The lncRNA TTN-AS1 Regulates the Proliferation, Apoptosis, and Migration of Gallbladder Cancer Cells Through the miR-138-5p/EGFR Axis

GBC (gallbladder carcinoma) is a common tumor of the biliary system, which seriously threatens human life and health. The aim of this study is to investigate the molecular mechanism of the lncRNA (long non-coding RNA) TTN-AS1 (titin antisense RNA1)/miR-138-5p (microRNA-138-5p)/EGFR (epidermal growth factor receptor) signaling axis in GBC. The relative expression levels of TTN-AS1, miR-138-5p and EGFR mRNA in GBC tissues and cell lines were detected by qRT-PCR, and their correlations in GBC tissues were analyzed. The GBC cell line GBC-SD was cultured and grouped into Control group, si-NC group, si-TTN-AS1 group, si-TTNAS1+in-miR-138-5p group and si-TTN-AS1+pc-EGFR group. qRT-PCR and Western blot were performed to cell transfection efficiency; CCK-8, EdU staining, flow cytometry and scratch healing assay were performed to measure the cell proliferation, apoptosis and migration; Western blot was performed to detect the cell proliferation (PCNA), apoptosis (Cleaved Caspase-3) and migration (MMP-2, MMP-9) related protein levels. In vivo xenograft tumor model was performed to study the impacts of TTN-AS1 on GBC tumor growth, Ki67 and PCNA positive expression, and miR-138-5p, EGFR mRNA expression. The results showed that, the expressions of TTN-AS1 and EGFR mRNA were both up-regulated in GBC tissues and cell lines, the expression of miR-138-5p was down-regulated, the level of TTN-AS1 in GBC tissue was negatively correlated with the level of miR-138-5p, and positively correlated with the level of EGFR mRNA; the level of miR-138-5p was negatively correlated with the level of EGFR mRNA (P<0.05), and the changes were greatest in GBC-SD cells. Bioinformatics analysis and dual luciferase confirmed the interaction of miR-138-5p with TTN-AS1 or EGFR 3′UTR; and RNA pull-down confirmed the binding of TTN-AS1 to miR-138-5p; qRT-PCR and Western blot demonstrated that TTN-AS1 might regulate EGFR expression through miR-138-5p (P<0.05). Knockdown of TTN-AS1 could significantly inhibit the proliferation and migration of GBC-SD cells and induce cell apoptosis (P<0.05); meanwhile, down-regulate the protein levels of PCNA, MMP-2 and MMP-9, and up-regulate the protein level of Cleaved Caspase-3 (P<0.05). Inhibition of miR138-5p or overexpression of EGFR partially reversed the impact of TTN-AS1 knockdown on the malignant behavior of GBC-SD cells. In vivo experiments confirmed that knockdown of TTN-AS1 could inhibit tumor growth, meantime up-regulate miR-138-5p level and down-regulate EGFR mRNA, Ki67 and PCNA positive expression level (P<0.05). In conclusion, TTN-AS1 may play a tumor-promoting role in the occurrence and development of GBC, and it may be achieved by regulating the miR-138-5p/EGFR signaling axis.

CSTR: 32200.14.cjcb.2023.01.0008