression of PFKFB3 can reverse the decline in the proliferation and metastasis of osteosarcoma caused by silencing ROCK2,while reducing the expression of PFKFB3 can inhibit the enhancement of the proliferation and metastasis of osteosarcoma caused by overexpression of ROCK2.4.The Co-IP results confirm that ROCK2 is directly combined with PFKFB3.Further using protease inhibitor MG132 to act on osteosarcoma cells,the expression of PFKFB3 increases with the accumulation of the action time of MG132.In the case of blocking proteasome degradation,silencing and over-expression of ROCK2,there was no significant change in PFKFB3 protein expression and the degradation rate of PFKFB3 in tumor cells was significantly reduced.We further found that ROCK2 can inhibit the ubiquitination degradation process of PFKFB3 in osteosarcoma cells.Conclusion:ROCK2 promotes the growth and metastasis of osteosarcoma by stabilizing the expression of PFKFB3.Our results provide a new theoretical basis for studying the mechanism of osteosarcoma growth and metastasis,and will provide a new research direction for the prevention and treatment of osteosarcoma.