MicroRNA-135b Expression Silences Ppm1e To Provoke AMPK Activation And Inhibit Osteoblastoma Cell Proliferation

Background: Osteoblastoma is one of the major causes of cancer-related mortalities among children and young teenagers around the World.Remarkable improvements have been achieved in the diagnosis and clinical treatment for osteoblastoma,and the five-year overall survival has been increased to over 70%.However,for those with advanced,malignant and/or recurrent osteoblastoma,the prognosis is still very poor.Our group has been dedicated to understanding the pathologic mechanisms of osteoblastoma tumorigenesis and progression,and developing novel anti-osteoblastoma agents.micro RNAs(miRNAs)are a group of endogenous small non-coding regulatory RNAs.Recent studies have been focusing on their role in OS progression.Objective:: Understanding the effect and underlying mechanism of miR-135b-induced inhibition on human osteosarcoma cell proliferation.To provide a molecular biological basis for the discovery of a novel and effective targeting therapy to treat this heterogeneous complex disease.Methods:1.Expression of miR-135b-5p,Ppm1 e RNA and Ppm1 e,p-AMPKα1 protein in human osteosarcoma tissues were tested by Western blotting assay and quantitative real-time(qRT-PCR)assay.2.A lentiviral miR-135b-5p expression vector was transfected to MG-63,U2 OS human osteoblastoma cells,and its effect on miR-135b-5p,Ppm1 e RNA and Ppm1 e p-AMPKα1 protein was studied,by Western blotting assay and quantitative real-time(qRT-PCR)assay.3.A lentiviral miR-135 b expression vector was transfected to human MG-63,U2 OS osteoblastoma cells,and its effect on osteosarcoma cell proliferation was tested,by cell counting kit,cell colony assay and Brd U ELISA.4.Ppm1 e shRNA was added to human MG-63 osteosarcoma cells,its effect on Ppm1e/p-AMPKα1 expression,and cell proliferation were tested,by cell counting kit,cellcolony assay and Brd U ELISA.5.AMPKα1 shRNA or dominant negative mutation vector was added to MG-63 human osteosarcoma cells to block AMPK activation,and the effect on miR-135b-5p-induced anti-osteosarcoma cell activity was tested.6.The stable U2 OS human osteosarcoma cells expressing miR-135b-5p or Ppm1 e shRNA,as well as the control human U2 OS osteosarcoma cells were inoculated into the nude mice to establish the mice model,to observe the growth of these tumors and the expression of miR-135b-5p,Ppm1 e RNA and Ppm1 e p-AMPKα1 protein was detected by quantitative real-time Western blotting and immunohistochemistry.Results:1.miR-135b-5p expression was significantly downregulated in human osteoblastoma tissues,which was correlated with Ppm1 e upregulation and AMPKα1de-phosphorylation.2.Forced-expression of miR-135b-5p in human MG-63,U2 OS osteoblastoma cells upregulated miR-135b-5p and depleted Ppm1 e,causing a profound AMPK activation(AMPKα1 phosphorylation at Thr-172).3.Human MG-63,U2 OS osteoblastoma cell proliferation was also inhibited with micro RNA-135b-5p expression.4.shRNA-mediated knockdown of Ppm1 e similarly activated AMPK signaling and inhibited human MG-63 osteoblastoma cell proliferation.5.AMPKα1 shRNA knockdown or dominant negative mutation(T172A)almost abolished miR-135b-5p’s actions in human MG-63 osteoblastoma cells.6.Compared with the nude mice model of U2 OS human osteosarcoma cells expressing Ppm1 e shRNA and Vec-miR-135 b and the control U2 OS cells,tumor growth was dramatically inhibited,the expression of Ppm1 e RNA and protein was significantly downregulated,the expression of p-AMPKα1 protein was significantly upregulated.Conclusions: miR-135b-induced Ppm1 e silence induces AMPK activation to inhibit osteoblastoma cell proliferation.