Down-Regulation of ZEB1 by miR-199a-3p Overexpression Restrains Tumor Stem-Like Properties and Mitochondrial Function of Non-Small Cell Lung Cancer

Abstract

Objective

MicroRNA-199a-3p (miR-199a-3p or miR-199b-3p) targeting of 3ʹ-UTR of ZEB1 was characterized as an important way to inhibit invasion and metastases in non-small cell lung cancer (NSCLC), one of the most common cancers around the world. Here we aimed to investigate the tumor-suppressive role of miR-199a-3p targeted ZEB1.

Materials and Methods

A549 cells were transfected with ZEB1 and/or miR-199a-3p. Then, tumor growth was investigated in xenograft mice. Stem-like property, proliferation and mitochondria injury were further validated in vitro.

Results

Overexpression of miR-199a-3p with premiRNAs significantly reduced tumor growth inhibited CD44 and Ki67 and increased Caspase-3 in A549 xenograft mice. Sphere formation and protein expression of stem-like markers showed that miR-199a-3p inhibited stemness of A549 cell. miR-199a-3p reduced proliferation of A549 cells, as showed with EdU staining and reduced expression of Ki67. Transfection of miR-199a-3p also promoted apoptosis, as indicated with increased apoptotic cells with flow cytometry, and increased cleaved Caspase-3/Caspase3 and Bcl-2/Bax. Apoptosis was further validated to be induced with mitochondria dysfunction, which indicated with JC-1 labeled loss of mitochondrial membrane potential, reduced activity of SOD, and increased MDA and LDH. All these effects were inverted with overexpression of ZEB1.

Conclusion

Altogether, the findings suggested that the up-regulation of miR-199a-3p significantly inhibited NSCLC growth in vivo, and reduced A549 cell proliferation and promoted mitochondrial-mediated apoptosis, through down-regulation of ZEB1. The findings supported ZEB1 down-expression with miR-199a-3p as a novel therapeutic target for NSCLC treatment.