Research Paper Volume 13, Issue 12 pp 16786—16803

Figure 5. MiR-27b-3p binds and negatively modulates Grb2, and Grb2 can reverse inhibition of GC cell promotion brought by miR-27b-3p. (A) The complementary sequence between miR-27b-3p and Grb2 3’UTR. (B) Luciferase reporter assay verifies a direct relationship between miR-27b-3p and Grb2 (P = 0.0041). (C) Across 60 GC tissue samples, Grb2 levels were higher compared to adjacent cancer (P < 0.0001). (D) Grb2 levels in GC cell lines is higher than that in gastric mucosa epithelial cells. AGS had the highest expression, while HGC-27 was relatively low. (E) Grb2 mRNA expression was lower in the miR-27b-3p mimics group (P = 0.0376) and was higher in the miR-27b-3p inhibitor group (P = 0.0034). (F) Grb2 expression in GC and adjacent tissues was detected through western blot (P < 0.0001). (G) There is a negative correlation between Grb2 and miR-27b-3p expression. (H–I) Up-regulation of Grb2 mRNA and protein levels were determined using qRT-PCR and WB after Grb2 ov in the miR-27b-3p mimics group (P = 0.0019). (J–K) CCK-8 and BrdU assays (P = 0.0128) detected GC cell viability and proliferation ability, respectively. Up-regulated Grb2 mRNA levels were positively associated with GC cell growth. (L) Colony formation assay detected the numbers of colony formation (P = 0.0075). (M) GC cell apoptosis was inhibited upon overexpression of Grb2, as examined by flow cytometry assay (P = 0.0009). (N–O) Accelerated cell migration (P = 0.0089) and invasion (P = 0.0125) abilities were found by transwell assays brought on by increased Grb2 levels. (P) Wound healing assay was utilized to discover GC cell metastasis, which indicated that high Grb2 levels promote GC cell metastasis (P = 0.0017).