LAPTM4B在胶质瘤组织中表达及对细胞增殖和侵袭力的影响

Expression of LAPTM4B in glioma tissues and its effect on cell proliferation and invasion

目的检测胶质瘤组织中溶酶体相关4次跨膜蛋白B(lysosome-associated protein transmembrane-4 beta,LAPTM4B)蛋白表达,探讨其对细胞增殖和侵袭力的影响以及与患者预后的相关性。方法选取2011年6月至2015年6月在新乡市中心医院行手术切除的胶质瘤患者86例,术后随访,随访截止时间2018年6月30日。选取同期正常脑组织25例,免疫组化(SP法)检测胶质瘤和正常脑组织中LAPTM4B蛋白表达,生存分析采用Kaplan-Meier法和Log-Rank检验,影响患者预后的风险因素分析采用Cox比例风险模型分析,培养U251细胞并分为siRNA-LAPTM4B组、siRNA-NC组和空白组,实时荧光定量PCR技术检测细胞中LAPTM4B表达,MTT法和克隆形成实验检测细胞增殖能力,Transwell法检测细胞迁移和侵袭力,Western blot检测细胞中LAPTM4B、E-cadherin、N-cadherin和Vimentin蛋白表达。结果LAPTM4B蛋白在胶质瘤组织中的阳性表达率为77.91%,高于正常脑组织中的28.00%(P<0.001);LAPTM4B蛋白阳性表达率在不同WHO分级差异有统计学意义(P<0.05);Kaplan-Meier生存分析结果显示,LAPTM4B蛋白阳性表达组患者中位生存时间和累积生存率低于LAPTM4B蛋白阴性表达组(P=0.002);Cox比例风险模型分析结果显示,WHO分级和LAPTM4B蛋白表达是影响胶质瘤患者预后的风险因素(P<0.05);与siRNA-NC组和空白组比较,siRNA-LAPTM4B组细胞中LAPTM4B mRNA和蛋白相对表达量降低(P<0.05),24、48、72、96 h时光密度值及细胞克隆数、迁移细胞数、侵袭细胞数、N-cadherin和Vimentin蛋白表达量均降低,而E-cadherin蛋白表达量升高(P<0.05)。结论LAPTM4B蛋白在胶质瘤组织中高表达,促进细胞增殖和侵袭,其机制可能与上皮-间质转化有关。LAPTM4B蛋白表达高低与胶质瘤患者生存有关。

Objective To detect the protein expression of lysosome-associated protein transmembrane-4 beta(LAPTM4 B)in glioma tissues,and to determine its effect on cell proliferation and invasiveness and its correlation with prognosis.Methods A total of 86 glioma patients who underwent surgical resection in our hospital from June 2011 to June 2015 were recruited in this study,and they were followed up till June 30,2018.In the same period,25 cases of normal brain tissues were selected.Immunohistochemical assay(SP)was used to detect LAPTM4 B protein expression in gliomas and normal brain tissues.Kaplan-Meier analysis and Log-Rank test were performed for survival analysis.Cox proportional hazards model analysis was carried out for risk factors affecting prognosis.U251 cells were cultured and divided into siRNA-LAPTM4 B group,siRNA-NC group and blank group.The expression of LAPTM4 B in the cells was detected by real-time fluorescent quantitative PCR.Cell proliferation was detected by MTT assay and colony formation assay.Cell migration and invasion was detected by Transwell assay.The protein levels of LAPTM4 B,E-cadherin,N-cadherin and Vimentin were detected by Western blotting.Results The positive rate of LAPTM4 B protein was 77.91%in glioma tissues,which was higher by 28.00%than that in the normal brain tissues(P<0.001).The positive expression rate of LAPTM4 B protein had statistical difference with different WHO grades(P<0.05).Kaplan-Meier survival analysis showed that the median survival time and cumulative survival rate were significantly lower in the LAPTM4 B-positive group than the negative group(P=0.002).Cox proportional hazard model analysis indicated that WHO classification and LAPTM4 B protein expression were risk factors for prognosis of glioma patients(P<0.05).Compared with the siRNA-NC group and the blank group,the relative expression of LAPTM4 B at mRNA and protein levels were decreased in the siRNA-LAPTM4 B group(P<0.05),and the cell proliferation at 24,48,72 and 96 h,the numbers of cellular clone,migrated cells and invasive cells,and protein levels of N-cadherin and Vimentin were decreased,while the level of E-cadherin protein was increased in the siRNA-LAPTM4 B group(all P<0.05).Conclusion LAPTM4 B protein is highly expressed in glioma tissues,and promotes cell proliferation and invasion,which may be associated with epithelial-mesenchymal transition.Its expression level is correlated with prognosis of the patients.