Xpression Of Dec1in Human Gliomas And Its Contribution To The Sensitivity Of Temozolomide-chemotherapy

Dec1(differentiated embryo chondrocyte expressed gene1), also named as Sharp2(split and hairy related protein2), BHLBH2(basic helix-loop-helix binding protein2) orStra13(stimulated with retinoic acid13), is a family member of bHLH (basichelix-loop-helix) transcriptional factors. This transcriptional factor has critical roles inmany cellular events such as cell differentiation and proliferation, cell-cycle arrest,inhibition of apoptosis, immuno-regulation, cellular metabolism and even circadianrhythms. Recently, increasing researches described the overexpression of Dec1in a varietyof human tumors and highlighted its contribution to oncogenesis. For example, it wasdemonstrated that stra13expression was significantly increased during the progressionfrom normal to in situ and invasive breast carcinoma and positively correlated with thetumor grade. Zheng Y et al. found that Dec1expression was significantly increased duringthe tumor progression from well differentiated to moderately differentiated, and poorlydifferentiated gastric cancer tissues. Moreover, Stra13gene was abundantly expressed in cancers of colon but not adjacent normal tissues. In renal carcinoma, Stra13acted as atarget of pVHL and its downregulated expression by pVHL indicated its role in renalcarcinogenesis. There was a closely relationship between Dec1overexpression andHIF1-α as well as carbonic anhydrase-9in non-small cell lung cancer. All these evidencessuggested crucial roles of Dec1in malignant progression. However, the profile of Dec1expression or its prognostic and therapeutic significances in human gliomas has neverbeen systematically analyzed.In the present study, we mainly investigated Dec1expression by immunohistochemistryand further analyzed the correlations of Dec1expression with clinical variables, prognosis,the response to temozolomide chemotherapy and temozolomide-induced apoptosis inhuman glioma specimens. Furthermore, we successfully constructed lentiviral expressionvector pLenti6.3-Dec1and established U87/U251cell lines with Dec1stable expression,and confirmed the effect of Dec1overexpression on response totemozolomide-chemotherapy in human gliomas in vivo.1. Expression of Dec1in human gliomas and its contribution to the sensitivity oftemozolomide-chemotherapyObjective: Dec1, a crucial cell differentiation mediator and apoptosis inhibitor, isabundantly expressed in a variety of human cancers and related to tumor malignantprogression. Because poor differentiation and low apoptosis were closely correlated withdismal survival and poor response to radio/chemo-therapy in cancer patients, we examinedprognostic value of Dec1expression and further analyzed its correlation with the responseto temozolomide-chemotherapy in glioma patients.Methods: Dec1expression was investigated by immunohistochemistry in157newlydiagnosed glioma patients and63recurrent glioblastoma patients who relapsed during thetemozolomide-chemotherapy era. Its correlations with clinical variables, prognosis and theresponse to temozolomide-chemotherapy were analyzed in the newly diagnosed gliomapatients. Dec1expression was also compared with the apoptosis index determined byTdT-mediated dUTP nick ending-labeling assay in the recurrent glioblastoma patients.Results: Dec1high-expression, which was significantly related to high pathologic grade and poor response to temozolomide-chemotherapy, could be an independentunfavorable prognostic factor and predicted a dismal survival in the newly diagnosedglioma patients. In the recurrent glioblastoma patients, there was a negative correlationbetween Dec1expression and the apoptotic index.Conclusion: Dec1was valuable both as a prognostic factor for the clinical outcome andas a predictive factor of the response to temozolomide-chemotherapy in glioma patients.2. Construction of lentiviral expression vector of Dec1by GatewayTMsystemObjective: To construct lentiviral expression vector of Dec1by GatewayTMtechnologyand establish stable expressing target gene of human glioma U251and U87cell lines.Method: Total RNA was extracted from human glioma tissues and reversed transcript toobtain object cDNA. Primers containing specific enzyme sites were designed and used toamplify full-length of target gene Dec1for recombination by PCR. The target fragment wascloned into entry vector pENTRTM3C to generate an entry clone. Target gene in entry clonewas transferred into destination vector pLenti6.3to generate an expression clonepLenti6.3-Dec1by GatewayTMtechnology. The lentiviral expression vector pLenti6.3-Dec1was packaged in293T cells and transfected into U251and U87cells. U251and U87cellswith Dec1stable expression were obtained by Blasticidin screening and Western blotidentification.Results: Obtaining of full-length of Dec1gene and constructing of lentiviral expressionvector pLenti6.3-Dec1were confirmed by PCR and sequencing. U251and U87cells withDec1stable expression were obtained by Blasticidin screening after being transfected withthe pLenti6.3-Dec1.Conclusion: Lentiviral expression vector pLenti6.3-Dec1was successfully constructedand Dec1stable expressing cells of U251and U87were established, which lays afoundation for further exploring molecular mechanism of Dec1.3. Effect of Dec1overexpression on the sensitivity of temozolomide chemotherapy inglioma cells in vivoObjective: Establish xenograft models of U87-cherry (control) and U87-Dec1gliomacells in nude mice. Following treatment of temozolomide, investigate the effect of Dec1 overexpression on chemosensitivity of temozolomide in xenograft models.Method: Tumor generation was initiated by s.c. injection of1×106proliferating stableU87cells of Dec1overexpression or cherry in each flank of a nude mouse. After generatingof palpable tumors approximately5-7days postinoculation, temozolomide was added intheir diet at50mg/kg for5consecutive days. The tumor size was measured with a slidecaliper and the tumor volume was recorded using the formula: volume=a×b2/2(a=the largerdimension, b=the smaller dimension). Tumors were removed, weighted and preparedpathological sections to detect the proliferative and apoptotic status by immunostaining ofproliferative marker Ki-67and TdT-mediated dUTP nick ending-labeling assay,respectively.Results: Following treatment of temozolomide for30days, the mean tumor weight andvolume in the U87-Dec1group (114±12.99mg and1257±382.7mm~3, respectively) wassignificantly higher than that of the U87-cherry group (44±2.63mg and468.8±193.4mm~3,respectively, P＜0.001for both). Results of the proliferative and apoptotic detectionindicated that the proliferative index in U87-Dec1group (51.6±3.74%) was significantlyhigher than that of the U87-cherry group （38.20±3.55%, t=2.600, P=0.032）; whereas theapoptotic index in U87-Dec1group (15.26±2.20%) was significantly lower than that of theU87-cherry group (44.10±2.69%, t=8.296, P＜0.0001).Conclusion: Dec1overexpression significantly attenuated the antiglioma potential oftemozolomide in vivo.