Research On Effects And Mechanism Of Rb In Human Osteosarcoma Proliferation,Migration And Invasion

Objective:To investigate the effect of Retinoblastoma(Rb)on human Osteosarcoma(OS)proliferation,migration and invasion,and to analyze its possible mechanism at the molecular level.Methods:(1)To detect the expression of Rb in normal osteoblast(hFOB1.19)and osteosarcoma cell lines(U2-OS,Saos-2 and MG-63),and to detect the the expression of Rb in normal bone tissues and human osteosarcoma tissues by using qRT-PCR.(2)To verify the biological function of Rb in virto,Saos-2 cells were transduced with lentivirus,and construct the Rb high expression and low expression models,and detect the function of affected cell proliferation(CCK-8 assay),migration(Scratch test)and invasion(Transwell assay,Western-blot assay)in the high-expression and low-expression models.(3)To investigate the function of Rb to osteosarcoma proliferation and metastasis in vivo,The tumor volume and weight of each group were measured and compared after tumor formation in nude mice.The expression levels of Rb and Ezrin genes in the tumor were verified by qRT-PCR respectively.Results:(1)The relative levels of Rb in Saos-2,MG-63 and U2-OS cells were similar and significantly lower than that in normal osteoblast(hFOB)(P <0.001).Similarly,the relative levels of Rb in OS tissues were significantly lower than that in the normal bone tissues(P <0.001).(2)Saos-2 osteosarcoma cells were transfected with a lentiviral vector,and the transfection efficiency was more than 90%.The expression of Rb was verified by using qRT-PCR(P < 0.05).(3)In vitro results revealed that Rb group could inhibit osteosarcomacell proliferation(p ≤0.001),migration(p <0.005)and invasion(p <0.005),while Sh-Rb could promote osteosarcoma cell proliferation(p <0.001),migration(p <0.05)and invasion(p <0.001).(4)The qRT-PCR results in vitro showed that the Rb group could inhibit Ezrin mRNA expression(P <0.05),and the Sh-Rb group could promote Ezrin mRNA expression(P <0.05).(5)Western-bolt results in vitro showed that the Rb group could inhibit Ezrin protein expression(P <0.05),and the Sh-Rb group could promote Ezrin protein expression(P <0.05).(6)The experimental results in vivo showed that the tumor volume(p <0.001)and weight(p <0.005)of Rb group were lower than those of Control group,and the weight(p ≤0.005)and volume(p <0.005)of nude mice in the Sh-Rb group were higher than those of Control group.(7)The expression of Ezrin in Rb group was lower than that in Control group(p <0.05),and the expression of Ezrin in Sh-Rb group was higher than that in Control group(p <0.05)by qRT-PCR.Conclusions:(1)The expression of Rb in osteosarcoma was closely correlated with the capacity of progression.(2)Rb may act as a tumor suppressor in inhibiting the invasion,migration and proliferation of osteosarcoma cells in vitro.(3)Rb may be a tumor suppressor in inhibiting the progression and metastasis in osteosarcoma in vivo.(4)Ezrin is closely related to the malignancy and invasion of osteosarcoma,and the high expression of Rb can inhibit the expression of Ezrin,while the low expression of Rb can promote the expression of Ezrin.Therefore,Rb may play a regulatory role in the upstream of Ezrin.