Specific Nucleolar Localization Of PHB2 And Its Promotion Of Cell Proliferation In Tumor Cells

Prohibitin 2(PHB2),as a conserved multifunctional protein,is widely expressed in eukaryotic cells and regulates the function of mitochondria by binding to PHB1,thus plays an important role in regulating various biological behaviors involving cell proliferation,and differentiation.At present,PHB2 has been shown to be involved in the tumorigenesis and tumor development.However,the biological fucntions and working mechanism of PHB2 in tumors is still not fully elucidated.Rhabdomyosarcoma(RMS)is one of the most prevalent pediatric soft tissue sarcoma,accounting for 50% of pediatric soft tissue sarcomas and it originates from mesenchymal progenitor cells.RMS expresses myogenic transcriptional factors but it is unable to undergo full-scale terminal myogenic differentiation.Our previous results show that PHB2 negatively regulates the activity of myogenic transcription factors(such as MyoD and MEF2)and thus inhibits the differentiation process of myoblasts.Does PHB2 play a role in the occurrence and development of RMS? In view of this question,our paper systematically studies the functions and underlying mechanisms of PHB2 in RMS.In addition,our preliminary results suggest that PHB2 may differ in subcellular localization and biological between certain cancer cells and their corresponding normal cells.To further investigate this difference,we execute preliminary comparative study on the biological functions of PHB2 in normal and cancer cells.To investigate the roles of PHB2 on proliferation and differentiation of RMS cells,we applied RNA interference technique to specifically knockdown the expression of PHB2 in RMS cells.Frist,we characterized influences of PHB2 knockdown on the viability of RMS cells.MTT assay showed that PHB2 knockdown significantly decreased the viability of RMS cells.Then to further delineate whether the decreased viability of RMS cells led by PHB2 knockdown is related with inhibition of cell proliferation,we used BrdU incorporation assay to detect the ability of DNA synthesis and immunofluorescence staining of Ki67,a common proliferative marker,to evaluate the rate of Ki67 positive cells and also performed plate colony formation assay.The results showed that down-regulation of PHB2 expression in RMS cells inhibited the ability of DNA synthesis,lowered Ki67 expression and positive cell rate and decreased the ability of plate colony formation.These findings revealed that down-regulation of PHB2 expression can significantly inhibit the proliferation of RMS cells.To further investigate if down-regulation of PHB2 expression affects the cycle and apoptosis of RMS cells,we performed flowcytometry and detected the levels of cell cycle-related proteins in RD cells by immunoblot analysis as well.The results showed that PHB2 knockdown inhibited the expression of cyclin E and induced G1 arrest while slightly increased proportion of apoptosis in RD cells.To further investigate detected the effect of PHB2 on the differentiation of RD cells,we detected the express level of myogenin by immunoblot and immunofluorescence staining.The results showed that PHB2 knockdown promoted the expression of myogenin and increased the rate of myogenin positive cells.Our previous work revealed that part of PHB2 seems likely localized in the nucleolus of RMS cells.To further confirm the nucleolus localization of PHB2 in RMS cells,we performed immunofluorescence to examine the co-localization of PHB2 and nucleophosmin(NPM),a nucleolus marker protein,in several RMS cell lines.The results verify that a substantial amount of PHB2 and NPM are co-localized in nucleolus in all RMS cell lines.Interestingly,there was no nucleolar localization in rat myoblast L6 cells.This findings suggest that the localization of PHB2 was abnormal in RMS cells.To investigate the universality and specificity of the nucleolar localization,we further identified subcellular localization of PHB2 in hepatocellular carcinoma(HCC)cells HepG2,human breast cancer cells MDA-MB-231,human ovarian cancer cells A2780,and human normal liver cells L02.The results demonstrated that there was no nucleolar localization of PHB2 in MDA-MB-231 and A2780 cells.Interestingly,partial nucleolar localization of PHB2 was also observed in HepG2 cells but not in L02 cells.These results indicates that partial PHB2 is specifically localized in nucleolus at least in some cancerous cells including RMS and HCC cells.Since a considerable amount of PHB2 appeared in the nucleolus of RD cells,we speculated that it may be involved in regulation of nucleolar morphology and function.To test this hypothesis,we observed the subcellular structures of siRNA-transfected RD cells by using transmission electron microscopy(TEM).As expected,a series of alterations could be found in the morphology and architecture of the nucleolus in PHB2 knockdown RD cells compared with negative control cells,i.e.nucleolar density increased,sponge-like nucleoli disappeared,and fuzzy nucleolus boundaries.To determine the effect of PHB2 knockdown on nucleolar function,we examined the transcription levels of 45 S rRNA and 18 S rRNA by real-time qPCR.The results show that both 45 S pre-rRNA and 18 S rRNA were down-regulated in siPHB2-transfected RD cells compared with control cells indicating that rRNA gene transcription was suppressed in siPHB2-transfected RD cells.It has been reported that the transcription factor proto-oncogene protein Myc and myoblast determination protein 1(MyoD)can participate in the regulation of cell proliferation by regulating the transcription of rDNA.To further investigate whether the regulation of PHB2 on rDNA transcription is related to the activity of these two transcription factors,we detect the binding of transcription factors c-Myc and MyoD to rDNA by a chromatin cross-linking and immunoprecipitation(ChIP)assay.The results indicated that occupancy of c-Myc at the rDNA promoter was attenuated by PHB2 knockdown,while more MyoD molecules bound to the rDNA promoter.These results are consistent with PHB2 knockdown to promote the differentiation of RMS cells and inhibit the proliferation of RMS cells.We further comparatively investigates the differential biological functions of PHB2 in normal and cancerous cells by using HepG2 cells and L02 cells.MTT assay showed that PHB2 knockdown significantly inhibited the viability of HepG2 cells,but not normal liver L02 cells.BrdU incorporation assay further showed that PHB2 knockdown inhibited the DNA synthesis ability of HepG2 cells,but not L02 cells.Consistently,PHB2 knockdown significantly inhibited the expression of Cyclin E while promoted the expression of p27 and p53 in HepG2 cells,but not in L02 cells.To clarify the different biological functions of PHB2 in tumor and normal cells are related to their different nucleolar localization,transmission electron microscopy was performed to observe the ultrastructure of HepG2 cells and L02 cells with PHB2 siRNA transfection.We observed that PHB2 knockdown induced a marked nucleolar pyknosis in HepG2 cells,but not in L02 cells.Moreover,real-time qPCR showed that PHB2 knockdown significantly inhibit rRNA synthesis in HepG2 cells,but not in L02,indicating that PHB2 is critical for rRNA synthesis in HCC cells.However unexpectedly,PHB2 knockdown inhibited the ability of ATP synthesis only in HepG2 cells,but not in L02 cells,though PHB2 exists in mitochondria both in HepG2 and L02 cells.In summary,for the first time our study identified a unique localization for PHB2 in RMS and other tumor cells and ectopic nucleolar-localized PHB2 in RMS,as a regulator of rDNA transcription,was found to support Myc-dependent rDNA transcription and maintain nucleolar structure which participates in sustaining the survival and proliferation of RMS cells.In addition,PHB2 via the regulation of the access of MyoD to rDNA adopts an additional mechanism to repress MyoD activities and thus keep RD cells less differentiated.Our preliminary data also showed that PHB2 might play differential roles in normal and cancerous cells.Altogether,the findings of our study suggest that PHB2 may be involved in the development of tumorigenesis and indicate a new mechanism for its function.