目的 探讨二氢嘧啶脱氢酶(DPYD)在抑郁状态小鼠脑胶质瘤中的表达及其在脑胶质瘤恶性进展中的作用.方法 采用慢性社会挫败应激实验(由10只14周龄CD-1小鼠轮流攻击C57BL/6J小鼠10 d)构建C57BI/6J小鼠(6周龄)抑郁模型(抑郁组,n=5),采用社会交互实验、糖水偏好实验和悬尾实验评价抑郁组与对照组(n=5)C57BL/6J小鼠,判断抑郁模型是否构建成功.采用立体定向技术将GL-261鼠源性胶质瘤细胞注射于两组小鼠尾状核头(各组n=5),构建脑胶质瘤原位模型,并通过7.0 T MRI观察肿瘤原位生长情况.通过RNA测序检测两组小鼠肿瘤的差异表达基因,将表达上调的差异基因匹配至中国脑胶质瘤基因组图谱计划(CGGA)(325例)和癌症基因组图谱(TCGA)(699例)数据库中,通过比较其在高级别胶质母细胞瘤与低级别脑胶质瘤中的表达差异确定目标基因.采用免疫组织化学染色法验证目标基因在小鼠脑胶质瘤组织中的表达情况.通过转染siRNA分别敲低LN229和HG9细胞株中目标基因的表达;根据转染的siRNA定义为敲低#1、#2、#3组及其对照组,并采用蛋白质免疫印迹实验检测各组目标基因的表达量.在LN229和HG9细胞株中应用CCK-8增殖实验、Transwell侵袭实验和划痕修复实验分别验证敲低目标基因对胶质瘤细胞增殖、侵袭和迁移能力的影响(n=3).结果 行为学实验结果显示,与无CD-1小鼠时比较,有CD-1小鼠时抑郁组小鼠在角落区域的停留时间延长,抑郁组较对照组糖水摄入比率下降、悬尾体位时静止时间延长(均P＜0.05),提示抑郁模型构建成功.种瘤后第20天的头颅MRI显示抑郁组脑胶质瘤体积[(292.41±8.88)cm3]较对照组[(223.22±41.44)cm3]明显增大(t=2.79,P=0.026)o两组鼠脑组织转录组测序结果显示,1 528个差异表达的基因中DPYD(差异倍数为14.51,P=0.012)等670个基因表达上调;经生物信息学分析,将DPYD作为目标基因.免疫组织化学染色结果显示,DPYD蛋白在抑郁组小鼠肿瘤中的阳性表达率高于对照组(t=4.44,P=0.016).在LN229和HG9细胞株中,与对照组比较,DPYD敲低#1、#2、#3组的DPYD表达量均下降(均P＜0.001),其中敲低#1组最为显著.在LN229和HG9细胞株中,与对照组相比,敲低#1组的划痕修复率均下降、60 h时的吸光度值均较低、穿膜细胞数量均减少(均P＜0.05).结论 脑胶质瘤进展可能与抑郁情绪相关,抑郁状态下DPYD的表达增高,敲低DPYD可显著降低胶质瘤细胞的侵袭、增殖和迁移能力.
Expression of dihydropyrimidinase deoxygenase in gliomas of depressed mice and its role in malig-nant progression of gliomas
Objective To investigate the expression of dihydropyrimidine dehydrogenase(DPYD)in brain gliomas of mice with depressive state and its role in the malignant progression of gliomas.Methods Chronic social defeat stress experiments(ten 14-week-old CD-1 mice were rotated to attack C57BL/6J mice for 10 days)were used to construct a depression model of 6-week-old C57BL/6J mice(depression group,n=5).The social interaction test,sugar water preference test,and tail suspension test were used to evaluate the C57BL/6J mice in the depression group and the control group(n=5)to determine whether the depression model was successfully constructed.GL-261 mouse-derived glioma cells were injected into the head of caudate nucleus of C57BL/6J mice in both groups using stereotactic technology(n=5 in each group)to construct an orthotopic brain glioma model,and the in situ growth of the tumor was observed by 7.0 T MRI.RNA sequencing was used to detect differentially expressed genes in the tumors of the two groups of mice,determine the target gene,and verify its expression by immunohistochemical staining.The expression of the target gene was knocked down in both LN229 and HG9 cell lines by transfection of siRNA.The transfected siRNA was defined as knockdown groups #1,#2,and #3 and their respective control groups.The effects of knocking down the target gene on glioma cell invasion,proliferation and migration were verified in LN229 and HG9 cell lines using CCK-8,Transwell chambers,and wound healing assays(n=3).Results Behavioral experiments showed that mice in the depression group spent more time in the corner,had reduced sugar water intake compared to the control group,and increased immobile time in the tail suspension position(all P＜0.05),indicating successful construction of the depression model.Head MRI on the 20th day after tumor implantation showed that the brain tumor volume in the depression group(292.41±8.88 cm3)was significantly larger than that in the control group(223.22±41.44 cm3)(t=2.79,P=0.026).Transcriptome sequencing of mouse brain tissue from both groups revealed that among 1 528 differentially expressed genes,670 genes,including DPYD(fold change:14.51,P=0.012;designated as the target gene),were upregulated.Immunohistochemical staining results showed that the positive expression rate of DPYD in tumors of mice in the depression group was higher than that in the control group(t=4.44,P=0.016).In both LN229 and HG9 cell lines,compared with the control group,the expression level of DPYD was decreased in the DPYD knockdown groups #1,#2,and #3(all P＜0.001),with the knockdown group #1 being the most significant.In both LN229 and HG9 cell lines,compared with the control group,the wound healing rate was decreased,the absorbance value at 60 h was lower,and the number of transmembrane cells was reduced in the knockdown group #1(all P＜0.05).Conclusions Glioma progression may be related to depressive mood,and increased expression of DPYD in a depressive state.The invasive,proliferative,and migratory abilities of glioma cells can significantly reduce by knocking down DPYD.
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