| Objective: Neuroblastoma(NB)is the most common malignant solid tumor in infancy.At present,chemotherapy resistance is a major problem in the therapy of refractory high-risk NB patients.The Akt inhibitor Perifosine can induce apoptosis of NB cells in vivo and in vitro,improve the sensitivity of NB cells to chemotherapy,and also has a promising effect in the clinical trials of NB treatment.Several studies have reported that Perifosine has other mechanism besides inhibiting Akt activation.Therefore,it is necessary to further understand the mechanism of Perifosine.Previously,we have performed global proteomics in AS cell upon Perifosine treatment.The result showed that Perifosine raised the expression of Rho-related GTP-binding protein B,RHOB.RHOB belongs to the RHO family,which are widely distributed in the cell,regulating cytoskeleton,cell adhesion,apoptosis,etc.The role of RHOB in malignant tumors is currently controversial,both tumor suppressor and carcinogenesis were reported,moreover,the effect of RHOB in NB remains elusive.The first part of this study explored the effect of RHOB on Perifosine and chemotherapeutics in AS cells.Ubiquitination is a common type of post-translational modifications and participates in almost every biological activity.In the second part of study,we performed the ubiquitylome analysis of the AS cells after Perifosine treatment,aims to get a better understanding of Perifosine mechanism,and to identify novel targets and pathways.Methods:The NB cell lines used in the first part of this study were AS,BE2,TB8 and TB3 cells.Western blot was used to mearsure the expression of RHOB in NB cells and tumor tissues upon Perifosine treatment.The immunohistochemical staining was used to detect the expression of RHOB in the tissue samples from NB and ganglioneuroma patients.Real-time PCR was used to detect RHOB mRNA in AS cells.Mice with AS-cell tumor were used for in vivo assay to detect RHOB expression in NB tissue upon Perifosine treatment.Through deep data mining of oncomine and R2 database,we compared the expression of RHOB in patients from different subgroups.To explore the effect of RHOB on Perifosine and chemotherapeutics,RHOB-siRNA or RHOB overexpression plasmid transfection was used to reduce or raise the expression of RHOB in AS cells.PI staining was used to analyse cell cycle and AnnexinV/PI staining was applied to mearsure cell apoptosis by flow cytometry.As for the cells transfected with RHOB-siRNA or RHOB overexpression plasid,we treated them with Perifosine or chemotherapeutics(Etoposide,VCR,ADR and Cisplatin)and after certain time,the cell confluence was observed dynamically by the Incucyte,and cell proliferation was detected in the MTS assay.SPSS19.0 was used to analyse data and P<0.05 was considered statistically different.The cell line used in the second part of this study was AS cells.In the ubiquitylome analysis,AS cells went through SILAC labeling,protein extraction and enzyme digestion,HPLC fractionation and affinity enrichment,and the liquid chromatography-mass spectrometry technology(LC-MS/MS)screening.We set the ratio of treatment/control group greater than 1.5 as up-regulated,the ratio less than 0.67 as down-regulated.Bioinformatic analysis was completed in differentialy expressed Kub proteins.Immunoprecipitation and western blot were used to detect the ubiquitylation level of hydroxymethyl glutaryl-coenzyme A reductase(HMGCR)in the AS cells after Perifosine treatment.Results:Part I: 1.Perifosine treatment increased the expression of RHOB in both NB cells and tissue.(1)We treated NB cells with Perifosine of different concentrations for the same duration or the same concentration but different durations.Western blot was used to detect expression of RHOB,the results showed that Perifosine increased RHOB expression,both time dependent and dose dependent.(2)Perifosine 24mg/kg/ day was administered to the mice with NB,after 8 days of Perifosine treatment,we collected the tumor tissue,and the expression of RHOB in the tumor tissue was 1.72 times of the control group(P<0.05).2.The analysis of RHOB and clinicopathological features in NB patients.(1)Ganglioneuroma,ganglioneublastoma and NB are all derived from original neuroblastic cells but differed in differentiation.The expression of RHOB in NB was relatively lower than other two tumors which were better differentiated.(2)When the clinical stage increased,the expression of RHOB gradually decreased.(3)The expression of RHOB in patients with non-amplified MycN was higher than those with MycN gene amplification.And in patients without MycN amplification,the expression of RHOB was higher in low-risk patients than high-risk ones.(4)Survival analysis showed that patients with a higher level of RHOB expression had better prognosis,while those with lower RHOB expression had significantly lower survival rate and overall survival rate.(5)In the tissue of ganglioneuroma and NB from patients in Shengjing hospital of China Medial University,we also found that the expression of RHOB was higher in ganglioneuroma than that in NB.3.Effect of RHOB on the proliferation and survival of AS cells.After silencing or overexpressing RHOB by transfection of RHOB-siRNA(#1,#2)or RHOB overexpression plasmid 24 h,we used Incucyte zoom to get dynamic observation of cell confluence and MTS assay to detect cell proliferation.The result showed that RHOB-siRNA transfection did not affect the confluence and proliferation of AS cells.RHOB overexpression could significantly inhibit the proliferation of AS cells.The confluence of RHOB overexpression group was decreased by 48%(P<0.01)and proliferation was decreased by 44.6%(P<0.01).After PI staining,cell cycle was detected and RHOB overexpression caused the increase of SubG1 phase cells by 6.7%(P<0.01).Annexin V/PI staining showed the percentages of cells in both early stage apoptosis and death were raised.In the whole,the percentage of dead cells was increased by 24.7%(P<0.01).4.The influence of RHOB on Perifosine.After transfection of RHOB-siRNA(#1,#2)or RHOB overexpression plasmid,we treated AS cells with Perfosine for 48 h and then calculated the confluence by incucyte,proliferation by MTS assay.(1)The result showed that after Perifosine treatment,the confluence of RHOB-siRNA #1 and RHOB-siRNA #2 were 55.9% and 53%,neither of them had statistical difference compared with the confluence of control group(56.3%,P>0.05).After Perifosine treatment,the proliferation of RHOB-siRNA #1 and RHOB-siRNA #2 were 42.7% and 44.9%,neither of them had statistical difference compared with the proliferation of control group(42.3%,P>0.05).(2)The result showed that after Perifosine treatment,the confluence of RHOB overexpression group was 43%,lower than the confluence of empty vector group(62.4%,P<0.01)and RHOB overexpression without Perfosine treatment group(52.0%,P<0.05).The proliferation after Perfosine treatment was 33.1%,lower than empty vector group(54.7%,P<0.01)and RHOB overexpression without Perfosine treatment group(55.4%,P<0.01).We proved that RHOB silencing was not able to block the cell death induced by Perfosine,while RHOB overexpression increased the cell death induced by Perfosine.5.Effects of RHOB on chemotherapy sensitivity of AS cells.After silencing or overexpressing RHOB,we treated AS cells with different chemotherapy drugs(etoposide,vincristine,adriamycin and cisplatin)with the duration of 48 h,through Incucyte dynamic observation,MTS assay to evaluate cells confluence and proliferation respectively.(1)After chemotherapeutics treatment,the confluence of RHOB-siRNA(#1,#2)was higher than control group(RHOB-siRNA #1,RHOB-siRNA#2 VS control group: etoposide treatment: 77.9%,60.8% VS 53.2%;VCR treatment: 70.3%,81.5% VS 55.9%;ADR treatment: 83.0%,81.0% VS 64.1%;Cisplatin treatment: 100.6%,79.2% VS 65.8%),with statistical difference(P<0.05).And the proliferation of RHOB-siRNA(#1,#2)was also higher than control group(RHOB-siRNA#1,RHOB-siRNA#2 VS control group: 0.5?g/ml etoposide treatment: 76.9%,62.6% VS 52.2%;0.5nM VCR treatment: 66.0%,74.9% VS 61.8%;0.5?M ADR treatment: 43.7%,32.4% VS 27.3%;0.25?g/ml Cisplatin treatment: 77.0%,61.1% VS 48.9%),with statistical difference(P<0.05).(2)After chemotherapeutics treatment,the confluence of RHOB overexpression group was lower than empty vector group and RHOB overexpression without chemotherapeutics treatment group(RHOB overexpression plus chemotherapeutics VS empty vector plus chemotherapeutics,RHOB overexpression without chemotherapeutics: etoposide treatment: 32.1% VS 48.5%,52.0%;VCR treatment: 32.1% VS 64.9%,52.0%;ADR treatment: 29.2% VS 50.1%,52.0%;Cisplatin treatment: 36.7% VS 79.5%,52.0%),with statistical difference(P<0.01).After chemotherapeutics treatment,the proliferation of RHOB overexpression group was lower than empty vector group and RHOB overexpression without chemotherapeutics group(RHOB overexpression plus chemotherapeutics VS empty vector plus chemotherapeutics,RHOB overexpression without chemotherapeutics: etoposide treatment: 31.1% VS 58.3%,55.4%;VCR treatment: 34.1% VS 63.4%,55.4%;ADR treatment: 20.7% VS 41.2%,55.4%;Cisplatin treatment: 35% VS 81.3%,55.4%),with statistical difference(P<0.01).We concluded that RHOB silencing promoted the chemotherapy resistance of AS cells while RHOB overexpression increased the cell death induced by chemotherapeutics.Part ii: We constructed ubiquitylation profiling of AS cells upon Perifosine treatment through LC-MS/MS and identified 3,974 lysine ubiquitylation(Kub)sites of 1,668 proteins,1658 proteins and 3,935 Kub sites were quantified.There was a total of 216 protein 297 Kub sites up-regulated and 176 protein 226 Kub sites down-regulated.2.We found six motifs that were most frequently ubiquitylated: DKub?EKub?Kub**E?Kub****D?D****Kub and D**Kub(*: any residue of amino acid,D:aspartic acid,E: glutamic acid).3.Those differentially quantified Kub proteins were mainly distributed in cytoplasm(32%)and nucleus(28%).GO analysis showed that they mainly participated in cellular process(16%),biological regulation(11%)and metabolic process(11%)with the function of binding(44%),catalytic activity(28%)and structural molecular activity(7%).In KEGG analysis,the signaling pathways of endocytosis,non-homologous end-joining,steroid biosynesis were enriched.CDC5 L and DNA-PK-Ku complex were enriched in the complex cluster analysis.We verified the result of ubiquitylome by the means of immunoprecipitation and Western blot and we found that Perifosine treatment could raise the ubiquitination level of HMGCR.Conclusions:1.Perifosine treatment increased the level of RHOB expression both in NB cells and tissue,consistent with the result of proteomics.The expression of RHOB in NB tissue was closely related with clinical stages and poor prognosis.RHOB overexpression induced apoptosis of NB cells.RHOB silencing did not affect the apoptotic induced by Perifosine while increasing RHOB expression promoted the death induced by Perifosine.RHOB silencing caused chemotherapy resistance(Etoposide,VCR,ADR and Cisplatin),while increasing RHOB expression promoted the death induced by chemotherapeutics.2.The ubiquitylation profiling of AS cells upon Perifosine treatment has been completed.Positions and proteins with differential ubiquitylation were identified,and we verified the result through immunoprecipitation and Western blot.Ubiquitylome expanded our understanding of Perifosine mechanism,paved the way for further studies. |
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