Development and characterization of a novel method for the analysis of gene expression patterns in lymphatic endothelial cells derived from primary breast tissues.
The combined application of laser capture microdissection (LCM) and gene expression microarray analysis has rarely been used to study lymphangiogenesis because of technical obstacles. In this study, a novel method using this combined approach was developed to analyze the gene expression patterns in lymphatic endothelial cells (LECs). First, LECs were identified in frozen sections using rapid immunostaining and isolated using LCM, and then intact RNA from the LECS was purified and amplified. The expression profile was analyzed using microarray analysis, and the expression of selected genes (Sema4C and C4orf7) was evaluated by quantitative RT-PCR (qRT-PCR) and immunofluorescence assays. These results indicate that the combination of RIHC-LCM, two-round linear amplification of the small sample RNA and genome-wide microarray analysis allows gene expression profiling of tumor LECs, which provides a powerful tool for the study of molecular details in human lymphangiogenesis-related diseases, such as lymphatic metastasis of human breast cancers. These findings also suggest that the LEC-specific genes Sema4C and C4orf7 may play an important role in the oncogenesis of human breast cancer.
Wu
Mingfu
M
Cancer Biology Research Center, Tongji Hospital, 430030 Wuhan, Hubei, People's Republic of China.
Han
Lingfei
L
Shi
Yanyan
Y
Xu
Gang
G
Wei
Juncheng
J
You
Luanyin
L
Chen
Yin
Y
Zhu
Tao
T
Li
Qiong
Q
Li
Shuang
S
Meng
Li
L
Lu
Yunpin
Y
Zhou
Jianfeng
J
Wang
Shixuan
S
Ma
Ding
D
D000328:Adult; D000368:Aged; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D018076:DNA, Complementary / Q000032:analysis; D042783:Endothelial Cells / Q000378:metabolism*; D005260:Female; D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D008198:Lymph Nodes / Q000378:metabolism* / Q000473:pathology; D008207:Lymphatic Metastasis; D046228:Microarray Analysis / Q000379:methods*; D042282:Microdissection / Q000379:methods; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D012334:RNA, Neoplasm / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012334:RNA, Neoplasm