Correlation Between Glycosyltransferase β3Gn-T8and Ranscription Factor C-Jun And Their Involvement In Nvasive Growth Of Gastric Cancer

Objectives:During the progression of tumorogenesis, the expressions of glycosyltransferasesare significantly altered, bringing about abnormal structures of glcoconjugates on thesurface of malignant cells. β1,3-N-acetylglucosaminyltransferases (β3Gn-Ts) familymembers are involved in synthesizing galactan backbones of β1,3-N-acetylglucosamine.Among them, β3Gn-T8engages in synthesis of poly N acetyl galactosamine of N-linked glycoproteins. Our previous study indicated that β3Gn-T8, through regulatingMMP-2expression, took part in proliferation, invasion and metastasis of gastric cancer.Thus, the role of β3Gn-T8on MMP-2expression might be a novel and promisingtherapeutic strategy for gastric cancer. β3Gn-T8contained the binding sites oftranscriptional factors Ets-1and c-Jun within several hundred base pairs upstream theTSS in this study，To further illustrate the correlation between β3Gn-T8and invasivegrowth of gastric cancer, this study was designed to explore the transcriptionalregulation on β3Gn-T8by transcription factor c-Jun and the influence of c-Jun genesilence on bioactivities of β3Gn-T8and gastric cancer cell line SGC7901. Then,expressions of c-Jun, CD147and Polylactosamine chain in human gastric cancer weredetected and their correlationships were discussed to probe new therapeutic method forgastric cancer.Methods:1. detect expression of c-Jun and β3Gn-T8in6tumor cell lines.2. Predict c-Jun binding site on β3Gn-T8promoter of gastric cancer cell lineSGC7901by using the bioinformatics software. Based on the prediction, truncatedβ3Gn-T8promoter series and expression vector of sense c-Jun were constructed. Transcription activity of truncated β3Gn-T8promoter series was analyzed by luciferasereporter gene expression analysis and then combination of c-Jun and β3Gn-T8geneswas detected by chromatin immunoprecipitation assay (ChIP).3.4gene silencing plasmids pGPU6/GFP/Neo-shRNA targeting c-Jun mRNA anda negative control were constructed and transected into SGC7901cells byLipofectamine. Then, the most effective siRNA was screened and identified by RT-PCRand Western-blot for further experiments. Before and after c-Jun gene silence, theexpression of β3Gn-T8was detected by RT-PCR and Western-blot; cell proliferationwas analyzed by CCK-8kit and expression of Polylactosamine chain was detected byflow cytometry.4. The expression of c-Jun, CD147and Polylactosamine chain on tissue microarrayof10cases of human gastric cancer and10adjacent non-tumor gastric tissues byimmunohistochemistry. Clinical and pathological parameters of gastric cancer werecollected for correlation analysis of c-Jun, β3Gn-T8, Polylactosamine chain, CD147andMMP-2expression.Results:1. Both c-Jun and β3Gn-T8were expressed in tumor cell lines, such as SGC7901,AGS, U87, LN229, MCF-7, MDA-MB-231. Gastric cancer cell line SGC7901expressed moderate level of c-Jun and high level of β3GnT8.2. According to bioinformatics prediction, many c-Jun binding sites were found inβ3Gn-T8promoter. Luciferase reporter gene expression analysis showed that c-Juncould activate transcription of truncated β3Gn-T8promoter series.(-561/+8) promoterregion had the highest transcription activity and was c-Jun dose dependant. Chip foundcombination of activated c-Jun and β3Gn-T8DNA.3. pGPU6/GFP/Neo-shRNA targeting c-Jun siRNA significantly inhibited mRNAand protein expression of c-Jun. After being transfected into SGC7901gastric cancercell line, the expression of β3Gn-T8mRNA and protein were down regulated bypGPU6/GFP/Neo-shRNA-1949by RT-PCR and Western-blot. Examined by CCK-8,cell proliferation ability decreased obviously and expression of Polylactosamine chain wasproved to be down regulated by FCM and luciferase reporter gene expression analysis. 4. The protein expression levels of c-Jun in gastric carcinoma tissues were higherthan those of the adjacent normal tissues.CD147which was highly expressed in gastriccarcinoma tissues could not be detected in adjacent normal tissues. The brown-dottedPolylactosamine chain positive products were detected on the membrane and in thecytoplasm of the gastric carcinoma cells. The expression level on adjacent normaltissues was obviously lower than that on carcinoma tissues. Moreover, the positive ratein poorly-differentiated denocarcinoma was significantly higher than that inwell-differentiated adenocarcinoma.Conclusions:1. Both c-Jun and β3Gn-T8were expressed in6tumor cell lines, among whichgastric cancer cell line SGC7901expressed moderate level of c-Jun and high level ofβ3Gn-T8.2. Bioinformatics prediction found many c-Jun binding sites in β3Gn-T8promoter.Luciferase reporter gene expression analysis showed that c-Jun could activatetranscription of truncated β3Gn-T8promoter series.(-561/+8) promoter region had thehighest transcription activity and was c-Jun dose dependant. Chip found combination ofactivated c-Jun and β3Gn-T8DNA. Taken together, c-Jun takes party in expressionregulation of β3Gn-T8in SGC7901.3. pGPU6/GFP/Neo-shRNA targeting c-Jun siRNA was successfully constructed.The expression of β3Gn-T8mRNA and protein were obviously down regulated bypGPU6/GFP/Neo-shRNA-1949. Cell proliferation ability and Polylactosamine chainswere inhibited obviously.4. The protein expression levels of c-Jun, CD147and Polylactosamine chain ingastric carcinoma tissues were higher than those of the adjacent normal tissues.In summary, transcription factor c-Jun takes party in positive regulation ofβ3Gn-T8promoter in SGC7901. shRNA targeting c-Jun significantly inhibitedβ3Gn-T8expression and ability of proliferation, invasion and metastasis of SGC7901.Similar to the expression pattern on SGC7901, the expression of c-Jun, CD147andPolylactosamine chain on10cases of human gastric cancer tissues were significantlyhigher than that on adjacent normal tissues. It was speculated that β3Gn-T8might be involved in tumor invasion and metastasis by regulating MMP expression throughCD147signal pathway. These findings provide new insight into β3GnT8targetinggastric cancer therapy.