酸性微环境下TSPAN9对胃癌SGC7901细胞侵袭、迁移的影响
发布时间：2018-09-09 12:53
【摘要】：目的:胃癌为最常见的消化道肿瘤之一,发病率及死亡率均高居消化道肿瘤之首。胃癌侵袭及迁移的生物学特征是导致胃癌患者死亡及预后差的主要原因。而肿瘤细胞侵袭、迁移的特性与肿瘤酸性微环境之间有着密不可分的关系。TSPAN9为四跨膜蛋白超家族中的一员,在胃癌中研究甚少,本研究拟探讨TSPAN9对胃癌SGC7901细胞在肿瘤酸性微环境下侵袭、迁移的影响。方法:用合成的TSPAN9基因连接至pIRES2-ZsGreen1载体从而获得重组质粒,然后转入TOP10克隆菌株中进行大规模基因扩增,转染至胃癌SGC7901细胞中从而构建TSPAN9过表达转染组,即SGC7901/TSPAN9(转染组)。同时培养SGC7901(空白组)及SGC7901/NC(对照组)。后用3%的HCL调节细胞培养基PH值为7.4及6.5分别培养72小时,形成7.4-SGC7901、6.5-SGC7901、7.4-SGC7901/NC、6.5-SGC7901/NC、7.4-SGC7901/TSPAN9、6.5-SGC7901/TSPAN9。应用实时荧光定量PCR(q RT-PCR)和Western Blot分别在RNA、蛋白质水平验证TSPAN9过表达。通过划痕实验、Transwell侵袭及迁移实验观察肿瘤酸性微环境对胃癌细胞侵袭、迁移的影响及TSPAN9在其中所发挥的作用。应用Western blot检测TSPAN9在肿瘤酸性微环境下对胃癌SGC7901细胞中u PA、MMP-9表达情况的影响。结果:SGC7901/TSPAN9转染组在PH=6.5即处于肿瘤酸性微环境下TSPAN9在RNA及蛋白质水平均高表达。通过划痕实验、Transwell实验,6.5-SGC7901较7.4-SGC7901可明显促进胃癌SGC7901细胞的侵袭及迁移能力,P0.05。而6.5-SGC7901、6.5-SGC7901/NC和6.5-SGC7901/TSPAN9组相比,转染组可明显促进u PA、MMP-9的表达,同时促进细胞的侵袭及迁移,P0.05。结论:肿瘤酸性微环境促进TSPAN9在RNA及蛋白质水平上的表达,肿瘤酸性微环境促进胃癌细胞的侵袭及转移。肿瘤酸性微环境通过上调TSPAN9促进胃癌SGC7901细胞的侵袭、迁移能力。将为胃癌在肿瘤酸性微环境下的治疗提供新靶点及新思路。
[Abstract]:Objective: gastric cancer is one of the most common digestive tract tumors. The biological characteristics of invasion and migration of gastric cancer are the main causes of death and poor prognosis of gastric cancer patients. However, the characteristics of invasion and migration of tumor cells are closely related to the acidic microenvironment of tumor. TSPAN9 is a member of the four-transmembrane protein superfamily. The purpose of this study was to investigate the effect of TSPAN9 on invasion and migration of gastric cancer SGC7901 cells in acidic microenvironment. Methods: the recombinant plasmid was obtained by ligating the synthesized TSPAN9 gene into the pIRES2-ZsGreen1 vector and then transferred into the TOP10 clone strain to amplify the gene. The recombinant plasmid was transfected into gastric cancer SGC7901 cells to construct the transfection group of TSPAN9 overexpression, that is, SGC7901/TSPAN9 (transfection group). SGC7901 (blank group) and SGC7901/NC (control group) were cultured at the same time. The cell culture medium PH value of 3% HCL was 7.4 and 6.5 for 72 hours, respectively, forming 7.4-SGC7901 6.5-SGC7901 + 7.4-SGC7901 / NC6.5-SGC7901 / SGC7901 / SGC7901 / TSPAN96.5-SGC7901 / TSPAN9. Real-time fluorescence quantitative PCR (q RT-PCR) and Western Blot were used to verify the overexpression of TSPAN9 at RNA, protein level, respectively. The effects of tumor acidic microenvironment on the invasion and migration of gastric cancer cells and the role of TSPAN9 in the invasion and migration of gastric cancer cells were observed by the scratch test and Transwell invasion and migration experiments. Western blot was used to detect the effect of TSPAN9 on the expression of u PA,MMP-9 in gastric cancer SGC7901 cells in acidic microenvironment. Results the expression of TSPAN9 in RNA and protein levels in the PH=6.5 group was significantly higher than that in the TSPAN9 / SGC7901 / TSPAN9 transfection group in the presence of tumor acidic microenvironment. Compared with 7.4-SGC7901, 6.5-SGC7901 significantly promoted the invasion and migration of gastric cancer SGC7901 cells (P0.05). Compared with 6.5-SGC7901/TSPAN9 group, 6.5-SGC7901 + 6.5-SGC7901 / NC could significantly promote the expression of u PA,MMP-9 and promote cell invasion and migration (P0.05). Conclusion: tumor acidic microenvironment promotes the expression of TSPAN9 at the level of RNA and protein, and tumor acidic microenvironment promotes the invasion and metastasis of gastric cancer cells. Tumor acidic microenvironment promotes the invasion and migration of gastric cancer SGC7901 cells by upregulating TSPAN9. It will provide new targets and new ideas for the treatment of gastric cancer in acidic microenvironment.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
本文编号：2232428
[Abstract]:Objective: gastric cancer is one of the most common digestive tract tumors. The biological characteristics of invasion and migration of gastric cancer are the main causes of death and poor prognosis of gastric cancer patients. However, the characteristics of invasion and migration of tumor cells are closely related to the acidic microenvironment of tumor. TSPAN9 is a member of the four-transmembrane protein superfamily. The purpose of this study was to investigate the effect of TSPAN9 on invasion and migration of gastric cancer SGC7901 cells in acidic microenvironment. Methods: the recombinant plasmid was obtained by ligating the synthesized TSPAN9 gene into the pIRES2-ZsGreen1 vector and then transferred into the TOP10 clone strain to amplify the gene. The recombinant plasmid was transfected into gastric cancer SGC7901 cells to construct the transfection group of TSPAN9 overexpression, that is, SGC7901/TSPAN9 (transfection group). SGC7901 (blank group) and SGC7901/NC (control group) were cultured at the same time. The cell culture medium PH value of 3% HCL was 7.4 and 6.5 for 72 hours, respectively, forming 7.4-SGC7901 6.5-SGC7901 + 7.4-SGC7901 / NC6.5-SGC7901 / SGC7901 / SGC7901 / TSPAN96.5-SGC7901 / TSPAN9. Real-time fluorescence quantitative PCR (q RT-PCR) and Western Blot were used to verify the overexpression of TSPAN9 at RNA, protein level, respectively. The effects of tumor acidic microenvironment on the invasion and migration of gastric cancer cells and the role of TSPAN9 in the invasion and migration of gastric cancer cells were observed by the scratch test and Transwell invasion and migration experiments. Western blot was used to detect the effect of TSPAN9 on the expression of u PA,MMP-9 in gastric cancer SGC7901 cells in acidic microenvironment. Results the expression of TSPAN9 in RNA and protein levels in the PH=6.5 group was significantly higher than that in the TSPAN9 / SGC7901 / TSPAN9 transfection group in the presence of tumor acidic microenvironment. Compared with 7.4-SGC7901, 6.5-SGC7901 significantly promoted the invasion and migration of gastric cancer SGC7901 cells (P0.05). Compared with 6.5-SGC7901/TSPAN9 group, 6.5-SGC7901 + 6.5-SGC7901 / NC could significantly promote the expression of u PA,MMP-9 and promote cell invasion and migration (P0.05). Conclusion: tumor acidic microenvironment promotes the expression of TSPAN9 at the level of RNA and protein, and tumor acidic microenvironment promotes the invasion and metastasis of gastric cancer cells. Tumor acidic microenvironment promotes the invasion and migration of gastric cancer SGC7901 cells by upregulating TSPAN9. It will provide new targets and new ideas for the treatment of gastric cancer in acidic microenvironment.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
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