Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, China.
Department of General Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, Shaanxi, China.
Sci Rep. 2021 Oct 25;11(1):20951. doi: 10.1038/s41598-021-98728-9.
Oxaliplatin resistance is the greatest obstacle to the management of local recurrence in gastric cancer patients after surgery. Accumulating evidence has suggested that inhibiting autophagy may be a novel approach for reversing resistance to oxaliplatin treatment. In this manuscript, we aimed to investigate the role of LINC00963 in regulating autophagy and oxaliplatin resistance. qRT-PCR, immunochemistry staining, and western blotting were used to detect gene expression. Plasmids were used to up- and downregulate the expression of LINC00963 and miR-4458. A caspase 3/7 activity kit and flow cytometry were used to detect the apoptosis rate. CCK8 and Transwell assays were used to test cell proliferation and migration, respectively. Transmission electron microscopy and a dual fluorescent lentivirus autophagy system were used to evaluate autophagic flux. Dual luciferase reporter gene assays and RNA pulldown assays were used to evaluate the potential crosstalk. LINC00963 was highly expressed in gastric cancer patients and cell lines. In addition, high LINC00963 expression was found to be associated with poor prognosis and local recurrence in gastric cancer patients, indicating that LINC00963 might be involved in oxaliplatin resistance. Moreover, we found that LINC00963 was aberrantly highly expressed in oxaliplatin-resistant SGC-7901 (SGC-7901-R) cells and promoted proliferation and migration and reduced the apoptosis rate in SGC-7901-R cells. Furthermore, among all potential target microRNAs, miR-4458 was found to be negatively regulated by LINC00963 both in vivo and in vitro. In addition, miR-4458 overexpression led to impaired proliferation and migration and enhanced cell apoptosis and G1 arrest in SGC-7901-R cells. Further RNA pulldown and dual luciferase reporter gene assays indicated the interaction between LINC00963 and miR-4458. Moreover, we found enhanced autophagic flux in SGC-7901-R cells compared with SGC-7901 cells; in addition, an inhibitor of autophagy induced apoptosis in SGC-7901-R cells. Then, we found that downregulation of LINC00963 expression and upregulation of miR-4458 expression significantly suppressed autophagic flux in SGC-7901-R cells. Based on starBase V3.0 and dual luciferase reporter gene assays, we predicted and confirmed that ATG16L1 might be the target of miR-4458 to regulate autophagy. In conclusion, LINC00963 and miR-4458 are potential biomarkers for predicting the overall survival of gastric cancer patients. Moreover, targeting LINC00963 to inhibit autophagic flux sensitizes gastric cancer cells to oxaliplatin treatment, suggesting that it is a potential novel therapeutic target for improving oxaliplatin sensitivity.
奥沙利铂耐药是胃癌患者手术后局部复发管理的最大障碍。越来越多的证据表明，抑制自噬可能是逆转奥沙利铂治疗耐药的一种新方法。在本手稿中，我们旨在研究 LINC00963 在调节自噬和奥沙利铂耐药中的作用。使用 qRT-PCR、免疫化学染色和 Western blot 检测基因表达。使用质粒上调和下调 LINC00963 和 miR-4458 的表达。使用 caspase 3/7 活性试剂盒和流式细胞术检测细胞凋亡率。使用 CCK8 和 Transwell 测定分别检测细胞增殖和迁移。使用透射电子显微镜和双荧光慢病毒自噬系统评估自噬通量。使用双荧光素酶报告基因测定和 RNA 下拉测定评估潜在的串扰。LINC00963 在胃癌患者和细胞系中高表达。此外，高表达 LINC00963 与胃癌患者的不良预后和局部复发有关，表明 LINC00963 可能参与奥沙利铂耐药。此外，我们发现 LINC00963 在奥沙利铂耐药的 SGC-7901（SGC-7901-R）细胞中异常高表达，并促进 SGC-7901-R 细胞的增殖和迁移，降低细胞凋亡率。此外，在所有潜在的靶microRNAs 中，miR-4458 在体内和体外均被发现受到 LINC00963 的负调控。此外，miR-4458 的过表达导致 SGC-7901-R 细胞的增殖和迁移受损，促进细胞凋亡和 G1 期阻滞。进一步的 RNA 下拉和双荧光素酶报告基因测定表明 LINC00963 和 miR-4458 之间存在相互作用。此外，我们发现与 SGC-7901 细胞相比，SGC-7901-R 细胞中自噬通量增强；此外，自噬抑制剂诱导 SGC-7901-R 细胞凋亡。然后，我们发现下调 LINC00963 的表达和上调 miR-4458 的表达显著抑制了 SGC-7901-R 细胞中的自噬通量。基于 starBase V3.0 和双荧光素酶报告基因测定，我们预测并证实 ATG16L1 可能是 miR-4458 调节自噬的靶标。总之，LINC00963 和 miR-4458 是预测胃癌患者总生存期的潜在生物标志物。此外，靶向 LINC00963 抑制自噬通量可使胃癌细胞对奥沙利铂治疗敏感，表明它是提高奥沙利铂敏感性的潜在新治疗靶点。
