Zhongguo aizheng zazhi (Mar 2022)
A study on mechanism of GOLM1 regulating PI3K/AKT/mTOR signaling pathway to promote proliferation, invasion and migration of lung adenocarcinoma cells
Abstract
Background and purpose: Golgi membrane protein 1 (GOLM1) plays the role of an oncogene in lung adenocarcinoma (LUAD), however, the effects of GOLM1 on the proliferation, invasion and migration of LUAD cells and its mechanism are not clear yet. This study investigated the effects of GOLM1 on the proliferation, invasion and migration of LUAD cells and its mechanism of action. Methods: We selected cancer tissues and corresponding paracancerous tissue specimens from 90 LUAD patients who underwent surgical resection in Karamay Central Hospital from April 2019 to April 2021. The expression of GOLM1 protein in LUAD tissues and paracancerous tissues was detected by immunohistochemistry, and the relationship between GOLM1 protein expression and clinicopathological characteristics of LUAD tissues was analyzed. Western blot was used to detect the expression of GOLM1 protein in human lung epithelial cells BEAS-2B and human lung adenocarcinoma H460, A549, PG49 and H1299 cells. Lung adenocarcinoma A549 cells in logarithmic growth stage were randomly divided into blank group (cells not transfected), GOLM1 small interfering RNA negative control (si-NC) group (cells transfected with si-NC), GOLM1 small interfering RNA (si-GOLM1) group (cells transfected with si-GOLM1), insulin-like growth factor-1 (IGF-1) group [10 μmol/L phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway activator IGF-1 for 30 min] and si-GOLM1+IGF-1 group (after treatment with 10 μmol/L IGF-1 for 30 min, si-GOLM1 was transfected). Cell counting kit-8 method was used to detect cell proliferation in each group of lung adenocarcinoma A549 cells. Scratch test was used to detect cell migration in each group of lung adenocarcinoma A549 cells. Transwell experiment was used to detect cell invasion in each group of lung adenocarcinoma A549 cells. Western blot was used to detect the expressions of GOLM1 and PI3K/AKT/mTOR signaling pathway related proteins in each group of lung adenocarcinoma A549 cells. Xenograft model was constructed by subcutaneously injecting A549 cells on the right side of BALB/c nude mice, which were divided into: nude mice blank group, nude mice si-NC group, nude mice si-GOLM1 group, nude mice IGF-1 group, nude mice si-GOLM1+IGF-1 group, with 6 mice in each group. The nude mice were sacrificed six weeks after injection, the tumor was collected, and the weight and volume of the tumor were measured. Results: The results of immunohistochemistry showed that the positive expression rate of GOLM1 protein was significantly higher in LUAD tissues than in adjacent tissues (P<0.05). The expression of GOLM1 protein was significantly correlated with the degree of tumor differentiation, lymph node metastasis, and clinical stage (P<0.05), but not significantly correlated with gender, age and smoking status of LUAD patients (P>0.05). Compared with BEAS-2B cells, the relative expression level of GOLM1 protein in human lung adenocarcinoma H460, A549, PG49 and H1299 cells was significantly increased (P<0.05), and the relative expression level of GOLM1 protein in A549 cells was the highest. Therefore, A549 cells were selected for subsequent experiments. Compared with the blank group and the si-NC group, OD value, scratch healing rate, number of invaded cells, GOLM1, p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR protein relative expression levels in the lung adenocarcinoma A549 cells of the si-GOLM1 group were significantly reduced (P<0.05). In the lung adenocarcinoma A549 cells of the IGF-1 group, there was no significant change in GOLM1 protein, and the other corresponding indicators were significantly increased (P<0.05). Compared with the si-GOLM1 group, the OD value, scratch healing rate, number of invaded cells, p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR protein relative expression levels of lung adenocarcinoma A549 cells in the si-GOLM1+IGF-1 group were significantly increased (P<0.05), and there was no significant difference in GOLM1 protein relative expression level (P>0.05). Compared with the nude mice blank group and nude mice si-NC group, the mass and volume of transplanted tumors in the nude mice si-GOLM1 group were significantly reduced, while the mass and volume of transplanted tumors in the nude mice IGF-1 group were significantly increased (P<0.05). Compared with the nude mice si-GOLM1 group, the mass and volume of transplanted tumors in the nude mice si-GOLM1+IGF-1 group were significantly increased (P<0.05). Conclusion: Silencing GOLM1 gene can inhibit the activation of PI3K/AKT/mTOR signaling pathway, thereby inhibiting the proliferation, migration and invasion of lung adenocarcinoma A549 cells.
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