乳腺癌易感基因1蛋白在脑胶质瘤中的表达及对替莫唑胺敏感性的影响

doi:10.3969/j.issn.1674-8115.2021.01.022

摘要/Abstract

摘要：

目的·探究乳腺癌易感基因1（breast cancer susceptibility gene 1，BRCA1）蛋白在脑胶质瘤组织中的表达及对替莫唑胺（temozolomide，TMZ）敏感性的影响。方法·选择2010年1月—2017年12月于山西省肿瘤医院神经外科行手术切除治疗的脑胶质瘤患者104例作为研究组，同时选择头颅损伤行内减压手术治疗的患者30例作为对照组；采用免疫组织化学法检测BRCA1蛋白的表达；采用χ²检验分析BRCA1蛋白表达与脑胶质瘤患者临床病理特征之间的关系；采用Kaplan-Meier法计算BRCA1蛋白阳性表达和阴性表达的脑胶质瘤患者的生存率，使用Log-Rank法进行组间差异显著性检验。将脑胶质瘤细胞U251随机分为3组，分别转染shRNA-BRCA1沉默载体（sh-BRCA1组）、shNC-BRCA1空表达载体（sh-NC组）及空白对照磷酸盐缓冲液（Blank组），使用定量聚合酶链反应（quantitative PCR，qPCR）检测其转染效果；采用CCK-8法检测3组细胞的增殖活性及其对TMZ的敏感性。结果·免疫组织化学分析显示，在30例对照组标本中有23例为BRCA1阳性表达（阳性表达率为76.67%），而在104例研究组标本中有33例呈阳性（阳性表达率为31.73%），阳性表达率的组间差异具有统计学意义（P=0.009）；研究组中病理分级为Ⅰ~Ⅱ级的肿瘤组织的BRCA1蛋白阳性表达率明显高于Ⅲ~Ⅳ级（P=0.022），且BRCA1蛋白表达与患者的年龄、性别、病理类型无显著相关性；BRCA1蛋白阳性表达患者的生存率与阴性表达患者间差异无统计学意义。体外研究显示，sh-BRCA1组BRCA1 mRNA表达水平明显低于sh-NC组（P=0.037）和Blank组（P=0.035）；经转染并培养48、72 h后，sh-BRCA1组细胞的增殖活性明显高于Blank组（P=0.043，P=0.037）和sh-NC组（P=0.046，P=0.037）；当TMZ浓度为100、200 μmol/L时，sh-BRAC1组的细胞增殖活性明显高于sh-NC组（P=0.041，P=0.040）和Blank组（P=0.038，P=0.042）。结论·BRCA1蛋白在脑胶质瘤中低表达，且BRCA1的阳性表达与肿瘤病理分级相关，而与患者的年龄、性别、病理类型及生存率无关。抑制BRCA1表达能够降低脑胶质瘤细胞对TMZ的敏感性。

关键词: 脑胶质瘤, 乳腺癌易感基因1, 替莫唑胺, 生存率, 药物敏感性

Abstract:

Objective·To investigate the expression of breast cancer susceptibility gene 1 (BRCA1) protein in brain glioma tissue and its influence on the sensitivity of temozolomide (TMZ).

Methods·A total of 104 patients with brain glioma (study group) who underwent surgical resection in the Department of Neurosurgery, Shanxi Provincial Cancer Hospital from January 2010 to December 2017 were collected. And 30 patients with head injury (control group) who underwent internal decompression surgery were collected. The BRCA1 protein expressions in brain tissues of the two groups were detected by immunohistochemistry. The relationship between BRCA1 protein expression and the clinicopathological characteristics of brain glioma patients was analyzed by χ² test. The survival rate of the glioma patients with BRCA1 protein positive or negative expression was calculated by Kaplan-Meier method, and the difference between the positive patients and the negative patients was tested by Log-Rank method. The U251 cells were randomly divided into three groups, and transfected with shRNA-BRCA1 silencing vector (sh-BRCA1 group), shNC-BRCA1 empty expression vector (sh-NC group) and blank control phosphate buffer saline (Blank group), respectively. The transfection efficiency of the three groups was detected by quantitative PCR (qPCR). CCK-8 method was used to detect proliferative activity of U251 cells in the three groups and the sensitivity of TMZ.

Results·Immunohistochemistry showed that 23 of the 30 control tissues were BRCA1 positive, with a positive expression rate of 76.67%, while 33 of the 104 glioma tissues were positive, with a positive expression rate of 31.73%; the difference in the positive expression rates between the two groups was statistically significant (P=0.009). In the study group, the expression of BRCA1 protein in the tumor tissues with pathological grade Ⅰ~Ⅱ was significantly higher than that in the tumor tissues with pathological grade Ⅲ~Ⅳ (P=0.022), and there was no significant correlation between BRCA1 protein expression and the patient's age, gender and pathological type. There was no significant difference in the survival rates between BRCA1 positive and negative patients. The results of in vitro studies showed that the BRCA1 mRNA expression level in the sh-BRCA1 group was significantly lower than those in the sh-NC group (P=0.037) and the Blank group (P=0.035). After transfection and 48 and 72 h of cell culture, the cell proliferation activity of the sh-BRCA1 group was higher than those of the Blank group (P=0.043, P=0.037) and the sh-NC group (P=0.046, P=0.037); when the TMZ concentration was 100 and 200 μmol/L, the cell proliferation activity of the sh-BRAC1 group was higher than those of the sh-NC group (P=0.041, P=0.040) and the Blank group (P=0.038, P=0.042).

Conclusion·BRCA1 protein is lower expressed in brain glioma, and the positive expression of BRCA1 is related to the tumor pathological grade, but not related to the patient's age, gender, pathological type and survival rate. Inhibition of BRCA1 expression can reduce the sensitivity of brain glioma cells to TMZ.

Key words: brain glioma, breast cancer susceptibility gene 1 (BRCA1), temozolomide (TMZ), survival rate, drug sensitivity

中图分类号:

R739.41

赵晓军, 乔志刚, 梁挺宇, 安艳玲. 乳腺癌易感基因1蛋白在脑胶质瘤中的表达及对替莫唑胺敏感性的影响[J]. 上海交通大学学报(医学版), 2021, 41(1): 118-122.

Xiao-jun ZHAO, Zhi-gang QIAO, Ting-yu LIANG, Yan-ling AN. Expression of breast cancer susceptibility gene 1 protein in brain gliomas and its influence on the sensitivity of temozolomide[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(1): 118-122.

图/表 7

表1 qPCR引物序列

Tab 1 Primer sequences for qPCR

Gene	Primer sequence
BRCA1	F：5'-GCCAAGGCAAGATCTAGAGG-3'
	R：5'-GTTGCCAACACGAGCTGA-3'
β-actin	F：5'-GCTTGGAATGAGACTGCTGA-3'
	R：5'-CTGGCCATATCCACCAGAGT-3'

图1 免疫组织化学法检测BRCA1蛋白表达阴性和阳性的镜下示例(×400)Note:A. Negative expression. B. Positive expression.

Fig 1 Microscopic examples of negative and positive BRCA1 protein expression detected by immunohistochemistry (×400)

表2 BRCA1蛋白表达与研究组患者临床病理特征之间的关系

Tab 2 Relationship between BRCA1 protein expression and clinicopathological characteristics of the patients in the study group

Clinicopathological characteristics	Positive rate of BRCA1 expression/%	χ² value	P value
Age/year		0.094	0.759
<50 (n=45)	33.33
≥50 (n=59)	30.50
Gender		0.559	0.455
Male (n=56)	28.57
Female (n=48)	35.41
Pathological type
Glioblastoma (n=30)	30.00
Astrocytoma (n=48)	35.41	0.244^①	0.622^①
Oligodendroglioma (n=26)	26.92	0.065^②	0.799^②
Pathological grade		5.232	0.022
Ⅰ~Ⅱ (n=62)	40.30
Ⅲ~Ⅳ (n=42)	19.05

图2 BRCA1蛋白表达与研究组患者生存率的关系

Fig 2 Relationship between BRCA1 protein expression and survival rate of the patients in the study group

图3 细胞转染后BRCA1 mRNA的表达水平Note:①P=0.037, ②P=0.035, compared with the sh-BRCA1 group.

Fig 3 Expression level of BRCA1 mRNA after transfection

图4 抑制BRCA1表达对3组U251细胞增殖活性的影响Note:①P=0.043, ②P=0.046, ③P=0.037， compared with sh-BRCA1 group.

Fig 4 Effect of inhibition of BRCA1 expression on proliferation activity of U251 cells in the three groups

图5 不同浓度TMZ对3组U251细胞增殖活性的影响Note:①P=0.041, ②P=0.038, ③P=0.040, ④P=0.042, compared with the sh-BRCA1 group.

Fig 5 Effects of different concentrations of TMZ on proliferation activity of U251 cells in the three groups

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