Falah Ghadeer, Sharvit Lital, Atzmon Gil
Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel.
Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel; Departments of Medicine and Genetics, Albert Einstein College of Medicine, Bronx, NY, USA.
Exp Gerontol. 2024 Oct 15;196:112586. doi: 10.1016/j.exger.2024.112586. Epub 2024 Sep 23.
The Growth Hormone Receptor (GHR) gene encodes a protein that is essential for mediating the biological effects of growth hormone (GH). A series of molecular events are set off when GH binds to its receptor, resulting in a variety of physiological reactions linked to development, growth, and metabolism. Recently a particular genetic variation, within the GHR gene that is labeled as the "d3GHR," which lacks exon 3 was associated with longevity. This specific deletion isoform was connected to changes in the structure of the GHR protein, which may have an impact on the GHR's function. To test in vitro the advantage of the d3 carrier that may link to longevity, we employed the CRISPR/Cas9 technique to produce two isoforms: the homozygotes isoform (d3/d3) and the heterozygotes isoform (d3/fl) using HEK293 cell line. The CRISPR editing effectiveness was >85 %, indicating that we had successfully built the Cas9-gRNA complex that is appropriate for the GHR gene. The viability of the resulted isoform cells was examined under three environmental stressors that mimic some aging processes. In addition, we examined the GHR signaling pathway by selecting potential downstream genes in the GHR signaling cascade. The results show that heterozygotes cells demonstrated higher survival rates under UV radiation compared with the WT cells (87 % compared with 67 % for the WT cells when exposed to 2 min of UV radiation), and in fasting conditions, the d3GHR cells showed a 15 % greater viability than the WT cells. Moreover, the baseline expression levels (without intervention) of the IGF1 and JAK/STAT genes signaling pathways significantly declined in the homozygotes cells compared with the WT (p < 0.05). This noteworthy finding might offer a practical approach to test illness prevention and give the scientific community critical new insights on mechanism associated with lifespan.
生长激素受体（GHR）基因编码一种蛋白质，该蛋白质对于介导生长激素（GH）的生物学效应至关重要。当GH与其受体结合时，会引发一系列分子事件，从而导致与发育、生长和代谢相关的各种生理反应。最近，GHR基因内一种被标记为“d3GHR”的特定基因变异（缺少外显子3）与长寿相关。这种特定的缺失异构体与GHR蛋白结构的变化有关，这可能会影响GHR的功能。为了在体外测试与长寿相关的d3携带者的优势，我们使用CRISPR/Cas9技术，利用HEK293细胞系产生了两种异构体：纯合子异构体（d3/d3）和杂合子异构体（d3/fl）。CRISPR编辑效率>85%，表明我们已成功构建了适用于GHR基因的Cas9-gRNA复合物。在模拟一些衰老过程的三种环境应激源下，检测了所得异构体细胞的活力。此外，我们通过选择GHR信号级联中的潜在下游基因来检测GHR信号通路。结果表明，与野生型（WT）细胞相比，杂合子细胞在紫外线辐射下表现出更高的存活率（暴露于2分钟紫外线辐射时，WT细胞的存活率为67%，而杂合子细胞为87%），并且在禁食条件下，d3GHR细胞的活力比WT细胞高15%。此外，与WT相比，纯合子细胞中IGF1和JAK/STAT基因信号通路的基线表达水平（无干预）显著下降（p<0.05）。这一值得注意的发现可能为测试疾病预防提供一种实用方法，并为科学界提供有关与寿命相关机制的重要新见解。
