Glycoprofiling Of Poorly Differentiated Gastric Adenocarcinoma

PURPOSEGlycosylation is a vital posttranslation modification of protein. Acting a significant role as informational molecule, glycan showed association with certain biological processes, such as cell adhesion, pathogen recognition and tumor metastasis. Recent studies revealed that the occurrences and developments of many types of tumor are accompanied by changes in structures and abundances of glycoproteins. Gastric cancer is a high incidence of malignant tumor,95%of the gastric cancers are gastric carcinoma. Poorly differentiated gastric adenocarcinoma is a major type of gastric adenocarcinoma, which was considered to be in low degree of cancer cell differentiation, poor prognosis and high mortality. Therefore, in this research, lectin microarray and lectin histochemistry were utilized to probe and validate the abnormal glycopatterns in different TNM staging of tumor tissues compared with tumor adjacent tissues and gastric ulcers. And we hope that this study will be helpful to the study of the molecular mechanism for the development of gastric cancer, and provide potential targets for the treatment of poorly differentiated gastric adenocarcinoma.METHODFirst,19cases of cancer patients with poorly differentiated gastric adenocarcinoma, their corresponding adjacent tissues, and2cases of gastric ulcer organization were obtained from the hospital. These poorly differentiated gastric adenocarcinoma can be divided into different TNM stages according to the1997UICC TNM staging principle. Second, extract and purify tissue proteins, label with Cy3fluorescent dye and then incubate with lectin microarray. Scan for chip image, and detect the fluorescent signal values with GenePix3.0software. Next, the data were conducted with Global normalization processing and Raito value analysis. Screen the abnormal glycopatterns in tumor tissues compared with tumor adjacent tissues and gastric ulcers. After that, lectin histochemistry was used to validate the glycan of abnormal glycoproteins which were screened. Eventually, a set of oligosaccharide structure which specific associated with poorly differentiated gastric adenocarcinoma were determined. RESULTS(1) Lectin microarray was utilized to analyze the glycoprofile of the poorly differentiated gastric adenocarcinoma tissue, the adjacent tissue and the gastric Gastric ulcer tissue. The results showed that:glycans which can be recognized by lectin GSL-I, STL, BPL and PNA have showed specifically high expressions in poorly differentiated gastric adenocarcinoma tissues according to the comparison between poorly differentiated gastric adenocarcinoma tissues in different TNM stages and adjacent tissues. Combined with the glycoprofiling of gastric ulcer, we found that only in low differentiated gastric adenocarcinoma,did alpha GalNAc, GalNAc alpha Ser/Thr(Tn) and alpha Gal, Oligomers of GlcNAc and Gal beta1-3GalNAc alpha Ser/Thr(T) showed high expression. Among these, the proportion of high expression of alpha GalNAc in the tested poorly differentiated gastric adenocarcinoma tissue samples was84.21%(16/19), in addition, highly expressed GalNAc alpha Ser/Thr(Tn) and alpha Gal, Gal beta1-3GalNAc alpha Ser/Thr(T) took73.68%(14/19) in the detected poorly differentiated gastric adenocarcinoma samples. At last, lectin histochemistry was used to validate the result of lectin microarrays, and we found that the glycan structures which recognized by GSL-I, STL and PNA have high expressions in poorly differentiated gastric adenocacinoma; At the same time, lectin histochemistry also showed the distribution of glycosidic residues in poorly differentiated gastric adenocacinoma tissues. As the results below, lectin PNA showed the binding had intensification in the cytoplasma membrane of pooly differentiated gastric adenocacinoma; Lectins STL and GSL-I showed the bindings had intensification in the cytoplasm of pooly differentiated gastric adenocacinoma.(2) Lectin microarray was utilized to analyze the difference of glycoprofile between poorly differentiated gastric adenocarcinoma tissue in different TNM stages and adjacent tissue, we found that there are differences of glycoprofiling in different TNM stages of the poorly differentiated gastric adenocarcinoma tissue. The results are as follows:①In Tl phase of poorly differentiated gastric adenocarcinoma tissue, aGalNAc, GalNAca-Ser/Thr(Tn), Poly-LacNAc LacNAc,(GlcNAc)n Oligomers of GlcNAc,and Galβ1-3GalNAc T antigen glycan structures showed increment in expression.②In T2phase of poorly differentiated gastric adenocarcinoma tissue, Oligomers of GlcNAc, aGalNAc, GalNAca-Ser/Thr(Tn), GlcNAc and galactosylated N-glycans, Galβ1-3GalNAc T antigen, Non-substituteda-1,6Man, Ga1β-1,4GlcNAc LacNAc glycan structures had high expressions.③In T3phase of poorly differentiated gastric adenocarcinoma tissue, Oligomers of GlcNAc (GlcNAc)n, aGalNAc, GalNAca-Ser/Thr(Tn), Galβ1-3GalNAc T antigen, Poly-LacNAc, Fucosea-1,3GlcNAc(core fucose), Sia-Lex and Lex Fucosea-1,6GlcNAc(core fucose). Bisecting GlcNAc and biantennary N-glycans glycan structures have highly expressed.CONCLUSION(1) The comparison among the poorly differentiated gastric adenocarcinoma tissue, the adjacent tissue and the gastric Gastric ulcer tissue revealed that aGalNAc, GalNAcα-Ser/Thr(Tn) and aGal, Oligomers of GlcNAc and Galβ1-3GalNAca-Ser/Thr(T) which can be recognized by lectin GSL-1, STL and PNA only showed specificity of high expressions in poorly differentiated gastric adenocarcinoma. This conclusion gives us potential therapeutic targets of poorly differentiated gastric adenocarcinoma.(2) By comparing the glycopatterns of the glycoprotein from poorly differentiated gastric adenocarcinoma tissues in different TNM stages and their corresponding adjacent tissues, we can draw the conclusion that among the samples in the T1phase,4kinds of glycan which can be recognized by7lectins(GSL-I, LEL, PNA, PTL-Ⅱ, STL, BPL, ECA) showed high specificity expressions in gastric cancer tissue. Furthermore, among the samples in the T2phase,6kinds of glycan which can be recognized by8lectins(GSL-I, GSL-Ⅱ, BPL, NPA, PNA, STL, MPL, MAL-I)showed high specificity expressions in gastric cancer tissue. Meanwhile, among the samples in the T3phase,7kinds of glycan which can be recognized by9lectins(STL, BPL, GSL-I, LEL, LTL, PHA-E+L, PNA, PSA, VVA)showed high specificity expressions in gastric cancer tissue, and the increment of the level of fucosylation (Fucose a-1,3/6) was found. The results given illustrated that with the increase of the degree of tumour infection, tendency of the rise of the type of glycan on glycoproteins can be expected to be determinate.