The Effects And Mechanisms Of PHB2 Silence On Tumoribiologic Characters Of Rhabdomyosarcoma Cells

PHB2(Prohibitin 2)is a highly conserved,ubiquitously expressed protein with homologues in bacteria,yeast,plant,Drosophila and mammal cells.PHB2 is involved in cell cycle regulation,cell proliferation,apoptosis,differentiation,senescence,and other important life events.Although it has been decades since its discovery,the functions of the PHB2 are still poorly understood.Rhabdomyosarcoma(RMS)is the most common pediatric soft tissue sarcoma originated from the skeletal muscle progenitor cells.It’s one of the most complicated pediatric tumors.Our previous study demonstrated that PHB2 is a negative regulator during skeletal muscle cell differentiation.PHB2 significantly inhibits the transcriptional activities of MyoD and MEF2 and then suppresses differentiation of skeletal muscle cells.While the main features of rhabdomyosarcoma’s are differentiation defects accompanied with over proliferation,we just speculate whether PHB2,a negative regulator of muscle differentiation,might play a role in the tumorigenesis and progression of rhabdomyosarcoma.Therefore,we applied RNA interference technique to induce PHB2 silence in RD cells and then observed the effects of PHB2 silence on RD cell proliferation,differentiation and migration and investigated its mechanisms as well.The main data were shown as follows:1.We confirmed that silencing of PHB2 could reduce RD cell viability by using the technology of Real-time cellular Analysis.Further FACS analysis showed that PHB2 silence could block cell cycle in the G1/S phase in RD cells.2.Soft agar colony formation assay showed that the ability of anchor-independent colony formation of RD cells was reduced following PHB2 silencing,which suggests that malignancy potential of RD cells decreased without PHB2.3.By using RT-PCR,we found that the 45 S rRNA level declined following PHB2 silencing.That indicates that nulcear PHB2 which seems to be located in nucleolus of RD cells might be involved in regulating nucleolus function.which might be another mechanism that PHB2 regulates RD cell proliferation.4.Western Blot analysis showed that the protein level of myogenin(the early marker of the muscle cell differentiation)increased after PHB2 was efficiently downregulated by siRNA,which indicates that PHB2 silence may partly enhance the differentiation of RD cells.Furthermore,reporter assay by using myogenin promoter responsive luciferase vectors showed that the myogenin promoter was activated following silencing of PHB2,which implies that PHB2 might downregulate RD cell differentiation through inhibiting the transcriptional activities of transcription factors essential for muscle differentiation,such as MyoD and MEF2.5.Transwell migration assay showed that the migration ability of RD cells wasn’t affected significantly after silencing of PHB2.