HER-2/neu (HER2) 是表皮生长因子受体家族的成员，编码具有酪氨酸激酶活性的蛋白质。基因扩增或 HER2 转录增加后，在某些癌症中观察到致癌作用。增强子序列中发生的遗传和表观遗传变化可以深刻影响下游基因的表达和转录调控，从而引起一些生理和病理变化，包括肿瘤进展。直接针对基因组序列改变的治疗方法非常重要，并且对健康细胞的副作用很小。在这里，我们采用 CRISPR/Cas9 方法从基因上敲除位于 HER2 基因 17q12：39,694,339-39,697,219 (UCSC-hg38) 内的表达推定增强子 (GH17J039694；我们将其称为 Her2-Enhancer1)。然后我们研究了 Her2-Enhancer1 对 HER2 和 HER2 相互作用基因的潜在调节作用。为了评估 Her2-Enhancer1 的顺式和反式效应，在 HER2 阳性和阴性乳腺癌细胞中对该区域进行了基因操作。我们的生物信息学和实时 PCR 数据表明，这个假定的增强子区域确实被表达，并且充当表达的增强子。对编辑和未编辑细胞的进一步功能分析揭示了 HER2 变体以及 HER2 的其他一些靶基因的表达发生显着变化。此外，编辑后的细胞中的凋亡率显着升高。正如我们预期的那样，Western blot 分析证实 PI3K/AKT 通路中 HER2、GRB7（与 HER2 相互作用的基因）和 P-AKT 的蛋白水平降低。总而言之，我们的研究结果揭示了 Her2-Enhancer1 对 HER2 和 HER2 相互作用基因的增强子调节作用；该区域具有针对 HER2 阳性癌症进行靶向治疗的潜力。© 2023。作者。

HER-2/neu (HER2) is a member of the epidermal growth factor receptors family, encoding a protein with tyrosine kinase activity. Following the gene amplification or increased HER2 transcription, carcinogenesis has been observed in some cancers. Genetic and epigenetic changes occurring in enhancer sequences can deeply affect the expression and transcriptional regulation of downstream genes, which can cause some physiological and pathological changes, including tumor progression. A therapeutic approach that directly targets the genomic sequence alterations is of high importance, with low side effects on healthy cells. Here, we employed the CRISPR/Cas9 method to genetically knockout an expressed putative enhancer (GH17J039694; we coined it as Her2-Enhancer1) located within the HER2 gene, 17q12: 39,694,339-39,697,219 (UCSC-hg38). We then investigated the potential regulatory effect of Her2-Enhancer1 on HER2 and HER2-interacting genes. To evaluate the cis and trans effects of Her2-Enhancer1, genetic manipulation of this region was performed in HER2-positive and -negative breast cancer cells. Our bioinformatics and real-time PCR data revealed that this putative enhancer region is indeed expressed, and acts as an expressed enhancer. Further functional analysis on edited and unedited cells revealed a significant alteration in the expression of HER2 variants, as well as some other target genes of HER2. Moreover, the apoptosis rate was considerably elevated within the edited cells. As we expected, Western blot analysis confirmed a reduction in protein levels of HER2, GRB7, the gene interacting with HER2, and P-AKT in the PI3K/AKT pathway. Altogether, our findings revealed an enhancer regulatory role for Her2-Enhancer1 on HER2 and HER2-interacting genes; and that this region has a potential for targeted therapy of HER2-positive cancers.© 2023. The Author(s).