The Role Of TPX2in Bladder Carcinoma And Its Preliminary Molecular Mechanisms

Bladder carcinoma is the seventh most frequently occurring tumors worldwidewith approximately13,000deaths annually in the United States, and the first leadingcause of death among genitourinary malignancies in China. Urothelial cell carcinomaof the bladder (UCB), as the most common subtype of bladder cancer, accounts formore than90%of all bladder cancers. Despite considerable advances in therapystrategies and the study of biomarkers during the development and progression ofUCB, more than30%of the patients with UCB will recur and eventually progress tomuscle-invasive disease. Therefore, it is necessary to seek and identify the novelmolecular biomarkers for diagnosis or therapy of the patients with bladder carcinoma.Malignant tumor is a kind of disease with abnormal cell proliferation,differentiation and apoptosis. Unlimited proliferation ability and abnormal regulationof cell cycle become its core characterics, which is main mechanism in the occurrenceand development of tumors. Recently, many researchers have focused on theregulation of cell cycle in a variety of tumors, especially centrosome, and thesestudies elucidated the essential role of centrosome in the regulation of cell cycle andcell cycle related proteins. Therefore, centrosome related proteins become a hotspotin tumor field after cell cycle, signaling transduction and cell apoptosis. Addtionally,more and more evidence has demonstrated that centrosome will be a novel moleculartarget for therapy of tumor, which will open up new avenues for therapy of tumors.TPX2is a centrosome-related protein, which often occurs in amplification of chromosome20q of many tumors. Additionally, TPX2, a microtubule-associatedprotein, is required for microtubule assembly and formation at kinetochores inmammalian cells, which is mediated through binding of the COOH-terminal domainof Xenopus kinesin-like protein2(Xklp2) to microtubules. TPX2is downstreamregulation gene of Ran-GTP and plays a central role in spindle formation. In the earlystages of mitosis, TPX2is released in a RanGTP-dependent manner, and interactswith Aurora A kinase. This results in the localization of Aurora A to the microtubulesof the mitotic spindle, which then initiates spindle assembly. TPX2expression istightly regulated during the stages of cell cycle, becoming detectable at the G1-Stransit and disappearing at the completion of cytokinesis. Therefore, TPX2expressionmight provide a more precise evaluation of the proliferative behavior of tumor cells.In recent years, TPX2has been verified to be abnormally expressed in a varietytumors, such as as a candidate oncogene from the amplicon on20q11.2showingcopy-number-driven overexpression in non-small-cell lung cancer, the high level ofTPX2mRNA and protein in pancreatic ductal adenocarcinoma (PDAC) as well ashigh expression of TPX2in over50%of patients afflicted with giant-cell tumor of thebone. These studies suggest that TPX2may be a novel molecular target for therapy ofmany tumors. However, to date, there were no reports about the role of TPX2in theoccurrence and development of bladder carcinoma worldwide, therefore, in thepresent study, firstly, the author detected the expressions of TPX2mRNA and proteinin bladder carcinoma tissues and normal tissues by immunohistochemistry,semi-quantitative RT-PCR and real-time quantitative PCR, meanwhile, expression ofTPX2was examined in different clinical staging and metastastic status, analyzed itscorrelation with clinicopathological features, and explored its association with theprognosis of bladder carcinoma patients by follow-up, which will preliminarilyexplore its underlying function in the occurrence and development of bladdercarcinoma. Furthermore, TPX2expression was upregulated or downreglated inbladder carcinoma cells by gene gain or gene depletion, investigated the effects of itsexpression change on cell proliferation, cell cycle and cell apoptosis in bladdercarcinoma cells, and explored its possible molecular mechanisms. Finally, the effectsof TPX2expression upregulation or downregulation on bladder carcinoma implanted in nude mice were investigated. The current study will provide the novelexperimental evidence for TPX2as molecular target for therapy of the patients withbladder carcinoma.Part I Expression of TPX2in bladder carcinoma tissues and itsvalue in prognosis of the patients with bladder carcinomaMethods(1) Immunohistochemistry was utilized to detect the expression of TPX2proteinin71cases of bladder carcinoma tissues and corresponding71cases of normaltissues.(2) Semi-quantitative RT-PCR was used to investigate the expressin of TPX2mRNA in randomly selected21cases of bladder carcinoma tissues and paired normaltissues.(3) Real-time quantitative PCR was used to investigate the expressin of TPX2mRNA in randomly selected8cases of bladder carcinoma tissues and paired normaltissues.(4) Kaplan-Meier survival curve was employed to investigate the association ofTPX2protein expression with the prognosis of the patients with bladder carcinoma.(5) Statistical analysis: Statistical analysis was performed by SPSS17.0software,all data were expressed as X±S. The results of immunohistochemistry was evaluatedby chi-square, the results of semi-quantitative RT-PCR and real-time quantitativePCR were carried out by One-way ANOVA, and the association of TPX2expressionwith the prognosis of the patients with bladder carcinoma was investigated byKaplan-Meier survival curve. A P value less than0.05was considered as statisticalsignificance.Results(1) TPX2was mainly localized in cytoplasm of bladder carcinoma cells. Theresult of immunohistochemistry showed that TPX2at high level was displayed inbladder carcinoma tissues, and no expression or low expression was exhibited in normal tissues. The positive expression ratio of TPX2in71cases of bladdercarcinoma tissues (83.10%,59/71) was significantly higher than that in normal tissues(12.68%,9/71), and the differences were statistical significance (P<0.05).(2) Relative level of TPX2mRNA in bladder carcinoma tissues was0.485±0.06,which was markedly higher than that in normal tissues (0.051±0.006), and thedifferences were statistical significance (P<0.05).(3) The result of real-time quantitative PCR demonstrated that relative level ofTPX2mRNA in bladder carcinoma was obviously higher than that in normal tissues,and the differences were statistical significance (P<0.05).(4) Expression of TPX2protein was not related to the gender and age of thepatients with bladder carcinoma (P>0.05), but closely correlated with clinical staging,histological grade and lymph node metastasis (P<0.05). Most notably, positiveexpression ratio of TPX2protein was100%in19cases of the patients with metastaticbladder carcinoma.(5) TPX2mRNA level in bladder carcinoma patients with pT2-T4staging wassignificantly higher than that with pTa-T1staging, and the differences were statisticalsignificance (P<0.05).(6) TPX2mRNA level in metastatic bladder carcinoma patients was significantlyhigher than that without metastasis, and the differences were statistical significance(P<0.05).(7) The survival time of the bladder carcinoma patients with high TPX2levelwas significantly lower than that with low TPX2level or no TPX2expression, andthe differences were statistical significance (P<0.05).Part II Expression of TPX2in bladder carcinoma cells and itsregulative role in cell proliferation, cell cycle and cell apoptosisMethods(1) The relative levels of TPX2mRNA and protein were detected in bladdercarcinoma cell lines including T24, J82,5637and RT4cells by real-time quantitativePCR and Western blotting methods. (2) Empty vector pcDNA3.1, pcDNA-TPX2eukaryotic expression vector, TPX2siRNA and control siRNA were transfected to bladder carcinoma T24cells bylipofectamine2000, and800μg/ml G418was utilized to select T24cell lines stablyexpressing TPX2and pcDNA3.1.(3) Real-time quantitative PCR and Western blotting were employed to identifythe T24cells untreated or transfected.(4) The effects of TPX2overexpression or its downregulation on theproliferation of bladder carcinoma T24cells were investigated by CCK-8proliferation experiment.(5) The effects of TPX2overexpression or its downregulation on cell cycle andapoptosis of bladder carcinoma T24cells were investigated by Flow cytometry.(6) The changes of cyclin D1, cdk2and p21protein expressions was detected inT24cells untreated or transfected by Western blotting method.(7) The change of caspase-3activity was detected in T24cells untreated ortransfected by caspase-3colorimetric assay kit.(8) Statistical analysis: Statistical analysis was performed by SPSS17.0software,all data were expressed as X±S. The comparison of mutiple sample tests werecarried out by One-way ANOVA and a P value less than0.05was considered asstatistical significance.Results(1) T24cells displayed the highest TPX2mRNA and protein levels, which wassignificantly higher than J82,5637and RT4cells, and the differences were statisticalsignificance (P<0.05). In addition, RT4cells exhibited the lowest TPX2mRNA andprotein levels, compared with the other cell lines, the differences were statisticalsignificance (P<0.05).(2) The levels of TPX2mRNA and protein in pcDNA-TPX2transfection groupwere significantly higher than those in untreated group and empty vector pcDNA3.1group, and the differences were statistical significance (P<0.05). However, there wasno difference in the levels of TPX2mRNA and protein between untreated group andempty vector pcDNA3.1group (P>0.05). (3) The levels of TPX2mRNA and protein in TPX2siRNA group weresignificantly lower than those in untreated group and control siRNA group, and thedifferences were statistical significance (P<0.05). However, there was no differencein the levels of TPX2mRNA and protein between untreated group and control siRNAgroup (P>0.05).(4) TPX2-overexpressing T24cells proliferated more rapidly than untreated T24cells or pcDNA3.1cells, and the differences were statistical significance (P<0.05),however, there was no difference in proliferation ability between untreated group andpcDNA3.1group (P>0.05). Besides, TPX2-depleted T24cells grew more slowly thanuntreated T24cells or control siRNA cells, and the differences were statisticalsignificance (P<0.05), however, there was no difference in proliferation abilitybetween untreated group and control siRNA group (P>0.05).(5) The percentage of cell number in G0/G1phase in pcDNA-TPX2group wassignificantly lower than that in untreated group and empty vector pcDNA3.1group,and the differences were statistical significance (P<0.05), whereas there was nodifference in the percentage of cell number in G0/G1phase between untreated groupand empty vector pcDNA3.1group (P>0.05). There was no difference in thepercentage of cell number in S phase among three groups (P>0.05). The percentageof cell number in G2/M phase in pcDNA-TPX2group was significantly higher thanthat in untreated group and empty vector pcDNA3.1group, and the differences werestatistical significance (P<0.05). In addition, the percentage of cell number in G0/G1phase in TPX2siRNA group was significantly higher than that in untreated group andcontrol siRNA group, and the differences were statistical significance (P<0.05),whereas the percentages of cell number in S and G2/M phases in TPX2siRNA groupwas significantly lower than that in untreated group and control siRNA group, and thedifferences were statistical significance (P<0.05), however, there was no difference ineach phase of cell cycle between untreated group and control siRNA group (P>0.05).(6) The upregulation of TPX2expression significantly increased the expressionsof cyclin D1and cdk2proteins, but reduced the expression of p21protein, and thedifferences were statistical significance (P<0.05), whereas there were no difference inexpressions of proteins above between untreated group and empty vector pcDNA3.1 group (P>0.05). Further investigation demonstrated that TPX2depletion markedlydecreased the expressions of cyclin D1and cdk2proteins, but enhanced theexpression of p21protein, and the differences were statistical significance (P<0.05),whereas there were no difference in expressions of proteins above between untreatedgroup and control siRNA group (P>0.05).(7) Compared with untreated group and empty vector pcDNA3.1group, theearly apoptotic rate, late apoptotic rate and total apoptotic rate in pcDNA-TPX2group were evidently reduced, and the differences were statistical significance(P<0.05), whereas there were no difference in the early apoptotic rate, late apoptoticrate and total apoptotic rate between untreated group and empty vector pcDNA3.1group (P>0.05). Additionally, compared with untreated group and control siRNAgroup, the early apoptotic rate, late apoptotic rate and total apoptotic rate in TPX2siRNA group were evidently increased, and the differences were statisticalsignificance (P<0.05), whereas there were no difference in the early apoptotic rate,late apoptotic rate and total apoptotic rate between untreated group and control siRNAgroup (P>0.05).(8) TPX2upregulation significantly reduced the caspase-3activity in bladdercarcinoma T24cells, and the differences were statistical significance (P<0.05),whereas there were no difference in the caspase-3activity between untreated groupand empty vector pcDNA3.1group (P>0.05). Further studies revealed that TPX2downregulation significantly increased the caspase-3activity in bladder carcinomaT24cells, and the differences were statistical significance (P<0.05), whereas therewere no difference in the caspase-3activity between untreated group and controlsiRNA group (P>0.05).Part III The role of TPX2in the growth of bladder carcinomaimplanted in nude miceMethods(1) Experimental grouping: TPX2overexpression grouping: untreated group(untreated T24cells were injected subcutaneously into nude mice); empty vector pcDNA3.1group (T24cells stably expressing pcDNA3.1were injectedsubcutaneously into nude mice) and pcDNA-TPX2group (T24cells stablyexpressing TPX2were injected subcutaneously into nude mice). TPX2depletiongroupsing: untreated group (not any treatment), control siRNA group (control siRNAwas utilized to inject into the tumors) and TPX2siRNA group (TPX2siRNA wasutilized to inject into the tumors).（2）Regarding TPX2overexpressin experiment: Tumor volume was recordedevery three days after subcutaneous inoculation of tumor cells, when the record wasfinished, the tumor was stripped and the weight was measured. As for the TPX2depletion experiment: When tumor volume reached55-80mm3, control siRNA andTPX2siRNA (100ng/μl) were injected into tumors every three days, meanwhile thetumor volume was recorded, when the record was finished, the tumor was strippedand the weight was measured.（3）Expression of TPX2mRNA was detected in different bladder carcinomaimplanted in nude mice by real-time quantitative PCR methods.（4）Expression of TPX2protein was detected in different bladder carcinomaimplanted in nude mice by Western blotting methods.（5） Statistical analysis: Statistical analysis was performed by SPSS17.0software, all data were expressed as X±S. The comparison of mutiple sample testswere carried out by One-way ANOVA and a P value less than0.05was considered asstatistical significance.Results(1) The expressions of TPX2mRNA and protein in nude mice tumor tissues inpcDNA-TPX2group were significantly higher than those in untreated group andempty vector pcDNA3.1group, and the differences were statistical significance(P<0.05).(2) The expressions of TPX2mRNA and protein in nude mice tumor tissues inTPX2siRNA group were significantly lower than those in untreated group andcontrol siRNA group, and the differences were statistical significance (P<0.05).(3) Tumor weight in pcDNA-TPX2group was significantly higher than that in untreated group and empty vector pcDNA3.1group, and the differences werestatistical significance (P<0.05).(4) Tumor weight in TPX2siRNA group was significantly higher than that inuntreated group and control siRNA group, and the differences were statisticalsignificance (P<0.05).(5) TPX2upregulation significantly accelerated nuce mice tumor growth,whereas TPX2depletion markedly suppressed nude mice tumor growth.Conclusion(1) The high TPX2expression is tightly associated with the occurrence anddevelopment of bladder carcinoma.(2) TPX2may be molecular biomarker for metastasis and prognosis of bladdercarcinoma.(3) TPX2overexpression accelerates the proliferation of T24cells, shortens thepercentage of cell number in G0/G1phase and inhibits cell apoptosis. Whereas TPX2depletion suppresses the proliferation of T24cells, increases the percentage of cellnumber in G0/G1phase and induces cell apoptosis.(4) TPX2mediated the changes of cell cycle and apoptosis may be tightlycorrelated with the expression or activity of cyclin D1, cdk2, p21and caspase-3protien.(5) TPX2overexpression promotes tumor growth in nude mice and increasestumor weight, whereas TPX2depletion suppresses tumor growth in nude mice andreduces tumor weight.