| ObjectiveGastric cancer and colon cancer are the common cancers found in gastrointestinal, which have high morbidity and mortality. Although clinical comprehensive therapy including surgery, chemotherapy, radiotherapy and biological therapy has been used, the treatment for gastrointestinal cancer is still poor. In addition, a considerable number of patients eventually die of recurrence and metastasis. And it brings a heavy financial and emotional burden to the family and society.β3GnT8, which uses UDP-GlcNA as donors, coud transfer GlcNAc to the nonreducing terminus of the Galβ1–4GlcNAc of tetraantennary N-glycan and form the polylactosamine. Here, in orded to investigate the regulation mechamisms of β3GnT8 in gastric cancer invasion and metastasis and colon cancer drug resistance, a depth and systematic study of β3GnT8 and polylactosamine was carried out from genes to glycans and from cell inside to cell outside.Method(1)Regulation mechamisms of β3GnT8 in gastric cancer invasion and metastasis(1)Gastric cancer tissue microarray was used and immunohistochemical staining of β3GnT8 and polylactosamine was observed. The clinicopathological parameters were analyzed to elucidate the relationship between the expression of β3GnT8 and polylactosamine and clinicopathological features of gastric cancer patients.(2)Three different tissue sources gastric cancer cells(AGS, SGC-7901, NCI-N87) were cultured. The invasive and metastatic ability of different groups was compared by transwell assay and wound healing assay. The expression of β3GnT8 was detected by quantitative RT-PCR and Western blot. The expression, structure and function of polylactosamine was analyzed by flow cytometry, lectin blot and lectin array.The above study was designed to clarify the relationship between the expression of β3GnT8 and polylactosamine and invasive and metastatic potential in the gastric cancer cells.(3)The expression of β3GnT8 in gastric cancer cells was regulated by gene transfection and RNA interference. The expression of polylactosamine was analyzed by flow cytometry and lectin blot. The invasive ability of different groups was compared by transwell assay. The glycosylation level of glycoprotein CD147 was detected by Western blot. The expression levels of matrix metalloproteinases(MMPs) were determined by quantitative RT-PCR and Western blot. The above study was designed to clarify the regulatory mechanisms of β3GnT8 in gastric cancer cell invasion and metastasis.(4)The binding proteins of β3GnT8 were enriched using immunoprecipitation assay in gastric cancer cells. The immunoprecipitates were separated on SDS-PAGE,then the binding proteins were cut from the gel and analyzed by mass spectrometry after bleaching, reduction, alkylation and enzymolysis. SWISS-PROT database was searched to screen and identify the target protein of β3GnT8 in gastric cancer cells.(5)The N-glycans of total glycoproteins in gastric cancer cells were hydrolysised and released using PNGase F. Then the N-glycans were purificated with Sep-Pak C18 column before fully methylated derivative. The glycan structures were analyzed by mass spectrometry to clarify the alteration pattern of N-glycans in gastric cancer cells.(2)Regulation mechamisms of β3GnT8 in colon cancer drug resistance(1)Quantitative RT?PCR was used to identify the expression of β3GnT8 in colon cancer 5-FU resistant cells SW620/5-FU and its parent cells SW620.Flow cytometry and lectin blot analysis were performed to detect the alteration of polylactosamine in SW620/5-FU cells and SW620 cells. The above study was designed to elucidate the correlation between β3GnT8 and polylactosamine expression and colon cancer drug resistance.(2)β3GnT8 expression in SW620/5-FU cells was inhibited by RNA interference. Flow cytometry and lectin blot analysis were performed to detect the alteration of polylactosamine. MTT assay was used to identify the change of drug sensitivity. Annexin V-FITC / PI double staining was used to determine cell apoptosis. PI staining was used to evaluate cell cycle. The above study was designed to clarify the regulatory mechanisms of β3GnT8 in colon cancer drugresistance.(3)The biosynthesis of polylactosamine was mediated by 3’?azidothymidine(AZT) in SW620/5-FU cells. MTT assay was used to detect the change of drug sensitivity. The above study was designed to elucidate the regulatory mechanisms of polylactosamine in colon cancer drug resistance.Result(1) Overexpressing β3GnT8 could induce the synthesis of polylactosamine and promote gastric cancer cell invasion and metastasis(1)The positive products of β3GnT8 were mainly located in the gastric cancer cell nuclei and cytoplasm, and appeared as brownish-yellow granules. Polylactosamine was not only mainly expressed in the cytoplasm of gastriccancer cells, which had brownish-yellow or tan particles, but also presented with a small amount of brownishyellow in cell membrane. The expression of β3GnT8 had no significant correlation with the age, sex and degree of differentiation, but was positively correlated with the pathological grade and clinical stage. The expression of polylactosamine in patients was positively correlated with the pathological grade and clinical stage, but had no correlation with the age, sex and degree of differentiation. The β3GnT8 and polylactosamine expression in gastric cancer tissues were positively correlated.(2)The invasive and metastatic ability of AGS cells was strongest, followed by SGC-7901 cells, and NCI-N87 was weakest. The gene and protein expression of β3GnT8 in AGS cells was highest, followed by SGC-7901 cells, and NCI-N87 was smallest. The expression of polylactosamine in AGS cells was most, followed by SGC-7901 cells, and NCI-N87 was least. The β3GnT8 and polylactosamine expression in gastric cancer cells have the consistency.(3)The overexpression of β3GnT8 in gastric cancer NCI-N87 cells could increase the content of polylactosamine, enhance cell invasion capacity, up-regulate high glycosylation(HG)-CD147 and promote gene and protein expression of MMP-14. Down-regulation of β3GnT8 expression in AGS cells had the ability to decrease the content of polylactosamine, suppress cell invasion capacity, down-regulate high glycosylation(HG)-CD147 and inhibit gene and protein expression of MMP-14.(4)In gastric cancer AGS cells, 45 kinds of binding protein interacted with β3GnT8 were isolated and identified. In gastric cancer NCI-N87 cells, 32 kinds of binding protein interacted with β3GnT8 were isolated and identified. And total 23 proteins interacted with β3GnT8 were obtained form AGS and NCI-N87 cells. Functional research combined with co-immunoprecipitation found that annexin A2(annexin A2, ANXA2) is the target protein of β3GnT8 in gastric cancer.(5)The kinds of N-glycans had no significant differences between AGS, SGC-7901, and NCI-N87 cells,but the amount of N-glycans had some differences. The content of fucose and sialic acid N-glycans in AGS cells were few. The proportion of fucose N-glycans in SGC-7901 cells was significantly higher than other cells. The proportion of sialic acid N-glycans in NCI-N87 cells was more than other cells.(2) Overexpressing β3GnT8 could induce the synthesis of polylactosamine and promote colon cancer cell drug resistance(1)The expression of β3GnT8 and the content of polylactosamine were significantly increased in SW620 /5-FU cells as compared with the parental SW620 cells.(2)Knockdown of β3GnT8 could inhibit the synthesis of polylactosamine in SW620/ 5?FU cells, increase 5-fluorouracil(5-FU) sensitivity, enhance apoptosis, decrease G1 phase and up-regulate G2 / M phase cells.(3)AZT could effectively reduce the synthesis of polylactosamine in SW620/ 5?FU cells and increase sensitivity to 5-FU.Conclusion(1) β3GnT8 and polylactosamine was both highly expressed in gastric cancer. Their expression was positively correlated with the pathological grade and clinical stage. The expression of β3Gn-T8 and polylactosamine had a positive correlation in gastric cancer tissues. With the enhancement of gastric cancer cell invasion and metastasis, the β3GnT8 expression and polyactosamine content was also increased. β3Gn-T8 was involved in the biosynthesis of polyactosamine carried on CD147 and could regulate the expression of MMP-14, thereby changing the invasive and metastatic potential of gastric cancer cells, which may require the synergy of ANXA2. In addition,the abnormal expression of N-glycans is associated with the malignant phenotype of gastric cancer cells.(2) β3GnT8 and polylactosamine was both highly expressed in colon cancer 5-FU resistant cells. Inhibition the expression of β3GnT8 and the biosynthesis of polyactosamine could enhance 5?FU sensitivity and reverse 5?FU resistance.In summary, β3GnT8 and polylactosamine plays a key role in the regulation of gastrointestinal tumor invasion and metastasis and resistance to chemotherapy. This study not only provide a novel target associated with molecule drug designing and screening for the treatment of gastrointestinal tumor, but also offer the basis for clinical individualized treatment and has important scientific significance and practical value. |
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