LncRNA FOXD2-AS1 Increased Proliferation and Invasion of Lung Adenocarcinoma via Cell-Cycle Regulation

Abstract

Background: Long non-coding RNA FOXD2 antisense RNA 1 (FOXD2-AS1) has been reported in many malignancies. However, the molecular mechanism of many actions is not clarified. This study was conducted to investigate the function of FOXD2-AS1 in lung adenocarcinoma and its molecular mechanism.

Methods: Bioinformatics and in vitro analysis including RT-qPCR, CFU, CCK8, Transwell, Cell Apoptosis and Cell Cycle Assay were used for the analysis of gene expression and related effects.

Results: It revealed increased expression of lncRNA FOXD2-AS1 in lung adenocarcinoma cell lines (A549 cells), and abundant expression of lncRNA FOXD2-AS1 was also observed in the acquired lung adenocarcinoma tissues. In vitro results showed that knockdown of lncRNA FOXD2-AS1 in A549 cells weakened cell proliferation, invasion and increased apoptosis. At the same time, we found that reducing the expression of lncRNA FOXD2-AS1 caused cell cycle arrest in the G1/S phase. Differential gene analysis of lung adenocarcinoma and adjacent normal tissues showed that the cell cycle and its related process regulation were significantly enriched. Gene Set Enrichment Analysis (GSEA) analysis showed that miR-206, miR-143, lL6-JAK-STAT3 signalling pathway, STAT3, E2F targets, EZH2, P53 signalling pathway and E2F3 targets interacting with lncRNA FOXD2-AS1 were also enriched.

Conclusion: This study demonstrates the role and mechanism of the lncRNA FOXD2-AS1 in lung adenocarcinoma and provides a better understanding for the treatment of lung adenocarcinoma, which indicates that interfering with lncRNA FOXD2-AS1 expression may be a novel strategy.

Keywords: cell cycle; lncRNA FOXD2-AS1; lung adenocarcinoma; non-small cell lung cancer; proliferation.

Figure 1

DEGs and LncRNA FOXD2-AS1 between lung adenocarcinoma and adjacent normal tissues. (A) The number of DEGs; (B) Top 5 up-regulation genes; (C) Top 5 down-regulation genes. (D) qRT-PCR was utilized to detect LncRNA FOXD2-AS1 in NSCLC. (E) The expression of LncRNA FOXD2-AS1 in lung adenocarcinoma from GEO datasets (GSE178466). ***p<0.001.

Figure 2

Downregulating LncRNA FOXD2-AS1 inhibits proliferation and invasion of A549 cells. (A) qRT-PCR detected the efficiency of downregulation and overexpression of lncRNA FOXD2-AS1 in A549 cells. (B) The representative picture of colony-forming unit assay. (C) Results of colony-forming unit assay quantification. (D) The proliferation of A549 cells on downregulation and overexpression of lncRNA FOXD2-AS1 at 48h via CCK8. *P < 0.05, ***P < 0.001 and ****P < 0.0001. (E) The representative picture of Transwell. (F) Results of Transwell assay quantification. ****P < 0.0001.

Figure 3

The effect of LncRNA FOXD2-AS1 on the apoptosis rate and cell cycle of lung adenocarcinoma cell line A549. Annexin V-PI Staining (A) and flow cytometry analysis (B) to determine the apoptotic effect of LncRNA FOXD2-AS1 against lung adenocarcinoma cell line A549. In the A549 cell line, downregulation of LncRNA FOXD2-AS1 led to a significant increase in apoptosis, however, overexpression of lncRNA FOXD2-AS1 led to a significant decrease in apoptosis (****P < 0.0001). (C) Cell-cycle analyses by flow cytometry. (D) Quantitative results of cell-cycle (*P < 0.05, ****P < 0.0001).

Figure 4

Enrichment Analysis Results. (A) The different levels of GO function analysis. (B) GO function analysis of DEGs at the levels of molecular function (MF). (C) GO function analysis of DEGs at the levels of biological process (BP). (D) GO function analysis of DEGs at the levels of cell components (CC). (E) Top 20 of KEGG enrichment. (F) Top 20 of reactome enrichment. (G) The circle diagram of KEGG enrichment in different levels. (H) The circle diagram of reactome enrichment in different levels. (I) The specific regulatory relationship of cell cycle (ko04110) analyzed by KEGG.

Figure 5

Potential relationship of LncRNA FOXD2-AS1 in lung adenocarcinomic cell proliferation and invasion. GSEA enrichment analysis was performed on the DEGs. In terms of miRNA target gene sets, result was significantly enriched in miR-206 and miR-143. In terms of hallmark gene sets, result was significantly enriched in lL6-JAK-STAT3 signaling, E2F targets and STAT3. In terms of transcription factor targets gene sets, result was significantly enriched in E2F3. In terms of KEGG gene sets, result was significantly enriched in P53 signaling pathway.