Department of Gastroenterology, Tianjin Nankai Hospital, Tianjin, 300100, PR China; Tianjin Key Laboratory of Acute Abdomen Disease Associated Organ Injury and ITCWM Repair, Tianjin, 300100, PR China.
Department of Gastroenterology, Tianjin Medical University General Hospital, Tianjin, 300052, PR China.
Surg Oncol. 2020 Dec;35:191-199. doi: 10.1016/j.suronc.2020.08.007. Epub 2020 Aug 14.
BRCA1-associated protein (BRAP) was first found to bind to the nuclear localization signal motifs of BRCA1. In this study, we investigated the role of BRAP in gastric cancer. The cancer genome atlas(TCGA) data were obtained from UALCAN. We downregulated and upregulated the level of BRAP in gastric cancer cells by transfection with shRNAs and plasmids. Then, we evaluated the expression of BRAP by qRT-PCR and investigated the expression of important proteins by Western blot analysis. We conducted a microarray analysis to identify the function of BRAP in gastric cancer cells. Then, we investigated the effect of BRAP on proliferation and migration by CCK-8 assays, colony formation assays, wound healing assays and an extreme limiting dilution analysis. The analysis of TCGA data showed that BRAP was significantly overexpressed in gastric cancer tissues compared to that in normal gastric mucosal tissues (P < 0.001). A hybridization-based microarray assay was used to analyze MGC-803 cells and BRAP-downregulated MGC-803 cells. We found 22,199 protein-coding RNAs that were differentially expressed. The genes in the two groups were analyzed with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and both the focal adhesion and MAPK pathways were significantly enriched. The results of Cell Counting Kit-8(CCK-8) assays, colony formation assays, wound healing assays and the extreme limiting dilution analysis showed that the knockdown of BRAP reduced gastric cancer cell proliferation and migration and inhibited the process of epithelial-mesenehymal transition (EMT). The overexpression of BRAP induced gastric cancer cell proliferation, migration and the process of EMT. To verify the function of the mitogen-activated protein kinase (MAPK) signaling pathway, we performed a Western blot analysis. The results showed that the downregulation of BRAP decreased the levels of p-ERK and p-Raf1, thereby decreasing the activity of the MAPK signaling pathway. The use of Honokiol increased the levels of p-ERK and p-Raf1, rescuing the function of BRAP downregulation in the MAPK pathway. Xenograft tumor transplantation experiments in nude mice further confirmed the role of BRAP in gastric cancer progression and metastasis.
BRCA1 相关蛋白（BRAP）最初被发现与 BRCA1 的核定位信号基序结合。在这项研究中，我们研究了 BRAP 在胃癌中的作用。从 UALCAN 获得癌症基因组图谱（TCGA）数据。我们通过转染 shRNA 和质粒下调和上调胃癌细胞中 BRAP 的水平。然后，我们通过 qRT-PCR 评估 BRAP 的表达，并通过 Western blot 分析研究重要蛋白的表达。我们进行了微阵列分析以确定 BRAP 在胃癌细胞中的功能。然后，我们通过 CCK-8 测定、集落形成测定、划痕愈合测定和极端极限稀释分析研究 BRAP 对增殖和迁移的影响。TCGA 数据分析表明，与正常胃黏膜组织相比，BRAP 在胃癌组织中显著过表达（P<0.001）。基于杂交的微阵列测定用于分析 MGC-803 细胞和 BRAP 下调的 MGC-803 细胞。我们发现 22199 个差异表达的蛋白质编码 RNA。对两组基因进行京都基因与基因组百科全书（KEGG）数据库分析，发现焦点黏附和 MAPK 途径均显著富集。Cell Counting Kit-8（CCK-8）测定、集落形成测定、划痕愈合测定和极端极限稀释分析的结果表明，BRAP 敲低降低了胃癌细胞的增殖和迁移，并抑制了上皮-间充质转化（EMT）过程。BRAP 的过表达诱导胃癌细胞增殖、迁移和 EMT 过程。为了验证丝裂原活化蛋白激酶（MAPK）信号通路的功能，我们进行了 Western blot 分析。结果表明，BRAP 下调降低了 p-ERK 和 p-Raf1 的水平，从而降低了 MAPK 信号通路的活性。使用 honokiol 增加了 p-ERK 和 p-Raf1 的水平，挽救了 BRAP 下调在 MAPK 通路中的功能。裸鼠异种移植肿瘤移植实验进一步证实了 BRAP 在胃癌进展和转移中的作用。
