Long non-coding RNA FAL1 functions as a ceRNA to antagonize the effect of miR-637 on the down-regulation of AKT1 in Hirschsprung's disease

Abstract

Objectives: Emerged evidence demonstrates that long non-coding RNAs (lncRNAs) may play quintessential regulatory roles in the cellular processes, tumourigenesis and the development of disease. Though focally amplified lncRNA on chromosome 1 (FAL1) has been identified to have crucial functions in many diseases, its biological mechanism in the development of Hirschsprung's disease (HSCR) still remains unknown.

Materials and methods: The expression levels of FAL1 in HSCR aganglionic tissues and matched normal specimens were detected by quantitative real-time PCR (qRT-PCR). Cell proliferation and migration were detected by Cell Counting Kit-8 (CCK-8) assay, Ethynyl-deoxyuridine (EdU) assay and transwell assay relatively. Cell cycle and apoptosis were assessed using flow cytometer analysis. Moreover, the novel targets of FAL1 were confirmed with the help of bioinformatics analysis and dual-luciferase reporter assay. Western blot assay as well as RNA immunoprecipitation (RIP) assay was conducted to investigate the potential mechanism.

Results: FAL1 expression was markedly down-regulated in HSCR aganglionic tissues and decreased FAL1 expression was associated with the diagnosis of HSCR. Cell functional analyses indicated that FAL1 overexpressing notably promoted cell proliferation and migration, while down-regulation of FAL1 suppressed cell proliferation and migration. Additionally, Flow cytometry assay demonstrated that knockdown of FAL1 induced markedly cell cycle stalled in the G0/G1 phase. Furthermore, FAL1 could positively regulate AKT1 expression by competitively binding to miR-637.

Conclusions: These results illuminated that FAL1 may work as a ceRNA to modulate AKT1 expression via competitively binding to miR-637 in HSCR, suggesting that it may be clinically valuable as a biomarker of HSCR.

Figure 1

The expression of FAL1 in HSCR.( )A, The expression of FAL1 was analysed in HSCR aganglionic tissues (n = 64) and relatively normal tissues (n = 64) by qRT‐PCR. FAL1 was significantly reduced in patients’ aganglionic tissues compared with control tissues. B, Receiver‐Operating Characteristic (ROC) curve analysis of the diagnostic potential of FAL1 in HSCR tissues. ***P < .0001, data represent the mean ± SD

Figure 2

FAL 1 regulates cell proliferation and migration in vitro.(A and B) EDU assay and CCK8 assay were performed to observe the effects on cell proliferation in human 293T and SH‐SY5Y cells after FAL1 overexpression plasmid and siRNAs transfection. C, Transwell migration assay showed that FAL1 regulated the migration capacity of 293T and SH‐SY5Y cells. The results indicated that up‐regulation of FAL1 promoted cell migration, and lower expression of FAL1 delayed the cell migration. Pictures were captured under a light microscope with the magnification, ×20. D, Cell cycle was detected by BD Biosciences FACS Calibur Flow Cytometry showed the cells mainly distribution in G0/G1 phase after treated with FAL1 siRNA in 293T and SH‐SY5Y cell lines. *P < .05, **P < .01, ***P < .0001, data represent the mean ± SD

Figure 3

FAL1 directly interacts with miR‐637.A, Levels of FAL1 from nuclear and cytoplasmic fractions of 293T and SH‐SY5Y cells analysed by qRT‐PCR showed that FAL1 was mainly enriched in the cytoplasmic fractions. B, The expression of miR‐637 in HSCR tissues and control tissues. MiR‐637 was significantly rose in patients’ tissues compared with control tissues. C, Bioinformatics evidence of binding of miR‐637 onto 3′‐UTR of FAL1. Bottom: mutations in the FAL1 sequence to create the mutant luciferase reporter constructs. D, Luciferase reporter assay in 293T and SH‐SY5Y cells after transfected with negative control or miR‐637mimics, renilla luciferase vector pRL‐SV40 and the reporter constructs. Both firefly and renilla luciferase activities are measured in the same sample. Firefly luciferase signals were normalized with renilla luciferase signals. E, RNA immunoprecipitation (RIP) experiments for the amount of FAL1 and miR‐637 in SH‐SY5Y cells. FAL1 and miR‐637 expression levels were detected using qRT‐PCR. **P < .01, ***P < .0001, data represent the mean ± SD

Figure 4

AKT1 is the direct target of miR‐637.A, The putative miRNA‐binding sites in the AKT1 sequence. The putative miRNAs recognition sites were cloned downstream of the luciferase gene and named pGL3‐AKT1‐Wild. Bottom: mutations in the AKT1 sequence to create the mutant luciferase reporter constructs named pGL3‐AKT1‐Mut. B, 293T, SH‐SY5Y cells were transfected with full‐length 3′‐UTR (wild type or mutant) of AKT1, and the luciferase reporter was performed to confirm the direct target sites. C, Relative expression of AKT1 in HSCR tissues in comparison with control tissues. AKT1 was significantly reduced in patients’ tissues. D, Protein level of AKT1 in HSCR tissues and normal control samples was detected by Western Blot. E, Bivariate correlation analysis of the relationship between FAL1 and AKT1 expression level. F, There was a significantly negative correlation between the expression level of FAL1 and the expression level of miR‐637 in the same paired intestinal samples (R = −.4527, P < .001, Pearson). G, There was a significantly negative correlation between the expression level of AKT1 and the expression level of miR‐637 in the same paired intestinal samples (R = −.4360, P < .001, Pearson). **P < .01, ***P < .0001, data represent the mean ± SD

Figure 5

FAL1‐miR‐637 regulatory loop is critical for the expression of AKT1.A, miR‐637 inhibitor with or without FAL1 siRNA was transfected into 293T and SH‐SY5Y cells and the mRNA level of AKT1 was evaluated by qRT‐PCR. B, Western blot analysis of AKT1 protein level following treatment of 293T and SH‐SY5Y cells with miR‐637 inhibitor or FAL1 siRNA. GAPDH was used as control. C, Two types of cells were transfected with miR‐637 with or without FAL1 overexpress plasmid and qRT‐PCR was used to detect the relative mRNA levels of AKT1 compared with controls. D, Relative protein level of AKT1 when transfected with miR‐637 mimics and reversed by FAL1 expression plasmid. E, Relative expression level of miR‐637 when transfected with FAL1‐WT overexpression plasmid or FAL1‐MUT overexpression plasmid. F, Relative mRNA level of AKT1 when transfected with FAL1‐WT overexpression plasmid or FAL1‐MUT overexpression plasmid. G, Relative protein level of AKT1 when transfected with FAL1‐WT overexpression plasmid or FAL1‐MUT overexpression plasmid. H, Western blot showing that knockdown of FAL1 down‐regulated the expression of phosphorylated AKT (p‐AKT) and Cyclin D1. GAPDH served as a loading control. *P < .05, **P < .01, ***P < .0001, ns, no significant difference, data represent the mean ± SD

Figure 6

FAL1 regulates cell function through miR‐637.(A and B) EdU assay and CCK8 assay were performed to determine the proliferation of miR‐637‐transfected cells and treated with miR‐637 mimics plus FAL1 expression plasmid. C, The migration ability with respect to changes of 293T and SH‐SY5Y cell lines after co‐transfection of miR‐637 mimics and FAL1 overexpression plasmids was qualified by transwell assay. D, EdU assay was performed to determine the proliferation of cells treated with FAL1‐MUT overexpression plasmid. E, The migration ability with respect to changes of 293T and SH‐SY5Y cell lines after transfection of FAL1‐MUT overexpression plasmid was qualified by transwell assay. F, Cell cycle was detected by BD Biosciences FACS Calibur Flow Cytometry after cells treated with FAL1‐MUT overexpression plasmid in 293T and SH‐SY5Y cell lines. *P < .05, **P < .01, ***P < .0001, ns, no significant difference, data represent the mean ± SD