To investigate the effects of LncRNA MIR4435-2HG on proliferation, invasion and migration of gastric cancer cells and its mechanism.

Normal human gastric mucosa epithelial cells (GES-1) and gastric cancer cell lines (including AGS, SGC7901, BGC823 and BGC803) were cultured in vitro. The levels of MIR4435-2HG and HMGA1 mRNA were detected by qRT-PCR and HMGA1 protein was detected by Western Blot. Dual luciferase experiment was used to verify the relationships between MIR4435-2HG and miR-138-5p, miR-138-5p and HMGA1 in AGS cells. AGS cells were divided into NC group (control), si-con group (transfected with random nonsense negative sequence), si-MIR4435-2HG group (transfected with MIR4435-2HG small interfering RNA), miR-con group (transfected with mimic control sequence), miR-138-5p group (transfected with miR-138-5p mimics), si-HMGA1 group (transfected with HMGA1 small interfering RNA), si-MIR4435-2HG+pcDNA group (co-transfected with MIR4435-2HG small interfering RNA and empty vector) and si-MIR4435-2HG+pcDNA-HMGA1 group (co-transfected with MIR4435-2HG small interfering RNA and HMGA1 overexpression vector). Then, in all the cell lines, cell proliferation was detected by MTT, and cell migration and invasion were detected by Transwell, at the same time, the protein levels of Ki67, CyclinD1, MMP2 and MMP9 were detected by Western blot. Another in vivo animal model was established, nude mice were divided into NC group (inoculated with normal cultured AGS cells), sh-MIR4435-2HG group (inoculated and transfected with AGS cells carrying the short hairpin RNA lentivirus of MIR4435-2HG gene) and sh-con group (inoculated with AGS cells transfected with shRNA lentiviruses carrying irrelevant sequence fragments). Then, the tumor weight was measured after 21 days, and the effect of MIR4435-2HG on the growth of transplanted tumors in nude mice was observed.

Compared with GES-1, the levels of MIR4435-2HG in AGS, SGC7901, BGC823 and BGC803 were significantly increased (P < 0.05), and the levels of HMGA1 mRNA and protein were significantly increased (P < 0.05). Thus, AGS cells were used to conduct follow-up experiments and the results were showed that MIR4435- 2HG negatively regulated by miR-138-5p, and miR-138-5p negatively regulated by HMGA1. Compared with the si-con group, 48 h later, the OD value in the si-MIR4435-2HG group significantly reduced (1.13±0.12 vs 0.86±0.09, P < 0.05), migration and invasion rate also significantly reduced (179.23±18.01 vs 60.15±6.05, 81.26±8.16 vs 25.64±2.59, respectively, P < 0.05), and the protein expression of Ki67, CyclinD1, MMP2 and MMP9 also significantly reduced (P < 0.05). Compared with the miR-con group, after 48 h, the OD value in the miR-138-5p group reduced (1.28±0.13 vs 0.85±0.09, P < 0.05), and migration and invasion rate also significantly reduced (178.26±17.59 vs 69.35±6.34, 81.02±8.06 vs 36.76±2.49, respectively, P < 0.05), and the protein expression of Ki67, CyclinD1, MMP2 and MMP9 significantly reduced (P < 0.05). Compared with the si-con group, the OD value in the si-HMGA1 group reduced at 48 h (1.28±0.13 vs 0.83±0.09, P < 0.05), migration and invasion rate reduced significantly (175.62±17.59 vs 63.26±6.34, 80.16±8.06 vs 24.56±2.49, respectively, P < 0.05), and the protein expression of Ki67, CyclinD1, MMP2 and MMP9 significantly reduced (P < 0.05). Compared with si-MIR4435-2HG+pcDNA group, the OD value in the si-MIR4435-2HG+pcDNA-HMGA1 group significantly increased at 48 h (0.80±0.09 vs 1.01±0.11, P < 0.05), and migration, invasion rate also significantly increased (60.45±5.79 vs 119.25±12.46, 23.46±2.34 vs 59.46±6.29, respectively, P < 0.05), and the protein expression of Ki67, CyclinD1, MMP2 and MMP9 increased (P < 0.05). Compared with the sh-con group, only the weight of transplanted tumors in nude mice of sh-MIR4435-2HG group was significantly reduced [(0.40±0.03)g vs (0.13±0.03)g, P < 0.05].

MIR4435-2HG was highly expressed in gastric cancer cells. Silencing MIR4435-2HG expression may inhibit the proliferation, migration and invasion of gastric cancer cells in vitro and inhibit tumor growth in vivo by regulating miR-138-5p/HMGA1 axis.