糖基转移酶β3Gn-T8与转录因子c-Jun的相关性及其与胃癌侵袭的关系
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摘要
目的：
根据已有文献报道：当细胞癌变时，细胞糖复合物上的糖链结构常发生变化，而这种变化来源于合成该糖链的相应糖基转移酶表达的改变。β1,3-N-乙酰氨基葡萄糖转移酶（β3Gn-Ts）家族是糖基转移酶的重要成员，β3Gn-Ts家族参与合成其产物中的β1,3-乙酰氨基葡萄糖连接键，其成员β3Gn-T8参与合成糖蛋白上N-连接型糖链中的多聚乳糖胺结构。本课题组先前研究证明，β3Gn-T8与胃癌的增殖侵袭转移密切相关，可能通过对基质金属蛋白酶MMP-2表达的调控促进胃癌的侵袭作用，因此，对MMP-2表达的调控通路有望作为控制胃癌侵袭转移的新靶点。本研究分析β3Gn-T8上游调控序列启动子区，发现β3Gn-T8启动子有五个c-Jun和一个Ets-1和结合位点。为了进一步阐明β3Gn-T8与胃癌的侵袭转移的相关性，本研究以体外培养的人胃癌SGC7901细胞株为研究对象，探讨胃癌SGC7901细胞株中转录因子c-Jun对糖基转移酶β3Gn-T8的转录调控；c-Jun基因沉默后对β3Gn-T8的表达以及对胃癌SGC7901细胞生物学行为的影响。同时在由10例临床标本制作成的组织芯片上检测c-Jun、CD147和多聚乳糖胺糖链的表达，对在临床胃癌标本中c-Jun、β3Gn-T8、多聚乳糖胺糖链和CD147的表达相关性进行探讨，以期为胃癌的治疗提供新的途径。
方法：
1. RT-PCR检测c-Jun和β3Gn-T8在SGC7901等六种肿瘤细胞株中的表达情况。
2.采用c-Jun中等量表达和β3Gn-T8高表达的人胃癌SGC7901细胞株为研究对象。应用三种生物信息学软件对β3Gn-T8启动子区的转录因子c-Jun基因的结合位点进行预测，根据预测结果构建β3Gn-T8启动子区系列截短体，并构建c-Jun基因的正义表达载体，经酶切、测序鉴定重组质粒的序列；应用双荧光素酶报告基因技术分析β3Gn-T8启动子区系列截短体的转录激活活性；并进一步采用染色质免疫沉淀（ChIP）检测分析c-Jun蛋白与β3Gn-T8基因DNA的结合。
3.设计并构建4条针对c-Jun mRNA靶点的siRNA真核表达质粒载体pGPU6/GFP/Neo-shRNA，同时设计shRNA-无关序列作为阴性对照（negativecontrol，NC），并构建表达载体；培养SGC7901细胞，以脂质体法转染细胞，荧光显微镜观察以最高转染效率和最低细胞死亡率为原则，优化转染条件，根据优化的条件将pGPU6/GFP/Neo-shRNA质粒转染胃癌SGC7901细胞，应用RT-PCR和western blot方法在mRNA水平和蛋白水平上检测四组pGPU6/GFP/Neo-shRNA对c-Jun基因表达的抑制作用，筛选出干扰效果最好的一组表达载体用于后续实验；设计空白对照组(Blank组，SGC7901细胞株)、阴性对照组（NC组，pGPU6/GFP/NC质粒转染的SGC7901细胞）、c-Jun-shRNA组（pGPU6/GFP/c-Jun-shRNA质粒转染的SGC7901细胞）为研究对象，经G418筛选，获得稳定转染表达载体pGPU6/GFP/c-Jun-shRNA的SGC7901细胞；应用RT-PCR和western blot方法在mRNA水平和蛋白水平上检测c-Jun基因沉默后β3Gn-T8的表达；应用CCK-8试剂盒检测三组细胞的增殖能力；应用凝集素细胞免疫荧光和FITC标志的凝集素流式细胞术检测三组细胞多聚乳糖胺糖链的表达。
4.通过免疫组织化学方法检测c-Jun、CD147和多聚乳糖胺糖链在由10例人胃癌组织和10例癌旁非肿瘤胃组织制作而成的组织芯片上的表达，以对在临床胃癌标本中c-Jun、β3Gn-T8、多聚乳糖胺糖链及与CD147和MMP-2表达相关性进行探讨。
结果：
1. C-Jun和β3Gn-T8在肿瘤细胞株中的表达：我们检测了胃癌细胞株SGC7901及AGS、恶性脑胶质瘤细胞株U87及LN229、乳腺癌细胞株MCF-7及MDA-MB-231中均有不同程度的表达，其中，胃癌SGC7901细胞株中c-Jun呈中等表达水平，β3Gn-T8呈高表达。
2．生物信息学分析：AliBaba2.1、PATCH和TESS三种在线软件预测结果分析β3Gn-T8启动子区存在着c-Jun基因的多个结合位点；双荧光素酶报告基因显示c-Jun对β3Gn-T8启动子区具有转录激活作用，其中启动子区－561/＋8片段的转录活性最高，且对c-Jun呈剂量依赖性；ChIP检测发现：活化的c-Jun蛋白与β3Gn-T8基因的DNA片段有相互结合。
3. shRNA表达载体的构建及其对胃癌生物学行为的影响：靶向c-Jun mRNA的四个pGPU6/GFP/Neo-shRNA重组质粒表达载体经酶切、测序鉴定，证实载体构建成功；重组质粒表达载体通过脂质体介导转染人胃癌SGC7901细胞，在荧光显微镜下观察到绿色荧光， RT-PCR和western blot结果证实pGPU6/GFP/Neo-shRNA-1949对c-Jun表达抑制的效果最好，因此，选择该shRNA载体，构建后续对c-Jun稳定下调的稳定细胞株；RT-PCR和western blot方法检测鉴定c-Jun基因沉默后SGC7901细胞株中β3Gn-T8基因在mRNA水平和蛋白水平表达均下降；CCK-8实验结果显示c-Jun基因沉默后SGC7901细胞增殖能力明显下降；凝集素细胞免疫荧光和流式细胞术检测结果显示c-Jun基因沉默后β3Gn-T8表达下降导致细胞上多聚乳糖胺糖链的表达量降低。
4. C-Jun、CD147和多聚乳糖胺链在胃癌组织的表达：结果显示c-Jun在胃癌组织的表达显著高于癌旁非肿瘤胃组织；CD147在胃癌组织高表达，而在癌旁非肿瘤胃组织几乎无表达；凝集素结合的多聚乳糖胺糖链表达在胃癌细胞和胃粘膜上皮细胞的胞浆中，棕黄色颗粒状。在弥漫型胃癌中的表达呈深棕色，而在肠型胃癌的表达为浅棕色，在某些肠型胃癌的癌细胞腺腔侧的胞浆及胞膜中呈深棕色颗粒状强阳性表达。在肠型胃癌的腺腔中和印戒细胞癌的粘液池中有强阳性颗粒。而在癌旁非肿瘤胃组织上皮细胞中的着色强度及范围明显弱于胃癌细胞。此外，在低分化腺癌中表达较高，而在高分化腺癌中表达较低。
结论：
1. C-Jun和β3Gn-T8在六种肿瘤细胞株中均有表达，胃癌SGC7901细胞株中c-Jun呈中等表达量，β3Gn-T8呈高表达；
2.生物信息学软件预测结果显示β3Gn-T8启动子区存在着转录因子c-Jun的多个结合位点；双荧光素酶报告基因结果显示c-Jun对β3Gn-T8启动子区具有转录激活作用，启动子区－561/＋8片段的转录活性最高，且对c-Jun呈剂量依赖性；ChIP结果显示c-Jun蛋白结合至β3Gn-T8基因的DNA片段。因此，c-Jun参与了胃癌SGC7901细胞株中β3Gn-T8基因的表达调控，至少是调控因素之一。且呈正性调控作用。
3.针对c-Jun基因的pGPU6/GFP/Neo-c-Jun-shRNA表达载体构建成功，经过检验，pGPU6/GFP/Neo-shRNA-1949对SGC7901细胞中c-Jun抑制效果最明显；并稳定转染SGC7901细胞株，获得c-Jun稳定下调的SGC7901细胞株，下调c-Jun基因表达后可降低胃癌SGC7901细胞株中β3Gn-T8基因在mRNA水平和蛋白水平上的表达；进而抑制胃癌SGC7901细胞的增殖能力以及细胞多聚乳糖胺糖链的合成。
4. C-Jun、CD147及多聚乳糖胺糖链结构在人胃癌组织的表达较癌旁非肿瘤胃组织显著增加。在弥漫型胃癌中多聚乳糖胺糖链结构的表达要强于肠型胃癌。
综上所述，转录因子c-Jun对胃癌SGC7901细胞株中糖基转移酶β3Gn-T8的启动子区有转录调控作用，且呈正性调控作用；c-Jun基因沉默后能够抑制胃癌SGC7901细胞株中糖基转移酶β3Gn-T8的表达，抑制胃癌细胞的增值能力，并且降低胃癌细胞上多聚乳糖胺糖链的合成，从而抑制胃癌细胞的侵袭转移能力；在10例胃癌病例的癌组织标本中，c-Jun、CD147及多聚乳糖胺糖链结构均明显高表达,其趋势与胃癌细胞SGC7901呈基本一致表现。综合本课题组相关研究结果，推导β3Gn-T8可能通过调控HG-CD147的表达，进一步调控MMP的表达而参与肿瘤的侵袭和转移，从而为β3Gn-T8作为胃癌的新兴标志物及基因治疗靶点奠定了一定的理论基础。
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.
根据已有文献报道：当细胞癌变时，细胞糖复合物上的糖链结构常发生变化，而这种变化来源于合成该糖链的相应糖基转移酶表达的改变。β1,3-N-乙酰氨基葡萄糖转移酶（β3Gn-Ts）家族是糖基转移酶的重要成员，β3Gn-Ts家族参与合成其产物中的β1,3-乙酰氨基葡萄糖连接键，其成员β3Gn-T8参与合成糖蛋白上N-连接型糖链中的多聚乳糖胺结构。本课题组先前研究证明，β3Gn-T8与胃癌的增殖侵袭转移密切相关，可能通过对基质金属蛋白酶MMP-2表达的调控促进胃癌的侵袭作用，因此，对MMP-2表达的调控通路有望作为控制胃癌侵袭转移的新靶点。本研究分析β3Gn-T8上游调控序列启动子区，发现β3Gn-T8启动子有五个c-Jun和一个Ets-1和结合位点。为了进一步阐明β3Gn-T8与胃癌的侵袭转移的相关性，本研究以体外培养的人胃癌SGC7901细胞株为研究对象，探讨胃癌SGC7901细胞株中转录因子c-Jun对糖基转移酶β3Gn-T8的转录调控；c-Jun基因沉默后对β3Gn-T8的表达以及对胃癌SGC7901细胞生物学行为的影响。同时在由10例临床标本制作成的组织芯片上检测c-Jun、CD147和多聚乳糖胺糖链的表达，对在临床胃癌标本中c-Jun、β3Gn-T8、多聚乳糖胺糖链和CD147的表达相关性进行探讨，以期为胃癌的治疗提供新的途径。
方法：
1. RT-PCR检测c-Jun和β3Gn-T8在SGC7901等六种肿瘤细胞株中的表达情况。
2.采用c-Jun中等量表达和β3Gn-T8高表达的人胃癌SGC7901细胞株为研究对象。应用三种生物信息学软件对β3Gn-T8启动子区的转录因子c-Jun基因的结合位点进行预测，根据预测结果构建β3Gn-T8启动子区系列截短体，并构建c-Jun基因的正义表达载体，经酶切、测序鉴定重组质粒的序列；应用双荧光素酶报告基因技术分析β3Gn-T8启动子区系列截短体的转录激活活性；并进一步采用染色质免疫沉淀（ChIP）检测分析c-Jun蛋白与β3Gn-T8基因DNA的结合。
3.设计并构建4条针对c-Jun mRNA靶点的siRNA真核表达质粒载体pGPU6/GFP/Neo-shRNA，同时设计shRNA-无关序列作为阴性对照（negativecontrol，NC），并构建表达载体；培养SGC7901细胞，以脂质体法转染细胞，荧光显微镜观察以最高转染效率和最低细胞死亡率为原则，优化转染条件，根据优化的条件将pGPU6/GFP/Neo-shRNA质粒转染胃癌SGC7901细胞，应用RT-PCR和western blot方法在mRNA水平和蛋白水平上检测四组pGPU6/GFP/Neo-shRNA对c-Jun基因表达的抑制作用，筛选出干扰效果最好的一组表达载体用于后续实验；设计空白对照组(Blank组，SGC7901细胞株)、阴性对照组（NC组，pGPU6/GFP/NC质粒转染的SGC7901细胞）、c-Jun-shRNA组（pGPU6/GFP/c-Jun-shRNA质粒转染的SGC7901细胞）为研究对象，经G418筛选，获得稳定转染表达载体pGPU6/GFP/c-Jun-shRNA的SGC7901细胞；应用RT-PCR和western blot方法在mRNA水平和蛋白水平上检测c-Jun基因沉默后β3Gn-T8的表达；应用CCK-8试剂盒检测三组细胞的增殖能力；应用凝集素细胞免疫荧光和FITC标志的凝集素流式细胞术检测三组细胞多聚乳糖胺糖链的表达。
4.通过免疫组织化学方法检测c-Jun、CD147和多聚乳糖胺糖链在由10例人胃癌组织和10例癌旁非肿瘤胃组织制作而成的组织芯片上的表达，以对在临床胃癌标本中c-Jun、β3Gn-T8、多聚乳糖胺糖链及与CD147和MMP-2表达相关性进行探讨。
结果：
1. C-Jun和β3Gn-T8在肿瘤细胞株中的表达：我们检测了胃癌细胞株SGC7901及AGS、恶性脑胶质瘤细胞株U87及LN229、乳腺癌细胞株MCF-7及MDA-MB-231中均有不同程度的表达，其中，胃癌SGC7901细胞株中c-Jun呈中等表达水平，β3Gn-T8呈高表达。
2．生物信息学分析：AliBaba2.1、PATCH和TESS三种在线软件预测结果分析β3Gn-T8启动子区存在着c-Jun基因的多个结合位点；双荧光素酶报告基因显示c-Jun对β3Gn-T8启动子区具有转录激活作用，其中启动子区－561/＋8片段的转录活性最高，且对c-Jun呈剂量依赖性；ChIP检测发现：活化的c-Jun蛋白与β3Gn-T8基因的DNA片段有相互结合。
3. shRNA表达载体的构建及其对胃癌生物学行为的影响：靶向c-Jun mRNA的四个pGPU6/GFP/Neo-shRNA重组质粒表达载体经酶切、测序鉴定，证实载体构建成功；重组质粒表达载体通过脂质体介导转染人胃癌SGC7901细胞，在荧光显微镜下观察到绿色荧光， RT-PCR和western blot结果证实pGPU6/GFP/Neo-shRNA-1949对c-Jun表达抑制的效果最好，因此，选择该shRNA载体，构建后续对c-Jun稳定下调的稳定细胞株；RT-PCR和western blot方法检测鉴定c-Jun基因沉默后SGC7901细胞株中β3Gn-T8基因在mRNA水平和蛋白水平表达均下降；CCK-8实验结果显示c-Jun基因沉默后SGC7901细胞增殖能力明显下降；凝集素细胞免疫荧光和流式细胞术检测结果显示c-Jun基因沉默后β3Gn-T8表达下降导致细胞上多聚乳糖胺糖链的表达量降低。
4. C-Jun、CD147和多聚乳糖胺链在胃癌组织的表达：结果显示c-Jun在胃癌组织的表达显著高于癌旁非肿瘤胃组织；CD147在胃癌组织高表达，而在癌旁非肿瘤胃组织几乎无表达；凝集素结合的多聚乳糖胺糖链表达在胃癌细胞和胃粘膜上皮细胞的胞浆中，棕黄色颗粒状。在弥漫型胃癌中的表达呈深棕色，而在肠型胃癌的表达为浅棕色，在某些肠型胃癌的癌细胞腺腔侧的胞浆及胞膜中呈深棕色颗粒状强阳性表达。在肠型胃癌的腺腔中和印戒细胞癌的粘液池中有强阳性颗粒。而在癌旁非肿瘤胃组织上皮细胞中的着色强度及范围明显弱于胃癌细胞。此外，在低分化腺癌中表达较高，而在高分化腺癌中表达较低。
结论：
1. C-Jun和β3Gn-T8在六种肿瘤细胞株中均有表达，胃癌SGC7901细胞株中c-Jun呈中等表达量，β3Gn-T8呈高表达；
2.生物信息学软件预测结果显示β3Gn-T8启动子区存在着转录因子c-Jun的多个结合位点；双荧光素酶报告基因结果显示c-Jun对β3Gn-T8启动子区具有转录激活作用，启动子区－561/＋8片段的转录活性最高，且对c-Jun呈剂量依赖性；ChIP结果显示c-Jun蛋白结合至β3Gn-T8基因的DNA片段。因此，c-Jun参与了胃癌SGC7901细胞株中β3Gn-T8基因的表达调控，至少是调控因素之一。且呈正性调控作用。
3.针对c-Jun基因的pGPU6/GFP/Neo-c-Jun-shRNA表达载体构建成功，经过检验，pGPU6/GFP/Neo-shRNA-1949对SGC7901细胞中c-Jun抑制效果最明显；并稳定转染SGC7901细胞株，获得c-Jun稳定下调的SGC7901细胞株，下调c-Jun基因表达后可降低胃癌SGC7901细胞株中β3Gn-T8基因在mRNA水平和蛋白水平上的表达；进而抑制胃癌SGC7901细胞的增殖能力以及细胞多聚乳糖胺糖链的合成。
4. C-Jun、CD147及多聚乳糖胺糖链结构在人胃癌组织的表达较癌旁非肿瘤胃组织显著增加。在弥漫型胃癌中多聚乳糖胺糖链结构的表达要强于肠型胃癌。
综上所述，转录因子c-Jun对胃癌SGC7901细胞株中糖基转移酶β3Gn-T8的启动子区有转录调控作用，且呈正性调控作用；c-Jun基因沉默后能够抑制胃癌SGC7901细胞株中糖基转移酶β3Gn-T8的表达，抑制胃癌细胞的增值能力，并且降低胃癌细胞上多聚乳糖胺糖链的合成，从而抑制胃癌细胞的侵袭转移能力；在10例胃癌病例的癌组织标本中，c-Jun、CD147及多聚乳糖胺糖链结构均明显高表达,其趋势与胃癌细胞SGC7901呈基本一致表现。综合本课题组相关研究结果，推导β3Gn-T8可能通过调控HG-CD147的表达，进一步调控MMP的表达而参与肿瘤的侵袭和转移，从而为β3Gn-T8作为胃癌的新兴标志物及基因治疗靶点奠定了一定的理论基础。
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.
引文
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