| ObjectivesCerebral glioma is one of common malignant tumor in the central nervous system, whichtake the first place in the incidence of all nervous system tumors, accounting for about35%-60%. With the continuous development of medical technology, operation-basedcombined with postoperative individualized comprehensive treatment such as radiotherapyand chemotherapy has become a major methods of glioma therapy, but the sensitivity ofconventional chemotherapy for quite a lot of patients is poorer, previously thought to be dueto the blood brain barrier that impacted chemotherapy drugs to reach tumor cells.Studies inrecent years showed, chemotherapy effect on cerebral glioma is affected by many reasons,and chemotherapy drug resistance is one of the important reasons that lead to chemotherapyfailure. Therefore, how to improve the sensitivity of gliomas to chemotherapy drugs, andlooking for more effective therapeutic targets has gradually become the focus of cerebralglioma postoperative adjuvant therapy. Molecular genetics studies on cerebral gliomas havefound that almost all types of cerebral glioma were associated with different levels of theactivation of oncogene and down regulation or missing of tumor suppressor genes, thescholars study that gene mutation is not only involved in cancer development, is also closelyrelated with tumor drug resistance. In recent years, more and more of the gene therapy havereached late phase clinical trials, and gene targeting therapy offers a new way for thetreatment of cerebral glioma.Programmed cell death-5(PDCD5) is a newly discovered factor of promoting apoptosis,are closely related to the construction of gene deletion mutant and its related functions, andmay participate in the coding process of function of promoting apoptosis activity structuredomain. At the same time, scholars found that PDCD5also plays an important role in influence the expression of tumor tissue. It has been tested that expression level of PDCD5decreased obviously in a wide variety of tumor diseases such as gastric cancer, ovarian cancer,kidney cancer, and is closely related to prognosis of the tumor, but its expression level incerebral glioma, impacts on the occurrence and development of cerebral glioma, whether canaffect cerebral glioma chemotherapy sensitivity and the feasibility of becoming gene therapytargets of cerebral gliomas, has not been proved.In order to define the expression and effect on chemosensitivity of tumor suppressorPDCD5in cerebral glioma, develop PDCD5into a relatively safe and effective gene therapytarget to provide the experimental basis,3aspects are being studied.1. Expression and effect of tumor suppressor PDCD5in cerebral glioma.2. Expression of tumor suppressor PDCD5in cerebral glioma cell growth andchemosensitivity.Methods1. Expression and effect of tumor suppressor PDCD5in cerebral glioma.(1)86paraffin specimens who received treatment from2009.1to2012.12in thedepartment of neurosurgery in our hospital were included, and30fresh specimens and1para-carcinoma tissue of cerebral glioma who received surgery from2013.1to2014.12in thedepartment of neurosurgery in our hospital were also included(2) RT-PCR, Western Blot and Immunohistochemistry were used respectively to detectthe expression of PDCD5mRNA and protein in glioma cell lines U87, U251and primarycerebral glioma.(3) The relationship between PDCD5expression and the characteristics ofpathophysiology in cerebral glioma was analyzed, Kaplan Meier-survival curve analysis wasperformed to analyze the correlation between PDCD5low expression or missing and theprognosis.2. Expression of tumor suppressor PDCD5in cerebral glioma cell growth andchemosensitivity. (1) Using different concentrations of chemotherapeutic drugs TMZ to process gliomacell line U87and U251, cell growth curves were determined by MTT method, influence ofPDCD5expression level on glioma cell line growth rate, and the relationship between theTMZ chemotherapy sensitivity with different concentration were analyzed.(2) Gene transfection technology was used to transfect PDCD5recombinant expressionvector into cerebral glioma cell line U87, RT-PCR, Western Blot and immunohistochemistrywere used to detect PDCD5expression after transfection. Steadily cloned U87-PDCD5wasscreened to compare the cell growth rate between U87-PDCD5and non-transfected U87byMTT. The impact of exogenous PDCD5overexpression on chemosensitivity of cerebralglioma was analyzed.(3) Synthesis PDCD5specific siRNA targets were designed according to the purposegene PDCD5, and siRNA was transfected into high expression PDCD5of glioma cell lineU251and stablily transfected U87-PDCD5, RT-PCR and Western Blot were used forevaluation of jamming effect, the influence of PDCD5expression on chemotherapysensitivity of glioma cell line after siRNAsilence was analyzed.(4) Nude mice tumor-bearing model of cerebral glioma cell line U87was established,and the model were randomly divided into4groups, which were given physiological saline(control group, n=5), TMZ (TMZ group, n=5), exogenous PDCD5recombinant expressionvector (PDCD5group, n=5), TMZ+exogenous PDCD5recombinant expression vector(combined group, n=5). All nude mice were broken neck to death after treatment for20d. Thevolume and weight of tumor isstue was measured, the PDCD5expression was detected byRT-PCR, Western Blot to analyze the impact of PDCD5plus TMZ on the growth of cerebralglioma.Results1. Expression and effect of tumor suppressor PDCD5in cerebral glioma(1) Expression of PDCD5mRNAin cerebral glioma cell line, primary cerebral gliomaRT-PCR showed that glioma cell line U87and U251express PDCD5mRNA in different degree, and the relative quantitative expression in U87significantly lower than that inU251(P<0.05). Compared with para-carcinoma tissue, the expression of PDCD5mRNA inprimary cerebral glioma reduced, or even missed (P<0.05).(2) Expression of PDCD5protein in cerebral glioma cell line, primary cerebral gliomaWestern Blot showed that U87had weaker expression of PDCD5protein, significantlylower than that in U251. PDCD5protein expression in para-carcinoma tissue was higher, andlower or even missed in primary cerebral glioma. Immunohistochemical results show that, inthe para-carcinoma tissue, PDCD5positive expression was mainly in the glial cell nucleusand cytoplasm, tan grains, its distribution was characterized by obvious heterogeneity, and incerebral glioma tissue, staining of PDCD5was weak, or even negative.(3) Relationship between expression of PDCD5in cerebral glioma and pathologicalstageRT-PCR showed that relative quantitative expression of PDCD5mRNA in high-gradecerebral glioma was significantly lower than that in low-grade cerebral glioma (P<0.05).Immunohistochemical results show that, PDCD5protein expression in cerebral gliomadecreased with increased pathology classification, and the differences were statisticallysignificant (P<0.05), and has nothing to do with age, sex, histological type (P>0.05).(4) Relationship between low expression or missing of PDCD5in cerebral glioma andprognosisKaplan-Meier survival analysis showed that average survival time of all patients was19.6months (95%CI:17.3-21.8), low expression group was17.5months (95%CI:15.1-19.9), andhigh expression group was23.7months (95%CI:19.5-27.8). Log-rank test showed thatsurvival time in low expression group was significantly shorter than that in high expressiongroup (χ2=4.94, P<0.05).2. Expression of tumor suppressor PDCD5in cerebral glioma cell growth andchemosensitivity(1) Relationship between expression of PDCD5in cell growth and chemosensitivity ofcerebral gliomaMTT showed that, when the concentration of TMZ in the range of0-110μmol/L, the cell survival rate dependent on the concentration of TMZ, with the increase of TMZ concentration,relative survival rates of U87and U251cells were reduced, and U251had more obviousruducation, illustrated that the sensitivity of U251to TMZ was higher than that of U87. WhenTMZ concentration was more than110μmol/L, the survival rate changed little.(2) Impact of exogenous PDCD5expression on chemosensitivity of cerebral gliomaRT-PCR showed that, the PDCD5mRNA expression in U87-PDCD5was significantlyhigher than non-transfected U87(P<0.05). Western Blot showed that, U87-PDCD5proteinlevels were significantly higher than PDCD5without transfection of U87.Determined byMTT, the results showed that when the concentration of TMZ in the range of0-110μmol/L,the cell survival rate dependent on the concentration of TMZ, with the increase of TMZconcentration, relative survival rates of U87and U87-PDCD5cells were reduced, andU87-PDCD5had more obvious ruducation, illustrated that the sensitivity of U87-PDCD5toTMZ was higher than that of U87. When TMZ concentration was more than110μmol/L, thesurvival rate changed little.(3) Impact of exogenous PDCD5expression after siRNA silence on cell growth andchemosensitivity of cerebral gliomaRT-PCR showed that, after transfected PDCD5siRNA, PDCD5mRNA expression inU251, U87-PDCD5in cerebral glioma were significantly reduced compared with those incontrole group (P<0.05). Western Blot showed that, after transfected PDCD5siRNA, PDCD5protein expression in U251, U87-PDCD5in cerebral glioma were significantly reducedcompared with those in controle group (P<0.05). MTT showed that, the relative cell growthrate of U87-PDCD5-siRNA, U251-siRNA reduced when the concentration of TMZ increased,and they were both higher than that in control group, that was the chemosensitivity to TMZreduced.(4) Impact of combination of PDCD5and TMZ for tumor tissue growth of cerebralgliomaThe tumor volume, weight in combination group was (1.78±0.07mm3,0.21±0.03g),were significantly lower than control group (3.46±0.06mm3,0.58±0.04g), TMZ group (2.73±0.13mm3,0.37±0.06g), PDCD5group (2.34±0.11mm3,0.34±0.05g), the difference was statistical significant (P<0.05). By compared in pairs, the tumor volume and weight were, inorder, control group> TMZ, PDCD5group> combination group. There was no significantdifference between TMZ group and PDCD5group(P>0.05).RT-PCR, Western Blot showedthat, the PDCD5mRNA and protein expression in combination group and PDCD5groupwhich carried PDCD5were higher than control group and TMZ group, manifested thatPDCD5successfully expressed in tumor-burdened model.Conclusions1. PDCD5mRNA and protein expression in patients with cerebral glioma is significantlyreduced or even missing, and the expression of PDCD5decreased with increased pathologyclassification, and has nothing to do with age, sex, histological types, survival time in low ormissing expression of PDCD5of cerebral glioma patients was significantly shorter than thatof high expression.2. PDCD5overexpression can obviously inhibit the growth of glioma cells, enhancecerebral glioma chemotherapy sensitivity of chemotherapy drugs TMZ. The sensitivity ofPDCD5expression after silence to the TMZ is significantly reduced. Application of PDCD5plus TMZ can better inhibit the growth of cerebra glioma, is expected to become a new kindof clinical therapeutic targets. |
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