Human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. Aberrant upregulation of GRB7 is frequently associated with the progression of human cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. Here, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. Methods: The upstream miRNA regulators were predicted by in silico analysis. Expression of GRB7 was examined by qPCR, immunoblotting and immunohistochemical analyses, while miR-193a-3p levels were evaluated by qPCR and in situ hybridization in ovarian cancer cell lines and clinical tissue arrays. MS-PCR and pyrosequencing analyses were used to assess the methylation status of miR-193a-3p. Stable overexpression or gene knockdown and Tet-on inducible approaches, in combination with in vitro and in vivo tumorigenic assays, were employed to investigate the functions of GRB7 and miR-193a-3p in ovarian cancer cells. Results: Both miR-193a-3p and its isoform, miR-193b-3p, directly targeted the 3' UTR of GRB7. However, only miR-193a-3p showed a significantly inverse correlation with GRB7-upregulated ovarian cancers. Epigenetic studies revealed that methylation-mediated silencing of miR-193a-3p led to a stepwise decrease in miR-193a-3p expression from low to high-grade ovarian cancers. Intriguingly, miR-193a-3p not only modulated GRB7 but also ERBB4, SOS2 and KRAS in the MAPK/ERK signaling pathway to enhance the oncogenic properties of ovarian cancer cells in vitro and in vivo. Conclusion: These findings suggest that epigenetic silencing of miR-193a-3p by DNA hypermethylation is a dynamic process in ovarian cancer progression, and miR-193a-3p may be explored as a promising miRNA replacement therapy in this disease.

中文翻译：

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甲基化相关的miR-193a-3p沉默通过靶向GRB7和MAPK / ERK途径促进卵巢癌侵袭性

人生长因子受体结合蛋白7（GRB7）是参与受体酪氨酸激酶信号传导并控制多种细胞过程的关键介质。GRB7异常上调通常与人类癌症的进展有关。但是，导致GRB7上调的分子机制仍然未知。在这里，我们建议在转录后水平上GRB7的表观遗传修饰可能是导致GRB7在卵巢癌中上调的关键因素。方法：通过计算机分析预测上游的miRNA调节子。通过qPCR，免疫印迹和免疫组化分析检查GRB7的表达，同时通过qPCR和原位评估miR-193a-3p水平卵巢癌细胞系和临床组织阵列中的杂交。MS-PCR和焦磷酸测序分析用于评估miR-193a-3p的甲基化状态。稳定的过表达或基因敲除和Tet-on诱导方法，与体外和体内致瘤性测定相结合，用于研究GRB7和miR-193a-3p在卵巢癌细胞中的功能。结果：两种的miR-193A-3P和其同种型，的miR-193b中-3P，直接针对GRB7的3' UTR。但是，只有miR-193a-3p与GRB7上调的卵巢癌呈显着负相关。表观遗传学研究表明，miR-193a-3p甲基化介导的沉默导致miR-193a-3p表达从低级到高级卵巢癌逐步降低。有趣的是，miR-193a-3p在MAPK / ERK信号通路中不仅调节GRB7，而且调节ERBB4，SOS2和KRAS，以增强体外和体内卵巢癌细胞的致癌特性。结论：这些发现表明，miR-193a-3p的DNA高甲基化沉默是卵巢癌进展的一个动态过程，miR-193a-3p 可以作为该疾病中有希望的miRNA替代疗法进行探索。