Study On The Role For Ubiquitin-conjugating Enzyme/UbcH10 In The Oncogenic Behavior Of Malignant Glioma Cells

Astrocytoma remains the most common primary neoplasm of the central nervous system and accounts for approximately 45～55%of all brain tumors.It represents a heterogenous group of diseases with different degree of malignancy from relatively indolent pilocytic astrocytomas to highly aggressive glioblastomas.Unfortunately,current therapeutic modalities including surgical resections,chemotherapy,radiotherapy or combinations can not ensure a cure and,the molecular mechanisms underlying the initiation,maintenance and progression of astrocytomas still remain largely unclarified. Hence,identification and characterization of the regulatory molecules that involved in the astrocytoma tumorigenesis may offer important targets for treatment strategies.Ubiquitin-proteasome proteolytic pathway is a major way for the post-transcriptional degradation of most proteins and is consisted of E1,E2,E3 protein and 26S proteasome. Ubiquitin-conjugating enzymes(E2) belong to a family of structurally related proteins that mediate the process of ubiquitin-dependent proteolysis,which pair with specific ubiquitin-ligase proteins(E3) and catalyze the ubiquitination of substrate proteins.As a member of the E2 family,ubiquitin-conjugating enzyme E2C(UbcH10) has drawn lots of attention lately owing to its involvement in the carcinogenesis of certain tumors.It is required for mitotic cyclins destruction and for cell cycle progression and,however,is significantly overexpressed in a variety of malignancies including breast,ovarian,thyroid, esophageal,and hepatocellular carcinomas.As a "hub gene" which has been involved in the carcinogenesis of various cancers,UbcH10 might play an important role in the malignant transformation of tumor cells.Hence,further investigation of the functional role of UbcH10 in the carcinogenesis of glioma cells may offer a better understanding of malignant behavior of glioma cells.The aim of this study is to clarify the exact role for UbcH10 in the oncogenic process of glioma cells by examining its expression pattern in glioma tissues of different grades and glioma cell lines and knocking down its expression level in glioma cell lines.This study is consisted of three main parts:the first part is to investigate the splice pattern and expression level of UbcH10 gene in astrocytomas of different grades and normal brain tissues,the second is to investigate the expression pattern of UbcH10 in glioma cell lines and its relationship with cell cycle process and finally,the effects of siRNA targeting human UbcH10 on tumor proliferation and apoptosis are evaluated in U251 glioma cells. Partâ… Expression of ubiquitin-conjugating enzyme E2C/UbcH10 in astrocytic tumorsObjective:To investigate the splice pattern and expression level of UbcH10 gene in astrocytomas of different grades and normal brain tissues.Methods:The splice pattern of UbcH10 mRNA was investigated by RT-PCR and direct sequencing in 32 astrocytic tumors and 6 normal brain tissue samples.The expression levels of UbcH10 mRNA were evaluated by real-time quantitative PCR.By using immunohistochemistry,expression levels of UbcH10 protein were assessed in 55 astrocytic tumors of different pathological grades and 7 normal brain controls.Further,the correlation between UbcH10 and Ki-67 immunoreactivity was examined with the Spearman's correlation coefficient.Finally,immunofluorescene labelling assay was employed to investigate the celluar location of UbcH10 protein in astrocytomas.Results:RT-PCR analysis revealed single bands corresponding to the calculated size of the amplified UbcH10 splice v1 in either astrocytic tumors or normal brain tissue samples using the sets of primers.Sequence analysis identified that each RT-PCR amplification product matched the published human UbcH10 v1 gene(GenBank accession nos. NM₀07019).Quantitative real time PCR analysis demonstrated elevated expression levels of UbcH10/β-actin in high-grade astrocytomas versus low-grade(64.33±60.98 vs 8.36±8.15,respectively;P=0.000) or normal brain tissues(64.33±60.98 vs 1.00±1.57, respectively;P=0.000).Further,UbcH10 immunoreactivity was predominantly detected in the cytoplasm of tumor cells,whereas no positive staining for UbcH10 was observed in normal brain tissues.Statistical analysis showed increased UbcH10 labelling index in high-grade astrocytomas versus low-grade tumors(10.53±5.79%vs 4.23±2.85%, respectively;P=0.000) or normal controls(10.53±5.79%vs 0.0±0.0%,respectively;P =0.000).Importantly,UbcH10 staining positively correlated with the proliferative activity indicated by Ki-67 staining(P＜0.001,r=0.63,by Spearman's correlation coefficient). Double immunofluorescene labelling assay demonstrated a close co-localization of UbcH10 and GFAP in the cytoplasm of glioma cells.Conclusion:In summary,we demonstrate that UbcH10 splice variant 1 is present in either astrocytomas or normal brain tissues examined,and its expression positively correlates with the degree of malignancy at both RNA and protein levels.These data suggest that overexpression of UbcH10 may serve as one important molecular mechanism that underlies the development and progression of astrocytic tumors. Partâ…¡Expression of ubiquitin-conjugating enzyme E2C/UbcH10 in U251 and SHG-44 glioma cell linesObjective:To investigate the expression pattern of UbcH10 in glioma cell lines and its relationship with cell cycle process.Methods:The splice pattern of UbcH10 mRNA was investigated by RT-PCR and direct sequencing in U251 and SHG-44 glioma cell lines.The expression level of UbcH10 mRNA was also evaluated by real-time quantitative PCR in these two glioma cell lines. Immunofluorescene labelling assay was employed to investigate the celluar location of UbcH10 protein in U251 and SHG-44 glioma cell lines.Further,UbcH10 protein expression pattern and its relationship with cell cycle were investigated at G1,S and G2/M phase which were blocked by no serum starve,thymidine and nocodazole,respectively.Results:RT-PCR analysis revealed single bands corresponding to the calculated size of the amplified UbcH10 splice variant 1(v1) in U251 and SHG-44 glioma cells.Sequence analysis identified that each RT-PCR amplification product matched the published human UbcH10 v1 gene(GenBank accession nos.NM₀07019).Quantitative real time PCR analysis demonstrated elevated expression levels of UbcH10/β-actin in U251 and SHG-44 glioma cells versus normal brain tissues(33.77±21.54,30.20±20.13 vs 1.00±1.57; P=0.000).UbcH10 immunoreactivity was predominantly detected in the cytoplasm of tumor cells by immunohistochemistry and immunofluorescene labelling assay.Western blot analysis showed increased UbcH10 expression levels in glioma cells versus normal brain tissues(0.40±0.23 vs 0.0±0.0;P＜0.05).Expression of UbcH10 protein was mainly detected in G₂/M phase of cell cycle instead of G₀ and S phase.Expression levels of UbcH10 increased in a time-dependant manner after release from double thymidine arrest, whereas expression levels of cyclin A and cyclin B1 decreased in an opposite way.After release from nocodazole arrest,expression levels of UbcH10 as well as cyclin A and cyclin B1 decreased in a time-dependant manner.Conclusion:UbcH10 splice variant 1 is present in glioma cell lines examined,and its expression levels significantly increase at both mRNA and protein levels versus normal brain tissues.The expression pattern of UbcH10 is regulated in a cell cycle-dependent manner,and its expression level reachs a peak value at G₂/M phase transition. Partâ…¢RNA interference targeting UbcH10 inhibits glioma cell proliferation,enhance cell apotosis in vitroObjective:To investigate the effects of siRNA targeting human UbcH10 on tumor proliferation and apoptosis of U251 glioma cells.Methods:Three siRNA targeting human UbcH10 were transfected into U251 glioma cells. Cell proliferative activity was assessed by MTT assay and,cell apoptosis was analyzed by flow cytometry and in situ TUNEL assay.The expression levels of cyclin A,cyclin B1, PCNA,Bax,Bcl-2 and P53 Protein were measured by western blot.Results:The expression level of UbcH10 protein was knocked down by UbcH10 siRNA as indicated by western blotting analysis.Proliferation of U251 glioma cells was significantly reduced compared with that of control at 24,48,72 and 96h post transfection.The flow cytometric analysis showed increased proportion of annexinâ…¤positive cells in UbcH10 siRNA group at 24 and 48h post transfection versus si-scrambled and untransfected groups(22.70±8.83%vs 8.00±1.67%,4.07±1.06%at 24h;12.37±2.14%vs 4.63±1.45%,4.60±1.01%at 48h post transfection).Similar results were obtained with TUNEL staining(26.75±7.56%vs 7.64±3.75%,4.33±2.10%;P＜0.05).Expression levels of cyclin A siginificantly increased at 24 and 48h after knocking down UbcH10 expression as well as cyclin B1.Expression levels of PCNA decreased significantly post transfection of UbcH10 siRNA.Expression levels of Bax siginificantly increased after knocking down UbcH10 expression,whereas expression levels of Bcl-2 decreased post transfection. Expression levels of P53 increased significantly post transfection of UbcH10 siRNA as well as the expression levels of p-P53.However,no significant difference for the expression levels of Akt,p-Akt,P38,p-P38,c-Jun and p-c-Jun was noted.Conclusion:Overall,our results indicate that siRNA transfection targeting UbcH10 can inhibit cell proliferation and induce apoptosis through a P53 dependant way.Given the strong upregulation of UbcH10 expression in malignant gliomas,knock-down of UbcH10 expression may therefore represent a potentially treatment strategy against glioma.