MiR-140-3p Impedes Gastric Cancer Progression and Metastasis by Regulating BCL2/BECN1-Mediated Autophagy

Abstract

Introduction: MiRNAs have been proven to modulate the progression of gastric cancer (GC). In this field, we evaluated the role and mechanism of miR-140-3p in GC.

Methods: Western blotting and qRT-PCR were used to detect the levels of miR-140-3p and BCL2. The interaction of miR-140-3p and BCL2 was confirmed by dual-luciferase reporter and miRNA pull-down assays. CCK-8, EdU, wound healing, and Transwell invasion assays were performed to evaluate cell proliferation, migration and invasion. Autophagy was analyzed using Western blot analysis of the LC3-II/I ratio and immunofluorescence staining. A xenograft model was established to reveal the role of miR-140-3p in tumorigenesis.

Results: In GC cell lines and tissues, miR-140-3p was highly expressed, and BCL2 was expressed at low levels. MiR-140-3p directly inhibited BCL2 expression and indirectly promoted BECN1 expression, and BCL2 inhibited BECN1 expression. MiR-140-3p overexpression or silencing restrained or facilitated migration, invasion and EMT in GC cells. Moreover, we noticed that overexpression or downregulation of miR-140-3p promoted or suppressed BECN1-dependent autophagy in GC cells. BCL2 introduction or BECN1 silencing in GC cells partially blocked the effects of miR-140-3p. In conclusion, miR-140-3p directly downregulated the expression of BCL2, BCL2 downregulation further activated BECN1-dependent autophagy, and autophagy activation further inhibited EMT.

Conclusion: miR-140-3p may act as a tumor suppressor by targeting BCL2 and regulating downstream BECN1-induced autophagy and metastasis in GC progression.

Keywords: BCL2; BECN1; EMT; autophagy; gastric cancer; miR-140-3p.

Figure 1

MiR-140-3p was downregulated in GC tissues and cell lines.

Figure 2

MiR-140-3p directly targeted BCL2 in GC cell lines.

Figure 3

BCL2 was negatively regulated by miR-140-3p in GC tissues and cell lines.

Figure 4

MiR-140-3p inhibits proliferation and promotes apoptosis by targeting BCL2.

Figure 5

MiR-140-3p inhibited migration and invasion and enhanced autophagy by targeting BCL2.

Figure 6

BECN1 interfered with autophagy regulated by miR-140-3p in GC cell lines.

Figure 7

BECN1 interfered with migration, invasion and EMT regulated by miR-140-3p. (A) A wound healing assay was used to detect the migration of BGC-823 and MKN45 cells; scale bar = 100 μm. (B) A Transwell assay was used to detect cell invasion; scale bar = 100 μm. (C) The protein expression of Vimentin, N-cadherin, E-cadherin, and LC3 II/I was determined using Western blot analysis. *P<0.05; **P<0.01; ***P<0.001.

Figure 8

MiR-140-3p regulated tumorigenesis in a nude mouse model.