MicroRNA-760 acts as a tumor suppressor in gastric cancer development via inhibiting G-protein-coupled receptor kinase interacting protein-1 transcription

Abstract

Background: Gastric cancer (GC) has become a serious threat to people's health. Accumulative evidence reveals that dysregulation of numerous microRNAs (miRNAs) has been found during malignant formation. So far, the role of microRNA-760 (miR-760) in the development of GC is largely unknown.

Aim: To measure the expression level of miR-760 in GC and investigate its role in gastric tumorigenesis.

Methods: Real-time quantitative polymerase chain reaction and Western blot analysis were used to measure the expression of miR-760 and G-protein-coupled receptor kinase interacting protein-1 (GIT1). Cell growth was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and cell colony formation assays. Apoptosis was assessed by flow cytometric analysis. The relationship between miR-760 and GIT1 was verified by luciferase reporter assay.

Results: The results showed that the expression of miR-760 was decreased in GC and associated with poor clinical outcomes in GC patients. Furthermore, miR-760 restrained cell proliferation and cell colony formation and induced apoptosis in GC cells. In addition, miR-760 directly targeted GIT1 and negatively regulated its expression in GC. GIT1 was upregulated in GC and predicted a worse prognosis in GC patients. We also found that upregulation of GIT1 weakened the inhibitory effect of miR-760 in GC.

Conclusion: In conclusion, miR-760 targets GIT1 to inhibit cell growth and promote apoptosis in GC cells. Our data demonstrate that miR-760 may be a potential target for the treatment of GC.

Keywords: G-protein-coupled receptor kinase interacting protein-1; Gastric cancer; Invasion; MicroRNA-760; Migration; Proliferation.

Figure 1

The expression of microRNA-760 is decreased in gastric cancer. A: MicroRNA-760 (miR-760) expression in gastric cancer tissues; B: MiR-760 expression in MKN-45, AGS, and GES-1 cells; C and D: Dysregulation of miR-760 was related to overall survival and disease-free survival in GC patients. ^bP < 0.01. GC: Gastric cancer; MiR-760: MicroRNA-760.

Figure 2

MicroRNA-760 regulates cell growth and apoptosis in gastric cancer. A: MicroRNA-760 (miR-760) expression in MKN-45 and AGS cells transfected with its mimic or inhibitor; B-D: Cell proliferation and colony formation regulated by miR-760 mimic or inhibitor; E: Flow cytometry analysis of MKN-45 and AGS cells transfected with miR-760 mimic or inhibitor. ^aP < 0.05, ^bP < 0.01. NC: Negative control; MiR-760: MicroRNA-760.

Figure 3

MicroRNA-760 suppresses G-protein-coupled receptor kinase interacting protein-1 expression in gastric cancer. A: The binding site between microRNA-760 (miR-760) and G-protein-coupled receptor kinase interacting protein-1 (GIT1); B: Luciferase reporter assay; C: A negative correlation between miR-760 and GIT1 in gastric cancer tissues; D and E: GIT1 expression in MKN-45 and AGS cells transfected with miR-760 mimic or inhibitor. ^bP < 0.01. GC: Gastric cancer; Mut: Mutant; Wt: Wild-type; 3'-UTR: 3'-untranslated region; NC: Negative control; MiR-760: MicroRNA-760; GIT1: G-protein-coupled receptor kinase interacting protein-1.

Figure 4

G-protein-coupled receptor kinase interacting protein-1 is upregulated in gastric cancer. A: G-protein-coupled receptor kinase interacting protein-1 (GIT1) expression in gastric cancer tissues; B: GIT1 expression in MKN-45, AGS, and GES-1 cells; C and D: High GIT1 expression was related to a shorter overall survival and disease-free survival in gastric cancer patients. ^bP < 0.01. GIT1: G-protein-coupled receptor kinase interacting protein-1.

Figure 5

MicroRNA-760 inhibits gastric cancer progression by inhibition of G-protein-coupled receptor kinase interacting protein-1. A: G-protein-coupled receptor kinase interacting protein-1 (GIT1) expression in MKN-45 and AGS cells transfected with GIT1 vector and microRNA-760 (miR-760) mimic; B-D: Cell proliferation and colony formation in MKN-45 and AGS cells transfected with GIT1 vector and miR-760 mimic; E: Flow cytometry analysis of MKN-45 and AGS cells transfected with GIT1 vector and miR-760 mimic. ^bP < 0.01. NC: Negative control; MiR-760: MicroRNA-760; GIT1: G-protein-coupled receptor kinase interacting protein-1.

Figure 6

Relevance of microRNA-760/G-protein-coupled receptor kinase interacting protein-1 axis in human gastric cancer. A: High expression of microRNA-760 was associated with a good prognosis in gastric cancer patients; B: G-protein-coupled receptor kinase interacting protein-1 (GIT1) expression in gastric cancer tissues; C: High expression of GIT1 was associated with a poor prognosis in gastric cancer patients; D: Up-regulation of GIT1 was related to cell proliferation and apoptosis in gastric cancer cells. HR: Hazard ratio; MiR-760: MicroRNA-760.