LncRNA FAS-AS1 upregulated by its genetic variation rs6586163 promotes cell apoptosis in nasopharyngeal carcinoma through regulating mitochondria function and Fas splicing

Abstract

Nasopharyngeal carcinoma (NPC) is a common head and neck malignant with a high incidence in Southern China. Genetic aberrations play a vital role in the pathogenesis, progression and prognosis of NPC. In the present study, we elucidated the underlying mechanism of FAS-AS1 and its genetic variation rs6586163 in NPC. We demonstrated that FAS-AS1 rs6586163 variant genotype carriers were associated with lower risk of NPC (CC vs. AA, OR = 0.645, P = 0.006) and better overall survival (AC + CC vs. AA, HR = 0.667, P = 0.030). Mechanically, rs6586163 increased the transcriptional activity of FAS-AS1 and contributed to ectopic overexpression of FAS-AS1 in NPC. rs6586163 also exhibited an eQTL trait and the genes affected by rs6586163 were enriched in apoptosis related signaling pathway. FAS-AS1 was downregulated in NPC tissues and over-expression of FAS-AS1 was associated with early clinical stage and better short-term treatment efficacy for NPC patients. Overexpression of FAS-AS1 inhibited NPC cell viability and promoted cell apoptosis. GSEA analysis of RNA-seq data suggested FAS-AS1 participate in mitochondria regulation and mRNA alternative splicing. Transmission electron microscopic examination verified that the mitochondria was swelled, the mitochondrial cristae was fragmented or disappeared, and their structures were destroyed in FAS-AS1 overexpressed cells. Furthermore, we identified HSP90AA1, CS, BCL2L1, SOD2 and PPARGC1A as the top 5 hub genes of FAS-AS1 regulated genes involved in mitochondria function. We also proved FAS-AS1 could affect Fas splicing isoform sFas/mFas expression ratio, and apoptotic protein expression, thus leading to increased apoptosis. Our study provided the first evidence that FAS-AS1 and its genetic polymorphism rs6586163 triggered apoptosis in NPC, which might have a potential as new biomarkers for NPC susceptibility and prognosis.

Figure 1

The role of FAS-AS1 rs6586163 in NPC. (a) The association of FAS-AS1 rs6586163 and overall survival of NPC patients in co-dominant model. (b) The association of FAS-AS1 rs6586163 and overall survival of NPC patients in dominant model. (c) The association of FAS-AS1 rs6586163 and overall survival of NPC patients in recessive model. (d) Relative expression of FAS-AS1 in NPC tissues with different genotypes of rs6586163. (e) The chromatin state at rs6586163. (f) Relative luciferase activity in NPC cells that transfected with rs6586163 C allele (MUT) reporter plasmid or rs6586163 A allele (WT) reporter plasmid. (g) The local centroid secondary structure (up) and the mountain plot indicating the positional entropy for each position (down) of the wild type FAS-AS1. (h) The local centroid secondary structure (up) and the mountain plot indicating the positional entropy for each position (down) of rs6586163 mutant FAS-AS1. (i) GO enrichment analysis of genes affected by FAS-AS1 rs6586163. (j) PPI network was constructed of the genes regulated by rs6586163. *P < 0.05, **P < 0.01.

Figure 2

FAS-AS1 was downregulated in NPC and correlated with prognosis of various cancers. (a) The gene expression profile of FAS-AS1 across all tumor samples and paired normal tissues. Each dots represent expression of samples. (b) Relative expression of FAS-AS1 in NPC tissues and rhinitis tissues were examined by RT-qPCR. (c) The expression level of FAS-AS1 was associated with clinical stage of NPC patients. (d) The expression level of FAS-AS1 was associated with short-term treatment efficacy of NPC patients. (e) Relative expression of FAS in NPC tissues and rhinitis tissues were examined by RT-qPCR. (f) The expression level of FAS was associated with clinical stage of NPC patients. (g) The expression level of FAS was not associated with short-term treatment efficacy of NPC patients. (h) Correlation analysis of FAS-AS1 and FAS in NPC tissues. (i–l) FAS-AS1 was associated with survival in breast cancer (i), ovarian cancer (j), lung cancer (k), and gastric cancer (l). *P < 0.05, **P < 0.01.

Figure 3

FAS-AS1 inhibits NPC cell viability. (a) Relative expression of FAS-AS1 in NPC cell lines (HNE1, HONE1) and normal nasopharyngeal epithelial cell line NP69. (b) Cell viability was measured by CCK-8 assay in HNE1 cells. (c) Cell viability was measured by CCK-8 assay in HONE1 cells. (d) The morphology of HNE1 and HONE1 cells 24 h after transfection with FAS-AS1 vector or control vector. (e) Cell viability was measured by CellTiter luminescent cell viability assay in HNE1 cells. (f) Cell viability was measured by CellTiter luminescent cell viability assay in HONE1 cells. (g) Cell cycle was determined by flow cytometry 24 h after transfection with FAS-AS1 vector or control vector. (h) Quantitative analysis of cells in each cell cycle phase in HNE1 cells. (i) Quantitative analysis of cells in each cell cycle phase in HONE1 cells.

Figure 4

FAS-AS1 promotes NPC cell apoptosis. (a) Representative picture of Annexin V-FITC cell apoptosis assay in FAS-AS1 overexpression group or control group. (b) Quantitative analysis of the apoptotic percentage of Annexin V-FITC cell apoptosis assay. (c) Representative image of Hoechst staining in FAS-AS1 overexpression group or control group. (d) Quantitative analysis of the apoptotic percentage of Hoechst staining. (e) Representative image of SYTO-9/PI staining in FAS-AS1 overexpression group or control group. (f) Quantitative analysis of the apoptotic percentage of SYTO-9/PI staining. *P < 0.05, **P < 0.01.

Figure 5

GSEA analysis of gene sets regulated by FAS-AS1. (a–h) The top eight biological process terms enriched by GSEA analysis. (i) The hub genes of mitochondria related DEGs regulated by FAS-AS1. (j) The heatmap of apoptosis related genes in FAS-AS1 overexpression group and control group was generated by an online platform named Bioinformatics (http://www.bioinformatics.com.cn/). (k) KEGG enrichment analysis of the apoptosis related DEGs was analyzed by Metascape (https://metascape.org/gp/index.html#/main/step1) and visualized by Bioinformatics.

Figure 6

FAS-AS1 promotes apoptosis through regulating mitochondria function and Fas splicing. (a) Representative picture of Mito-Tracker red labeled mitochondria in FAS-AS1 overexpressed cells or control cells. (b) Quantitative analysis of the representative picture’s fluorescence intensity. (c) Microstructure of mitochondria was observed by TEM in FAS-AS1 overexpressed cells or control cells. (d) Relative expression of sFas was determined by RT-qPCR 24 h after transfection with FAS-AS1 vector or control vector. (e) Relative expression of mFas was determined by RT-qPCR 24 h after transfection with FAS-AS1 vector or control vector. (f) The expression of several cell apoptosis signal regulators (cleaved-caspase 3, Bcl-2 and Bax) were examined by western blotting after transfection with FAS-AS1 vector or control vector. (g) The quantification of cleaved-caspase 3. (h) The quantification of Bax/Bcl-2. *P < 0.05, **P < 0.01.

Figure 7

The regulatory mechanism of rs6586163 and FAS-AS1 in NPC. FAS-AS1 rs6586163 increases the transcriptional activity of FAS-AS1 and contributed to ectopic overexpression of FAS-AS1 in NPC. Overexpression of FAS-AS1 induces apoptosis by regulating mitochondrial function and Fas alternative splicing, which is followed by activation of caspases 3, up-regulation of bax and down-regulation of bcl2.