miR-106a-5p inhibits the proliferation and migration of astrocytoma cells and promotes apoptosis by targeting FASTK

Abstract

Astrocytomas are common malignant intracranial tumors that comprise the majority of adult primary central nervous system tumors. MicroRNAs (miRNAs) are small, non-coding RNAs (20-24 nucleotides) that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs. In our previous studies, we found that the downregulation of miR-106a-5p in astrocytomas is associated with poor prognosis. However, its specific gene target(s) and underlying functional mechanism(s) in astrocytomas remain unclear. In this study, we used mRNA microarray experiments to measure global mRNA expression in the presence of increased or decreased miR-106a-5p levels. We then performed bioinformatics analysis based on multiple target prediction algorithms to obtain candidate target genes that were further validated by computational predictions, western blot analysis, quantitative real-time PCR, and the luciferase reporter assay. Fas-activated serine/threonine kinase (FASTK) was identified as a direct target of miR-106a-5p. In human astrocytomas, miR-106a-5p is downregulated and negatively associated with clinical staging, whereas FASTK is upregulated and positively associated with advanced clinical stages, at both the protein and mRNA levels. Furthermore, Kaplan-Meier analysis revealed that the reduced expression of miR-106a-5p or the increased expression of FASTK is significantly associated with poor survival outcome. These results further supported the finding that FASTK is a direct target gene of miR-106a-5p. Next, we explored the function of miR-106a-5p and FASTK during astrocytoma progression. Through gain-of-function and loss-of-function studies, we demonstrated that miR-106a-5p can significantly inhibit cell proliferation and migration and can promote cell apoptosis in vitro. The knockdown of FASTK induced similar effects on astrocytoma cells as those induced by the overexpression of miR-106a-5p. These observations suggest that miR-106a-5p functions as a tumor suppressor during the development of astrocytomas by targeting FASTK.

Figure 1. Differentially regulated genes in cells with increased or decreased expression of miR-106a-5p.

(A) Overexpression or knockdown of miR-106a-5p. U251 cells were seeded into 6-well plates and transfected the following day using Lipofectamine 2000. For each well, 100 pmol of pre-ncRNA, pre-miR-106a-5p, anti-ncRNA or anti-miR-106a-5p was transfected. The intercellular levels of miR-106a-5p were evaluated by qRT-PCR at 24 h after transfection. For comparison, the expression levels of miR-106a-5p in pre-ncRNA- or anti-ncRNA-transfected cells were arbitrarily set at 1. The results are presented as the mean ± SD of three independent experiments (*** p<0.001). (B) The scatter plot of altered genes that were inversely expressed with increased or decreased expression of miR-106a-5p. Left: downregulated genes when miR-106a-5p is upregulated; Right: upregulated genes when miR-106a-5p is downregulated. (C) A Venn diagram of the overlap of altered genes with increased or decreased miR-106a-5p expression. The differentially expressed genes are depicted as two overlapping circles. The green circle indicates the number of genes that are downregulated when miR-106a-5p is upregulated, whereas the red circle indicates the number of genes that are upregulated when miR-106a-5p is downregulated. The number in the overlapping area indicates the number of mRNAs that belong to the intersecting sets.

Figure 2. FASTK is a direct target gene of miR-106a-5p.

(A) A schematic description of the hypothesized duplexes formed by interactions between the FASTK 3′-UTR binding sites and miR-106a-5p. The predicted free energy of each hybrid is indicated. The complementary seed sites are marked in red, and all of the nucleotides in these regions are completely conserved across several species. (B) Representative western blots showing FASTK protein levels in U251 cells treated with pre-ncRNA, pre-miR-106a-5p, anti-ncRNA and anti-miR-106a-5p. (C) Statistical analysis of three independent experiments. (D) Quantitative real time-PCR analysis of FASTK mRNA expression levels in U251 cells treated with pre-ncRNA, pre-miR-106a-5p, anti-ncRNA and anti-miR-106a-5p. The results shown represent data from three independent experiments. (E) Direct recognition of the FASTK 3′-UTR by miR-106a-5p. Firefly luciferase reporters containing either wt or mut FASTK 3′-UTRs were co-transfected into U251 cells with pre-miR-106a-5p, anti-miR-106a-5p and their corresponding negative controls. The parental luciferase plasmid was also transfected as a control. At 24 h post-transfection, the cells were assayed using luciferase assay kits. The results are presented as the mean ± SD of three independent experiments (** p<0.01; *** p<0.001). (F) Relative miR-106a-5p expression levels in NAT samples and WHO grade I-IV astrocytomas. (G) Representative western blots showing FASTK protein levels in NAT samples and WHO I-IV astrocytomas. (H) Statistical analysis of three independent experiments. (I) Relative FASTK mRNA expression levels in NAT samples and WHO grade I-IV astrocytomas. (J) The relationship between miR-106a-5p expression and astrocytoma patient survival time. (K) The relationship between FASTK expression and astrocytoma patient survival time.

Figure 3. The role of miR-106a-5p and FASTK in cell proliferation, migration and apoptosis.

FASTK siRNA interference assay (A–B). Three siRNA sequences targeting different sites of human FASTK cDNA and a scrambled control siRNA (si-NC) were transfected into U251 cells using Lipofectamine 2000. Total protein or total RNA was isolated at 48 h or 24 h post-transfection. FASTK protein levels were determined by western blot analysis (A), and FASTK mRNA levels were assessed by qRT-PCR (B). The siRNA eliciting the most optimal interfering effect (siRNA-1, named si-FASTK) was used in further studies. (C) The role of miR-106a-5p and FASTK on cell proliferation. An MTT cell viability assay was performed at 12, 24, 48, 72 and 96 h after transfection of U251 cells with equal concentrations of pre-ncRNA, pre-miR-106a-5p, si-NC and si-FASTK. (D) Transwell assays of U251 cells treated with equal concentrations of pre-ncRNA, pre-miR-106a-5p, si-NC and si-FASTK. The images shown are representative images from three independent experiments, and a statistical analysis was performed (mean ± SD; * p<0.05, ** p<0.01). (E) The role of miR-106a-5p and FASTK on apoptosis. U251 cells were transfected with equal concentrations of pre-ncRNA, pre-miR-106a-5p, si-NC and si-FASTK. The experiment was repeated three times, and representative data are shown.