ROCK2 Promotes The Growth Of Osteosarcoma By Regulating Autophagy Level

Background and Objective:Osteosarcoma is the most common of primary bone tumors.It usually occurs in children and adolescents.It has a high degree of malignancy and a poor prognosis.At present,clinical treatment is still based on tumor resection combined with multi-drug chemotherapy,but the 5-year survival rate of patients with poor chemotherapy response or metastasis is less than 20%.Therefore,finding effective molecular targets has very important clinical significance for the treatment of osteosarcoma.ROCK2 plays an important role in the development and progression of tumors,and participates in regulating cell contraction,adhesion,migration,proliferation and apoptosis.YAP1 is the main effector of the Hippo signaling pathway.It has a key regulatory effect on cell proliferation and apoptosis,and is closely related to the occurrence of tumors.In recent years,a large number of studies have shown that the occurrence and progression of tumors are closely related to autophagy.At the same time,some studies have shown that both ROCK2 and YAP1 can participate in the regulation of autophagy,but the effects and mechanisms of both on autophagy and tumor growth in osteosarcoma are still unclear.This study focused on the expression of ROCK2 and YAP1 in osteosarcoma and the correlation between the two,and further explored the specific mechanisms of their effects on the autophagy level and growth of osteosarcoma.Method:1.qRT-PCR and Western blot were used to detect the level of ROCK2 in each osteosarcoma cell line and normal osteoblasts.The constructed shROCK2 lentivirus was infected with the osteosarcoma cell line with high ROCK2 expression level,and select the shROCK2 lentiviral cell line with the best down-regulation effect.CCK8,Ed U and cell colony formation experiments were used to detect the changes in cell proliferation ability.Then use flow cytometry to detect cell cycle and apoptosis changes.Finally,the constructed stable osteosarcoma cell lines shNC/U2-OS and shROCK2/U2-OS were respectively injected into the hips on both sides of nude mice to perform tumor formation experiments in nude mice,and observe the tumor formation and tumor growth in nude mice.2.Change the expression of ROCK2 and observe the changes in the autophagy level of osteosarcoma cells.Then,the autophagy agonist Rapamycin was added to the osteosarcoma cells whose ROCK2 was stably down-regulated,and the autophagy inhibitor 3-MA was added to the osteosarcoma cells whose ROCK2 was stably overexpressed.The expression of ROCK2,autophagy and apoptosis-related proteins were detected by Western blot,and use the Ed U experiment to analyze the changes in cell proliferation.3.Change the expression of ROCK2 and observe the change of YAP1 expression in osteosarcoma cells.Then,an osteosarcoma cell line stably transfected with MRFP-GFP-LC3 lentivirus was constructed.In the cell line,ROCK2 expression was inhibited while YAP1 was overexpressed,and ROCK2 expression was overexpressed while YAP1 expression was down-regulated.The expressions of YAP1,ROCK2 and autophagy related proteins were detected by Western blot.The changes of autophagy level were observed by double fluorescence MRFP-GFP-LC3 system and transmission electron microscopy.Finally,Co-IP was used to verify whether ROCK2 can directly bind to YAP1,thereby regulating its expression.4.qRT-PCR,Western blot and immunohistochemical experiments to analyze the expression of ROCK2 and YAP1 in osteosarcoma tissue and its corresponding adjacent tissues,and analyze the correlation between the two expressions in osteosarcoma tissue.Result:1.Down-regulate the level of ROCK2 in osteosarcoma cells U2-OS and MG-63.The results of CCK8,Ed U and cell colony formation experiments showed that the cell growth ability was weakened(p<0.01).Flow cytometry cell cycle and apoptosis experiments found that the cell cycle was blocked in the G1 phase,and the proportion of cell apoptosis increased significantly(p<0.01).The tumor formation experiment in nude mice also confirmed that the tumor size was significantly smaller than the control group after reducing the expression of ROCK2(p<0.001).2.Down-regulate the expression of ROCK2 in osteosarcoma cells and reduce the level of autophagy.On the contrary,the level of autophagy increased after overexpression of ROCK2.Our further study found that adding autophagy agonists to osteosarcoma cells with stable low expression of ROCK2 can reverse the inhibitory effect of ROCK2 downregulation on cell proliferation,while autophagy inhibitors can inhibit the enhancement of cell proliferation caused by ROCK2 overexpression(p<0.01).3.In osteosarcoma cells,the expression of ROCK2 was inhibited,and the expression level of YAP1 also decreased.On the contrary,after ROCK2 was overexpressed,the expression level of YAP1 of the cells also increased correspondingly.In osteosarcoma cells stably transfected with m RFP-GFP-LC3 lentivirus,down-regulate the expression of ROCK2 while overexpression of YAP1.Western blot,dual-fluorescence m RFP-GFP-LC3 system and transmission electron microscopy experiments proved that overexpression of YAP1 can restore the decline in autophagy caused by the decline of ROCK2.On the contrary,ROCK2 was overexpressed in osteosarcoma cells stably transfected with m RFP-GFP-LC3 lentivirus while inhibiting YAP1.Western blot,double-fluorescence m RFP-GFP-LC3 system analysis and transmission electron microscopy analysis experimental results show that YAP1 inhibition can reverse the increase in autophagy level caused by ROCK2 overexpression.Finally,a Co-IP experiment was used to verify whether ROCK2 can directly bind to YAP1 and regulate its expression.It was found that ROCK2 and YAP1 could not directly bind to each other.4.qRT-PCR and Western blot experiments showed that the expression levels of ROCK2 and YAP1 in osteosarcoma tissues were significantly higher than those in the corresponding adjacent tissues(p<0.001).The results of immunohistochemistry showed that ROCK2 was highly expressed in 71.88%(23/32)of osteosarcoma tissue specimens,while only 6.25%(2/32)of the corresponding adjacent tissues were highly expressed.YAP1 was highly expressed in 81.25%(26/32)of the osteosarcoma tissue samples,while only 9.38%(3/32)of the corresponding paracancerous tissues were highly expressed.Further analysis found that the expression levels of ROCK2 and YAP1 in osteosarcoma tissue were positively correlated(p<0.01).Conclusion:ROCK2 regulates the autophagy level of osteosarcoma by mediating the expression of YAP1,thereby promoting the growth of osteosarcoma.Our research provides a new theoretical basis for the growth of osteosarcoma and the theoretical research direction of targeted therapy.