miR-196b-5p Promotes Proliferation, Migration and Invasion of Lung Adenocarcinoma Cells via Targeting RSPO2

Abstract

Objective: To explore the biological role of miR-196b-5p/RSPO2 in the occurrence and development of lung adenocarcinoma (LUAD) and to provide a basis for finding new therapeutic targets for LUAD.

Methods: Differentially expressed genes were analyzed based on LUAD microarray, and the target gene of the target miRNA was predicted. qRT-PCR was used to detect the expression levels of miR-196b-5p and RSPO2 mRNA in normal human bronchial epithelial cell line BEAS-2B and LUAD cell lines A549, NCI-H1792 and NCI-H226. Western blot was used to evaluate protein expression. Cell proliferative, migratory and invasive abilities were detected by CCK-8 and transwell assays. Dual-luciferase assay was conducted to verify the targeting relationship between miR-196b-5p and RSPO2.

Results: The results of qRT-PCR showed that miR-196b-5p was significantly highly expressed in LUAD cells, and the expression level of its downstream target gene RSPO2 was significantly decreased. The results of CCK-8 and transwell assays exhibited that miR-196b-5p promoted proliferation, migration and invasion of LUAD cells, while RSPO2 inhibited the malignant progression of LUAD cells. Dual-luciferase assay confirmed the targeted binding relationship between miR-196b-5p and RSPO2. Overexpression of RSPO2 partially reversed the promotion of miR-196b-5p on proliferation, migration and invasion of LUAD cells.

Conclusion: miR-196b-5p promoted proliferation, migration and invasion of LUAD cells by targeting and down-regulating RSPO2, which provided ideas for searching new targets for the diagnosis and treatment of LUAD.

Keywords: RSPO2; invasion; lung adenocarcinoma; miR-196b-5p; proliferation.

Figure 1

miR-196b-5p is highly expressed in LUAD tissue and cells. (A) Volcano map of DEmRNAs in TCGA-LUAD. (B) The box plot of miR-196b-5p expression in normal tissue (blue) and tumor tissue (red). (C) The expression of miR-196b-5p in human normal bronchial epithelial cell line and three LUAD cell lines was detected by qRT-PCR.. All results were representative of three independent experiments. *p<0.05, **p<0.01.

Figure 2

miR-196b-5p can promote proliferation, migration and invasion of LUAD cells. (A) miR-196b-5p expression in A549 cells transfected with miR-196b-5p mimic or miR-196b-5p inhibitor. (B and C) Cell viability of cells in the miR-196b-5p mimic and miR-196b-5p inhibitor groups was detected by CCK-8 assay. (D and E) Transwell migration and invasion assays detected the effects of miR-196b-5p on migratory and invasive abilities of A549 cells (100×). All results were representative of three independent experiments. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

miR-196b-5p targets and down-regulates RSPO2. (A) The target genes of miR-196b-5p predicted by miRDB, mirDIP, TargetScan and starBase databases were intersected with down-regulated DEmRNAs. (B) Pearson correlation of miR-196b-5p and the candidate target genes TGFBR3, ERG and RSPO2. (C) Pearson correlation of miR-196b-5p and RSPO2. (D) The box plot of RSPO2 expression in normal tissue (green) and tumor tissue (red). (E) The correlation of RSPO2 and clinical stage. (F) The mRNA expression of RSPO2 in human normal bronchial epithelial cell line and three LUAD cell lines was detected by qRT-PCR. (G) The putative binding sites of RSPO2 3ʹ-UTR and miR-196b-5p. (H) The luciferase activity was detected by dual-luciferase reporter gene assay. (I and J) The mRNA and protein expression levels of RSPO2 were analyzed by qRT-PCR and Western blot in A549 cells transfected with miR-196b-5p mimic or inhibitor. All results were representative of three independent experiments. *p<0.05, **p<0.01.

Figure 4

Silencing RSPO2 promotes proliferation, migration and invasion of LUAD cells. (A and B) The mRNA and protein expression levels of RSPO2 in A549 cells transfected with RSPO2 siRNA were detected by qRT-PCR and Western blot. (C) Cell viability was determined by CCK-8 after RSPO2 was silenced. (D and E) Transwell migration and invasion assays detected migratory and invasive capacities of A549 cells (100×). All results were representative of three independent experiments. *p<0.05, ***p<0.001.

Figure 5

Overexpression of RSPO2 reverses the promotion of miR-196b-5p on proliferation, migration and invasion of LUAD cells. (A and B) qRT-PCR and Western blot were used to analyze RSPO2 mRNA and protein expression levels in NC mimic+oe-NC, miR-196b-5p mimic+oe-NC and miR-196b-5p mimic+oe-RSPO2 groups. (C) CCK-8 detected the proliferative capacity of cells in each group. (D and E) Transwell migration and invasion assays detected the migratory and invasive capacities of A549 cells in each group (100×). All results were representative of three independent experiments. *p<0.05, **p<0.01.