The proteome approach, applied in this study is of clinical importance to identify the differentially expressed proteins in NLN and SLNMM of CRC, since these proteins can be potentially used as tumor markers and anticancer targets.
We marked SLN using isosulfan blue and identified micrometastasis of CRC with HE staining and CK-IHC. The total positive rate was 42.85%, which is consistent with the reported data[20
]. A total of 40 proteins were differentially expressed in NLN and SLNMM. Of these 40 proteins, 15 were up-regulated and 25 were down-regulated. The 15 proteins with their expression up-regulation were then divided into 3 groups according to their functions. Western blotting and immunohistochemistry analysis showed that the expression and distribution of 4 metastasis-concerned proteins in NLN and SLNMM were significantly different.
The Annexins are a family of calcium-regulated phospholipid-binding proteins with a diverse role in cell biology[21
]. To date, 12 Annexins have been found in higher vertebrates. Although no exact physiological function of Annexins has been described, there is evidence that they are differentially expressed in various carcinomas. For example, expression of Annexins at mRNA and protein level is sharply up-regulated in many cancers[22,23
], while some data indicate that declined expression of Annexins may play a significant role in tumorigenesis and metastasis[24
]. So, the precise role of Annexin expression in pathogenesis of tumors is still unknown. In this study, Western blotting and IHC showed that the expression level of Annexin A1 was significantly higher in SLNMM than in NLN, suggesting that up-regulated expression of Annexin A1 may contribute to early CRC metastasis.
Ezrin, a membrane-cytoskeleton anchor, can affect cell adhesion and regulate tumor cell invasion and metastasis. Wang et al[25
] reported that Ezrin expression level is obviously higher in CRC tissue than in normal colorectal mucosa tissue, which is closely related to CRC invasion and metastasis. Elzagheid et al[26
] found that intense Ezrin immunoreactivity in cytoplasm can predict poor survival of CRC patients, thus providing clinically valuable information on the biological behavior of CRC. In this study, Ezrin was expressed on cell membrane surface or in cytoplasm, but not uniformly expressed in cytoplasm, which is consistent with the reported findings in pancreatic cancer[27
], indicating that membrane translocation of Ezrin may also play an important role in early CRC metastasis.
Cell locomotion, including cancer cell invasion, is closely associated with dynamics of cytoskeletal structures. Tubulin isotype composition may affect polymerization properties and dynamics of microtubules. Portyanko et al[28
] showed that tubulin β (III) is associated with tumor budding grade, and changes in tubulin isotypes can modulate the invading activity of CRC cells. In our study, the expression of tubulin β-2C was about 2-fold higher in SLNMM than in NLN, and IHC showed that the staining of tubulin β-2C was weak and mostly gathered around nuclei of NLN but stronger and diffused in cytoplasm of SLNMM, suggesting that the expression and distribution of tubulin β-2C are different in NLN and SLNMM of CRC, and the increased expression of tubulin β-2C is associated with early lymph node micrometastasis, thus leading to poor prognosis of CRC.
HnRNP is most abundantly expressed in nuclear protein of mammalian cells, which is associated with pre-mRNA processing and other aspects of mRNA metabolism and transport[29
]. As a class of protein family, many of its subtypes are related to the occurrence of different tumors, and hnRNP A2/B1 subtype is now used as an indicator in early diagnosis of lung cancer[30
]. In our study, Western blotting and IHC showed the expression level of hnRNP A1 was higher in SLNMM than in NLN, indicating that hnRNP A1 plays an important role in the occurrence and development of CRC[31,32
] and can thus be considered a potential molecular indicator/biomarker of tumorigenesis in CRC.
In summary, comparative proteomics technologies can be used in study of protein profiles in NLN and SLNMM and in identification of early CRC metastasis-related proteins. Increased expression of hnRNP A1, Ezrin, tubulin β-2C and Annexin A1 in SLN suggests a significantly elevated incidence of early CRC metastasis. However, further study is needed to verify their role in therapeutic target of CRC.