High Expression of lncRNA AFAP1-AS1 Promotes the Progression of Colon Cancer and Predicts Poor Prognosis

Abstract

Long non-coding RNAs (lncRNAs) are dysregulated in various cancers. However, the clinical relevance and functional roles of AFAP1-AS1 in colon cancer (CC) have not been clarified. We analyzed the lncRNA expression patterns in Gene Expression Omnibus (GEO) datasets and the Cancer Genome Atlas (TCGA) RNA-seq datasets, and found that the expression level of AFAP1-AS1 was significantly elevated in CC tissues. High levels of AFAP1-AS1 were associated with poor disease-free survival and overall survival in CC patients. In vitro experiments demonstrated that AFAP1-AS1 knockdown significantly inhibited the cell invasive and migration capability in CC cell line HT-29. AFAP1-AS1 knockdown also increased the expression of E-cadherin and ZO-1 while inhibited the expression of Vimentin, MMP9, ZEB1 and β-catenin, suggesting that AFAP1-AS1 is involved in the epithelial-mesenchymal transition (EMT) process of CC. Further studies confirmed that AFAP1-AS1 knockdown also affected the actin-cytokeratin signaling pathway. Thus, AFAP1-AS1 might be a potential novel diagnostic marker and therapeutic target for CC.

Keywords: AFAP1 antisense RNA 1 (AFAP1-AS1); colon cancer (CC); long non-coding RNA (lncRNA); metastasis; prognosis.

Figure 1

AFAP1-AS1 is elevated in CC tissues relative to normal tissues. The AFAP1-AS1 expression data in CC tissues were obtained from the GEO datasets in accession GSE39582 (A) and GSE37364 (B), Analysis revealed that CC tissues have a higher AFAP1-AS1 expression level than normal tissues. (C) Comparison of AFAP1-AS1 expression levels in CC tissues and normal tissues from the TCGA-COAD RNA-Seq dataset within the Cancer RNA-Seq Nexus database.

Figure 2

High AFAP1-AS1 is associated with poor prognosis in CC patients. CC patients with high AFAP1-AS1 have shorter overall survival (OS, A) and disease-free survival (DFS, B). Data were obtained from the TCGA-COAD RNA-Seq dataset within the GEPIA database. The Kaplan-Meier method was used to calculate curves.

Figure 3

AFAP1-AS1 knockdown inhibits cell migration and invasion. (A) AFAP1-AS1 expression levels across four CC cell lines were measured by qRT-PCR. (B) Expression levels of AFAP1-AS1 in HT-29 cells were measured by qRT-PCR after transfected with siRNAs targeting AFAP1-AS1 (siRNA1 and siRNA2), or a scrambled negative control (NC). qRT-PCR results indicated that the siRNA2 had the strongest inhibitory effect. (C) Representative images of cells that migrated into the scratched area and (D) the gap distance of each groups at 0 h, 24 h and 48 h. (E) Representative images of invasive cells transfected with NC or with siRNA2. (F) Measurement of the relative number of invaded cells in each group.*P< 0.05, **P < 0.01, ***P < 0.001.

Figure 4

AFAP1-AS1 knockdown influences EMT-associated gene expression and the actin-cytokeratin signaling pathway. (A) qRT-PCR-measured mRNA expression of E-cadherin, vimentin, MMP9 and ZEB1 in siRNA2-transfected HT-29 cells. (B) The protein expression of E-cadherin, Vimentin, MMP9, ZEB1, β-catenin and ZO-1 were detected by Western blotting. (C) Western blotting analysis showed that CTTN, RHOA and RAB1B were elevated while MPRIP decreased after silencing AFAP1-AS1.