MiR-96-5p Facilitates Lung Adenocarcinoma Cell Phenotypes by Inhibiting FHL1

Abstract

Objective: FHL1 is understood as a tumor repressor gene in various cancers and a possible target for cancer treatment. We investigated the influences of FHL1 on cell functions as well as its molecular mechanisms in lung adenocarcinoma (LUAD) cells.

Methods: The miRNA-mRNA modulatory axis was predicted by bioinformatics. The expression levels of FHL1 mRNA and protein in LUAD cells were, respectively, analyzed by qRT-PCR and western blot. Dual luciferase analysis was introduced to verify the interaction between miR-96-5p and FHL1. CCK-8, cell colony formation, and Transwell assays were utilized to analyze proliferation, colony formation, migration, and invasion of A549 cells.

Results: Expression of FHL1 mRNA and protein in LUAD tissue and cells was downregulated, which was linked with poor prognoses of patients. In addition, FHL1 overexpression could hamper colony formation, proliferation, invasion, and migration of LUAD cells. In addition, dual-luciferase analysis verified miR-96-5p as an upstream regulator of FHL1. Overexpression of miR-96-5p suppressed FHL1 expression in LUAD cells and promoted proliferation, invasion, and migration of LUAD cells, while overexpression of FHL1 could simultaneously restore the above-mentioned promoting effect.

Conclusion: MiR-96-5p fostered cell malignant behaviors by targeting FHL1. This research uncovered the regulatory mechanism of FHL1 in LUAD and offered optional therapeutic targets for LUAD patients.

Figure 1

FHL1 expression is notably decreased in LUAD cells. (a) Violin plot of FHL1 level in normal group (green; n = 59) and tumor group (orange; n = 535); (b) survival curves of high-FHL1 (red) and low-FHL1 (blue) groups. Abscissa represents time (year), and ordinate represents survival rate; (c) FHL1 mRNA expression in A549, H1650, H441, H1299, and BEAS-2B cells; (d) FHL1 protein level in cells; ^∗p < 0.05.

Figure 2

Overexpression of FHL1 hinders cell phenotype progression of LUAD. (a) FHL1 level in LUAD A549 cells in oe-NC and oe-FHL1; (b) proliferative efficiency of A549 cells in various transfection groups; (c) colony formative capability of A549 cells in various transfection groups; (d and e) migratory and invasive properties of two groups of A549 cells (100×); ^∗p < 0.05.

Figure 3

MiR-96-5p downregulates FHL1 expression in LUAD cells. (a) Volcano map of differential miRNAs in normal and tumor groups in TCGA database. Red indicates differentially upregulated miRNAs, and green indicates differentially downregulated miRNAs; (b) Venn diagram of predicted upstream miRNAs of FHL1 and differential miRNAs; (c) Pearson correlation analysis of FHL1 and its predicted upstream miRNAs; (d) violin plot of miR-96-5p expression in normal (green; n = 46) and tumor (orange; n = 521) tissue; (e) MiR-96-5p level in BEAS-2B and A549, H1650, H441, and H1299 cell lines; (f) diagram of binding of miR-96-5p and FHL1-WT and FHL1-MUT sequences; (g) luciferase activity of A549 cells in treatment groups (miR-NC and miR-mimic); (h) FHL1 mRNA level in A549 cells; (i) FHL1 protein expression in A549 cells; ^∗p < 0.05.

Figure 4

MiR-96-5p facilitates cell malignant behaviors via suppressing FHL1. (a) FHL1 mRNA and protein levels in LUAD A549 cells in varying groups; (b) A549 cell proliferative properties in varying groups; (c) cell colony formation of A549 cells in varying groups; (d and e) migratory and invasive abilities of A549 cells in varying groups (100×); ^∗p < 0.05.