Potentiated lung adenocarcinoma (LUAD) cell growth, migration and invasion by lncRNA DARS-AS1 via miR-188-5p/ KLF12 axis

Abstract

Lung adenocarcinoma (LUAD) is the most common histological type of non-small cell lung cancer (NSCLC). Due to the nonspecific early symptoms, the majority of the diagnosed LUAD patients are in the middle and late stages, with multiple metastases, and have missed the optimal period for treatment. Current studies have reported lncRNA DARS-AS1 as a cancer-promoting gene that expedites tumorigenesis. This is the first study demonstrating that DARS-AS1 is involved in the mediating process of LUAD. Cell functional experiments revealed that lncRNA DARS-AS1 participated in enhancing LUAD proliferation, invasion, and migration by inhibiting miR-188-5p. The investigation on DARS-AS1/miR-188-5p led to the discovery of KLF12 as a downstream target of miR-188-5p, and the regulatory pathway was established as DARS-AS1/miR-188-5p/KLF12. According to western blot results, DARS-AS1 promoted LUAD cell growth, migration, and invasion via stimulation of the PI3K/AKT pathway, activating the EMT process, and up-regulating the CyclinD1 and Bcl-2 proteins. This was the first report on the DARS-AS1/miR-188-5p/KLF12 axis and offered a novel strategy for early diagnosis, a new therapeutic method, and an improved prognosis for LUAD.

Keywords: DARS-AS1; Krüppel-like factor 12; lncRNA; lung adenocarcinoma; miR-188-5p.

Figure 1

LncRNA DARS-AS1 was highly expressed in LUAD tissues and cell lines. (A) The top 50 differential expression lncRNA of LUAD identified by a bioinformatics screen in the TCGA database. (B) Expression of lncRNA DARS-AS1 was higher than usual in LUAD tissues from the TCGA database. (C) Higher expression of DARS-AS1 was identified in 50 LUAD tissues compared to normal tissues (p < 0.001). (D) Higher expression of DARS-AS1 was identified in LUAD cell lines compared to the HBE cell line (p < 0.01). (E) High expression of DARS-AS1 in LUAD patients was associated with a poor prognosis (Log-Rank: p = 0.03).

Figure 2

Knockdown of DARS-AS1 resulted in alleviated LUAD progression. (A) The DARS-AS1 expression level was significantly decreased in A549 cells compared to the si-NC group after transfection with si-DARS-AS1 (p < 0.05). DARS-AS1 knockdown resulted in a lower proliferation rate of LUAD cells according to the (B) CCK-8 (p < 0.05), (C) BrdU (p < 0.01), and (D) colony formation (p < 0.01) assays compared to the si-NC group. (E) An increased apoptosis rate was observed in the si-DARS-AS1 group compared to the si-NC group (upper right quadrant + lower right quadrant) (p < 0.01). A549 cells transfected with si-DARS-AS1 exhibited less (F) migrating (p < 0.05) and (G) invading (p < 0.01) cells compared to the si-NC group.

Figure 3

Overexpression of DARS-AS1 resulted in accelerated LUAD progression. (A) Expression of DARS-AS1 was significantly higher in PC-9 cells transfected with DARS-AS1 ov compared to the NC group (p < 0.01). (B) PC-9 cells with DARS-AS1 ov transfection exhibited greater proliferative capability according to the (B) CCK-8 (p < 0.05), (C) BrdU (p < 0.01), and (D) colony formation (p < 0.01) assays compared to the NC group. (E) Flow cytometry results showed a significantly lower apoptosis rate in the DARS-AS1 ov transfection group (upper right quadrant + lower right quadrant) compared to the NC group (p < 0.01). PC-9 with DARS-AS1 ov transfection exhibited a higher number of (F) migrating (p < 0.01) and (G) invading (p < 0.01) cells compared to the NC group.

Figure 4

DARS-AS1 promoted LUAD growth in vivo. (A) LUAD transfected with si-DARS-AS1 had smaller tumors, while (B) the DARS-AS1 ov transfection group had larger tumors. The average tumor volume of LUAD transfected with (C) si-DARS-AS1 (p < 0.05 compared to si-NC) or (D) DARS-AS1 ov (p < 0.05 compared to NC group) was continuously measured over 21 days. After 21 days, the average tumor weight of LUAD transfected with (E) si-DARS-AS1 (p < 0.01) or (F) DARS-AS1 ov (p < 0.01) was scored. (G) The si-DARS-AS1 group had smaller tumors and fewer spreading lesions in the lung than the si-NC group. (H) The DARS-AS1 ov group had larger tumors and more spreading lesions in the lung than the NC group.

Figure 5

MiR-188-5p is a target gene of DARS-AS1. (A) The binding site between DARS-AS1 and miR-188-5p. (B) The dual-luciferase assay results confirmed a direct interaction between DARS-AS1 and miR-188-5p (p < 0.05). (C) The expression level of miR-188-5p was lower than usual in LUAD tissues compared to normal tissues (p < 0.01). (D) The expression level of miR-188-5p was lower than usual in LUAD cell lines compared to the HBE cell line (^**p < 0.01).

Figure 6

Inhibition of miR-188-5p altered the suppression of A549 cell progression mediated by si-DARS-AS1. (A) Three groups were transfected with si-DARS-AS1, si-DARS-AS1+miR-188-5p inhibitor, and si-NC in the A549 cell line. The cell proliferative capacity of the three groups was measured using the (B) CCK-8, (C) BrdU, and (D) colony formation assays (^*p < 0.05). (E) Inhibition of miR-188-5p resulted in a significant reduction in the apoptosis ratio (upper right quadrant+lower right quadrant). The transwell chamber results showed that inhibition of miR-188-5p significantly facilitated cell migration (F) p < 0.01 and invasion (G) p < 0.01, which were suppressed by si-DARS-AS1.

Figure 7

KLF12 is a negative regulatory target of miR-188-5p. (A) The binding site between KLF12 and miR-188-5p. (B) The dual-luciferase assay results confirmed the existence of direct interaction between KLF12 and miR-188-5p (p < 0.05). (C) High expression of KLF12 in LUAD tissues was detected by western blot.

Figure 8

Overexpression of KLF12 promoted LUAD progression. (A) Three groups were transfected using miR-188-5p mimics, miR-188-5p mimics+KLF12 ov, and NC mimics in the A549 cell line. The cell proliferative capacity of the three groups was measured using the (B) CCK-8 (p < 0.05), (C) BrdU (^*p < 0.05), and (D) colony formation (p < 0.01) assays. (E) Overexpression of KLF12 resulted in a significant reduction in the apoptosis ratio (upper right quadrant+lower right quadrant). The transwell chamber results indicated that overexpression of KLF12 significantly promoted cell (F) migration (p < 0.01) and (G) invasion (p < 0.01), which were suppressed by miR-188-5p.

Figure 9

Western blot results demonstrated up-regulation of KLF12, p-PI3K, p-AKT, Bcl-2, Vimentin, and CyclinD1 in the DARS-AS1 overexpression group, while the proteins were down-regulated in the si-DARS-AS1 group.