MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

Abstract

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells.

Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells.

Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration.

Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

Keywords: AQP4; invasion; lung adenocarcinoma; miR-196b; migration.

Figure 1.

Differential and survival analyses of miR-196b and AQP4 in LUAD tissue and cells. (A) Volcano plot of DEmiRNAs in TCGA-LUAD (the red dots represent differentially up-regulated genes and the blue dots refer to differentially down-regulated genes); (B) relative expression of miR-196b in tumor tissue (n = 521) and normal tissue (n = 46) in the TCGA-LUAD dataset; (C) survival significance of miR-196b; (D) volcano Plot of DEmRNAs in TCGA-LUAD (the red dots represent differentially up-regulated genes and the blue dots refer to differentially down-regulated genes); (E): Venn diagram of differentially down-regulated mRNAs in TCGA and predicted target genes on miRDB, mirDIP and TargetScan databases; (F) correlation analysis of miR-196b and 4 candidate target genes; (G) negative correlation between miR-196b and AQP4; (H) relative expression of AQP4 in tumor tissue (n = 535) and normal tissue (n = 59) in the TCGA-LUAD dataset; (I) survival significance of AQP4.

Figure 2.

MiR-196b is up-regulated in LUAD cells and promotes the invasion and migration of cancer cells. (A) The expression of miR-196b in human normal lung fibroblasts IMR-90 and LUAD cells NCI-H1568, NCI-H1563 and NCI-H2030; (B) The expression of miR-196b in LUAD cell line NCI-H2030 after transfection with miR-196b mimic and NC mimic, respectively; (C) the effect of miR-196b overexpression on the invasion of NCI-H2030 cells; (D) the effect of miR-196b overexpression on the migration of NCI-H2030 cells. Note: “*” in Figure A refers to comparison with IMR-90, p < 0.05; “*” in Figure B, C, and D refer to comparison with NC mimic, p < 0.05.

Figure 3.

AQP4 is down-regulated in LUAD cells and inhibits the invasion and migration of cancer cells. (A) The expression of AQP4 in human normal lung fibroblasts IMR-90 and LUAD cells NCI-H1568, NCI-H1563 and NCI-H2030; (B) the expression of AQP4 in LUAD cell line NCI-H2030 after transfection with oe-NC and oe-AQP4, respectively; (C) the effect of AQP4 overexpression on the invasion of NCI-H2030 cells; (D) the effect of AQP4 overexpression on the migration of NCI-H2030 cells. note: “*” in Figure A refers to comparison with IMR-90, p < 0.05; “*” in Figure B, C and D refer to comparison with NC mimic, p < 0.05.

Figure 4.

MiR-196b targets and inhibits the expression of AQP4. (A) Putative binding sites of miR-196b on AQP4 mRNA 3′UTR; (B) results of dual-luciferase reporter assay; (C) the effect of miR-196b overexpression on AQP4 protein expression. Note: “*” refers to comparison with NC mimic, p < 0.05.

Figure 5.

Overexpression of AQP4 reverses the promotive effect of miR-196b on the invasion and migration of LUAD cells. (A) The expression of miR-196b in cells of each group (NC mimic + oe-NC, miR-196b mimic + oe-NC, miR-196b mimic + oe-AQP4); (B) the protein expression of AQP4 in cells of each group; (C): invasive ability of cells in each group; (D) migratory ability of cells in each group. Note: “*” refers to comparison with NC mimic + oe-NC group, p < 0.05; “#” refers to comparison with miR-196b mimic + oe-NC group, p < 0.05.