儿童伯基特淋巴瘤中差异表达基因的鉴定及临床应用

doi:10.16150/j.1671-2870.2020.03.009

摘要/Abstract

摘要：

目的： 分析儿童伯基特淋巴瘤（Burkitt lymphoma, BL）组织与正常淋巴结组织中磷脂酰肌醇3-激酶（phosphatidylinositide3-kinase, PI3K）信号通路、核因子κB（nuclear factor kappa B,NF-κB）信号通路及RAS/RAF信号通路等相关基因在mRNA及蛋白水平上的表达差异,探讨这些基因及相关信号通路与BL发生、发展间的关系,鉴定可用于BL临床诊断、治疗的特异性分子靶标。方法： 收集18例儿童BL的组织切片和20例无癌变儿童淋巴结组织（对照）切片,用实时荧光定量反转录聚合酶链反应（reverse transcription polymerase chain reaction,RT-PCR）分析上述信号通路相关基因mRNA水平的表达情况,并用免疫组组化学技术进行蛋白水平表达的分析验证。结果： 与对照相比,MYC、TCF3、ID3、CCDN3、CDK6、CDK4、NRAS、RAF1等基因及相应的蛋白在肿瘤组织中高表达,而一些NF-κB信号转导通路目的基因（如BCL2、CD44、CCND2、C-FLIP、A20）表达则受到抑制（P<0.05）。c-MYC基因易位伴BCL2表达（BCL2⁺）的儿童BL患者其血清乳酸脱氢酶（lactate dehydrogenase,LDH）水平升高（P<0.05）。结论： MYC、TCF3、CCND3、CDK6、NRAS和RAF1等基因在儿童BL中高表达,可作为临床BL诊断和治疗监控的分子靶点;c-MYC基因易位伴BCL2⁺在儿童BL中与高LDH水平相关,其提示患儿具有较晚的肿瘤分期、较大的肿瘤负荷及不良的预后,可作为临床辅助诊断BL的重要分子指标。

关键词: 儿童伯基特淋巴瘤, 临床分子靶标, 荧光定量反转录聚合酶链反应, 免疫组织化学

Abstract:

Objective: To identify the differentially expressed genes of phosphatidylinositide3-kinase(PI3K), nuclear factor kappa-B(NF-κB) and RAS/RAF signal pathways at mRNA and protein levels in pediatric Burkitt lymphoma（BL） for exploring the correlation of these genes with the development and progression of BL and determining the specific target genes for diagnosis and treatment monitoring. Methods: Tumor tissues from 18 cases of pediatric BL and tissues of normal lymphnodes of 20 pediatric cases were collected. The expression levels of mRNA of related target genes in the pathways were analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction and the protein levels were determined by immunohistochemistry. Results: The expressions of MYC, TCF3, ID3, CCDN3, CDK6, CDK4, NRAS, RAF1 at mRNA and protein levels were up-expressed significantly in BL and some target genes and its protein of NF-κB pathway such as BCL2, CD44, C-FLIP, CCND2 and A20 were suppressed in BL when compared with normal tissues (P<0.05). Cases carrying c-MYC translocation and BCL2 expression (BCL2⁺) had higher serum LDH levels (P<0.05). Conclusions: Expressions of MYC, TCF3, CCND3, CDK6, NRAS and RAF1 genes are high,which might be used as molecular markers for the diagnosis and treatment monitoring of pediatric BL. c-MYC gene translocation accompanied with BCL2⁺ might be an important molecular indicator in BL, which is correlated with high serum LDH level, indicating advanced tumor staging, high tumor burden, and poor prognosis.

Key words: Pediatric Burkitt lymphoma, Clinical molecular target, Fluorescence quantitative reverse transcription polymerase chain reaction, Immunohistochemistry

中图分类号:

R733.4

孟磊俊, 张晶, 王雪莉, 李治, 张泓, 曾乃燕. 儿童伯基特淋巴瘤中差异表达基因的鉴定及临床应用[J]. 诊断学理论与实践, 2020, 19(03): 248-257.

MENG Leijun, ZHANG Jing, WANG Xueli, LI Zhi, ZHANG Hong, ZENG Naiyan. Identification of differentially expressed target genes in pediatric Burkitt lymphoma and its clinical application[J]. Journal of Diagnostics Concepts & Practice, 2020, 19(03): 248-257.

图/表 13

表1

实时荧光定量RT-PCR的引物序列

基因	引物	序列（5'→3'）	扩增子长度(bp)
MYC	正向反向	TAGTGGAAAACCAGCAGCCT GTCGCAGTAGAAATACGGCTG	107
TCF3	正向反向	AAGCACTGGCCTCGATCTACTC GAGGCCACTGTGACGTTCCT	111
ID3	正向反向	ATCCAGACAGCCGAGCTCAC GCGTTCTGGAGGTGTCAGGA	96
CCND3	正向反向	CAGTGCAAGGCCTGGGTG CTGAGTGCAGCTTCGATCTGCT	124
CDKN2A	正向反向	TGCCCAACGCACCGAATA CACGGGTCGGGTGAGAGTG	137
CKD6	正向反向	AAATCTTGGACGTGATTGGACTC AACACTTCAGAAGTAGGTCTTTGCCTA	153
CDK4	正向反向	ACACCCGTGGTTGTTACACTCT ACAGAAGAGAGGCTTTCGACG	132
TP53	正向反向	GTGTGGTGGTGCCCTATG CAGTGTGATGATGGTGAGGAT	128
PTEN	正向反向	TTGCAGAGTTGCACAATATCCT TCTTCACTTAGCCATTGGTCAAG	99
PTPN6	正向反向	GGAGGAGTTTGAGAGTTTGCAGA GCTGTGGTCAAAGGGGAGAA	121
A20	正向反向	AGGTTCCAGAACACCATTCC GGCTCGATCTCAGTTGCTC	151
ABIN-1	正向反向	GAAGCAAGTGGAGAAGCTGC CTCTCCTGAGGCCTTTGCT	127
ABIN-2	正向反向	GCAGATTCTCGCTTACAAGGA TCCTGTCTCCAGGACACCT	126
ABIN3	正向反向	CGCATCAGAGGCAGAGAAAGG TTCCTTCTCCTCCCGCTGC	92
BCL2	正向反向	GATAACGGAGGCTGGGATG AGCCAGGAGAAATCAAACAGAG	74
CCND2	正向反向	CTGGCCTCCAAACTCAAAGAG GCACCACCAGTTCCCACTC	109
CD44	正向反向	CAGCTCCACCTGAAGAAGATTG GGTGCCATCACGGTTAACAA	95
C-FLIP	正向反向	GACCTGCTCAAACGTATCTTGAAG CTCTGCCATCAGCACTCTATAGTC	102
IRF4	正向反向	CTTTGAGGAACTGGTTGAGCG GCTGCTTGGCTCCTTTTTTG	98
CCR2A	正向反向	GCGTTTAATCACATTCGAGTGTTT CCACTGGCAAATTAGGGAACAA	77
CD69	正向反向	CCACCAGTCCCCATTTCTCAA TTGGCCCACTGATAAGGCAAT	125
TLR6	正向反向	AACAAGTACCACAAGCTGAAG CTCTAATGTTAGCCCAAAAGAG	100
IκBɑ	正向反向	CTGAAGGCTACCAACTACAATGG CACCAAAAGCTCCACGATG	84
KRAS	正向反向	TGAGGGACCAGTACATGAGGA CCTACTAGGACCATAGGTACATCTTCAG	145
NRAS	正向反向	AGCTTGAGGTTCTTGCTGGTGT CAAAGTGGTTCTGGATTAGCTGGAT	110
RAF1	正向反向	AGAACAGTGGTCAATGTGCGA TTTACCTTTGTGTTCGTGGAGAAG	126
18S	正向反向	TGACTCAACACGGGAAACC TCGCTCCACCAACTAAGAAC	114

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名称	品牌	货号	稀释比例	抗原修复
ID3	Abcam,美国	ab2365045	1∶50	pH=6.0
TCF3	Abcam,美国	ab69999	1∶50	pH=9.0
CFLAR/C-FLIP	Proteintech,日本	10394-1-AP	1∶50	pH=6.0
BCL2	Proteintech,日本	12789-1-AP	1∶00	pH=9.0
CD44	Proteintech,日本	15675-1-AP	1∶200	pH=9.0
CDK6	Proteintech,日本	19117-1-AP	1∶200	pH=9.0
MYC	Proteintech,日本	10828-1-AP	1∶200	pH=6.0
RAF1	Proteintech,日本	51140-1-AP	1∶50	pH=6.0
CDK4	Proteintech,日本	11026-1-AP	1∶300	pH=9.0
NRAS	Proteintech,日本	10724-1-AP	1∶200	pH=9.0
Cyclin D3	Proteintech,日本	26755-1-AP	1∶200	pH=6.0
SHP1	Proteintech,日本	24546-1-AP	1∶200	pH=9.0
TNFAIP3 A	Bioss,中国	bs-2803R	1∶200	pH=6.0
Anti-Cyclin D2	Bioss,中国	bs-1148R	1∶200	pH=6.0

序号	年龄（岁）	性别	发病部位	分期	骨髓	中枢神经系统	预后	c-MYC基因易位	肿瘤溶解综合征
1	3	男	颈部	Ⅱ	未累及	未累及	良好	+	未发生
2	11	男	鼻咽部	Ⅲ	未累及	未累及	良好	-	未发生
3	4	男	鼻咽部	Ⅲ	未累及	未累及	不良	+	未发生
4	10	女	回盲部	Ⅲ	未累及	未累及	良好	-	未发生
5	4	男	十二指肠	Ⅲ	未累及	未累及	良好	+	未发生
6	5	男	鼻咽部	Ⅱ	未累及	未累及	良好	-	未发生
7	7	女	卵巢	Ⅲ	未累及	未累及	良好	-	未发生
8	5	男	腹股沟	Ⅲ	未累及	未累及	良好	+	发生
9	1	女	腹股沟	Ⅲ	未累及	未累及	良好	+	未发生
10	4	男	腹腔	Ⅲ	未累及	未累及	良好	+	未发生
11	6	男	椎管内	Ⅲ	未累及	未累及	良好	-	未发生
12	6	男	肺部	Ⅳ	累及	未累及	不良	+	未发生
13	9	男	回盲部	Ⅲ	未累及	未累及	良好	+	未发生
14	10	男	结肠	Ⅲ	未累及	未累及	不良	+	未发生
15	10	男	结肠	Ⅲ	未累及	未累及	良好	+	未发生
16	6	男	颈部	Ⅱ	未累及	未累及	良好	+	未发生
17	2	男	回盲部	Ⅳ	累及	未累及	不良	+	未发生
18	2	男	回盲部	Ⅲ	未累及	未累及	/	+	未发生

相关基因	P值
MYC^*	0.015
TCF3^*	0.007
ID3^*	0.017
CCND3^*	0.048
CDK6^*	0.001
CDK4^*	0.039
PTPN6^#	0.043
CDKN2A	0.845
PTEN	0.755
TP53	0.743

相关基因	P值
A20^#	0.016
BCL2^#	0.003
CCND2^#	0.001
C-FLIP^#	0.030
CD44^#	0.029
IRF4	0.467
ABIN1	0.531
ABIN2	0.134
ABIN3	0.873
CD69	0.157
CCR2A	0.955
IκB-ɑ	0.363
TLR6	0.614

临床特征及指标	c-MYC⁺伴BCL2⁺	c-MYC^+/-伴BCL2^+/-	P值
性别
男	5	10	0.485
女	1	2
分期
Ⅰ或Ⅱ	0	3	0.270
Ⅲ或Ⅳ	6	9
预后
良好	3	10	0.091
不良	3	1
LDH
升高	5	3	0.030
正常	1	9
UA
升高	4	3	0.264
正常	4	7
其他
LDH和UA均升高	4	1	0.022
LDH或UA任意一个指标升高或均正常	2	11