GPR78 Regulates Autophagy and Drug Resistance in Non-small Cell Lung Cancer.

ABSTRACT

Context Lung cancer is one of the most common forms of cancer. Autophagy and apoptosis play an important role in the development of lung cancer. Researchers have found upregulation of GRP78 expression in cancer cells of various types. Objective: The study intended to explore the mechanism of G protein-coupled receptor 78(GPR78) in regulating autophagy and drug resistance in non-small cell lung cancer (NSCLC). Design: The research team performed a laboratory study. Setting: The study took place in the Department of Thoracic Surgery at Hainan General Hospital of the Hainan Affiliated Hospital of Hainan Medical University in Haikou, Hainan, China. Intervention The research team cultured immortalized, normal, human bronchial epithelial cells C3 (HBEC3) lines and HBEC4 lines in a serum medium without keratinocytes and infected the expression of GPR78 in knockdown A549 cells using lentiviral agents. The team divided the cells into a control group and a shRNA-GPR78 group, the intervention group. The lentiviral silencing vector expressing shRNA targets human GPR78#1 and GPR78 #2aadam10. Outcome Measures: The research team analyzed the mRNA expression of GPR78 in the NSCLC cell lines H1975, H1299, and A549 and in HBEC3 and HBEC4 using a real time-polymerase chain reaction (RT-PCR) and measured the proliferation of A549 cells at 0h, 24h, 48h, 72h, and 96h using yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The team also analyzed the migration and invasion ability of cells using wound healing and Transwell tests as well as measured the protein expression of the autophagy-related factors Beclin-1, microtubule-associated protein light chain 3-I/II (LC3-I/LC3-II), ubiquitin-binding protein p62 and c-Jun N-terminal kinase (JNK) using a Western blot test. The team also analyzed the protein expressions of caspase-9, caspase-3, and caspase-12 related to apoptosis using a Western blot. To detect the cell viability induced by cisplatin, the team used a Cell Counting Kit 8 (CCK-8) at the concentrations of 1µM, 3µM and 10µM. Results: The mRNA expression of GPR78 in the H1975, H1299, and A549 cell lines was significantly higher than that in the HBEC3 and HBEC4 cell lines (P < .05). At 48h, 72h, and 96h, the A549 cell proliferation in the shRNA-GPR78 group was significantly lower than that of the control group (P < .05). The cell migration and invasion of cells in the shRNA-GPR78 group was significantly lower than that in the control group (P < .05), and the cell viability of the shRNA-GPR78 group was significantly lower than that of control group (P < .05). The expression of Beclin-1 and JNK protein in shRNA-GPR78 group was significantly higher than that in the control group (P < .05), and the expression of LC3-I/LC3-II and p62 protein in shRNA-GPR78 group was significantly lower than that in the control group (P < .05). The protein expressions of caspase-9, caspase-3, and caspase-12 in the shRNA-GPR78 group were significantly higher than those of the control group (P < .05), and the protein activities of RhoA and Rac1 in the shRNA-GPR78 group were significantly lower than those in the control group (P < .05). Conclusion: NSCLC upregulated GPR78. The knockdown of GPR78 can attenuate the proliferation, migration, and invasion of NSCLC cells and increase the apoptosis and autophagy of NSCLC cells that cisplatin has induced. Therefore, targeting GPR78 may be a promising treatment strategy for NSCLC patients.