Multiple lines of evidence indicate that aberrant activation of Hedgehog (Hh) signaling plays an important role in tumorigenesis in human glioma. However, the underlying molecular mechanism and crucial downstream targets of glioma-associated oncogene (Gli), a primary transcriptional regulator of Hh signaling, are not fully understood. Here, we report the identification of miR-124 as a novel downstream target of the transcriptional factor Gli2, which is important for proliferation and tumor growth in human glioma cells. Blockade of Hh signaling leads to a remarkable increase in miR-124 expression in glioma cells, whereas overexpression of Gli2 suppresses miR-124 expression by increasing the direct binding of Gli2 to the upstream region of the transcriptional start site for miR-124. Furthermore, we found that miR-124 potentially interacts with the 3′-UTR region of AURKA. Overexpression of miR-124 significantly decreased the expression of AURKA in glioma cells. In contrast, the loss of miR-124 led to the increased expression of AURKA mRNA and protein. In addition, cell proliferation and colony formation ability were significantly decreased following Gli2 knockdown in human glioma cells, while transfection with a miR-124 inhibitor rescued the proliferative ability of cells. These results demonstrate that miR-124 is an important downstream target gene of Hh signaling, and the Gli2/miR-124/AURKA axis is essential for the proliferation and growth of human glioma cells.
Glioma is one of the most common and aggressive human malignancies worldwide (
Hh signaling transduction is initiated by the binding of Hh proteins (sonic Hh, Shh; Indian Hh, Ihh; and Desert Hh, Dhh) to the 12-pass transmembrane protein Patched (PTCH), which abrogates the repressive activity of PTCH, allowing the 7-pass transmembrane protein Smoothened (Smo) to transduce the signal to the nucleus. Specifically, Smo promotes nuclear translocation of the 5-zinc-finger transcription factors glioma-associated oncogenes (Glis) and subsequently activates target gene transcription (
In humans, the Hh signaling pathway is critical for embryonic development and adult homeostasis, and Hh signaling activity normally ceases after embryogenesis. However, aberrant activation of the Hh signaling cascade has been shown to be associated with oncogenesis and maintenance of the malignant phenotype in multiple types of human cancers (
MicroRNAs (miRNAs) are a class of short, single-stranded endogenous non-coding RNAs (approximately 20–22 nucleotides in length) that post-transcriptionally control gene expression via either translational repression and/or mRNA degradation in multicellular eukaryotes (
It is noteworthy that as a regulatory element, miRNA itself often acts as downstream effector of transcription factors including p53, HIF-1, and c-myc, which have been verified to regulate the expression of several miRNAs (
In this study, we performed a set of experiments to elucidate the molecular mechanisms by which the Hh signaling pathway regulates cancer cell proliferation and tumor growth. Our findings indicate that inhibition of Hh signaling suppresses cell proliferation, at least in part, via the Gli2/miR-124/AURKA axis in human glioma cells.
Primary antibodies were purchased from Millipore (GAPDH, mAb374) and Cell Signaling Technology (Aurka, 4718). A PrimeScript™ RT reagent kit with gDNA Eraser, SYBR®Premix Ex Taq™ II and a One Step PrimeScript miRNA cDNA Synthesis kit were purchased from Takara (Tokyo, Japan). The miR-124 inhibitor and control oligonucleotides were purchased from Ribobio (Guangzhou, China) and prepared as 50
Human glioma cell lines (H4 and U87) and a human embryonic kidney cell line (HEK293T) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 50 mg/ml penicillin (100 U/ml) and streptomycin (100
Total RNA, including miRNA, was isolated using TRIzol (Ambion, Austin, TX, USA). The integrity of the total RNA was analyzed by gel electrophoresis. Then, 200 ng of the isolated total RNA was labeled using an Illumina Total Prep-96 RNA Amplification kit (PN:4393543, Ambion Life Technologies, Grand Island, NY, USA), and 750 ng of cRNA was generated and hybridized into a Human HT-12 V4 BeadChip. Then, the BeadChip was washed and stained as per the Illumina protocol and scanned on an iScan (Illumina, San Diego, CA, USA). Data analysis was performed with Genespring GX 12.0 Software (Agilent Technology, Inc., Santa Clara, CA, USA). Raw data were filtered by percentile (lower cut-off: 20). An unpaired t-test was used to identify significant (P<0.05) gene expression changes with multiple testing correction (Benjamini-Hochberg) to control the false discovery rate and obtain statistically reliable results.
Real-time PCR analysis of the expression of all mRNA and miRNA was analyzed using a SYBR Green kit (Takara) according to the manufacturer's instructions. Briefly, for the detection of mRNA, 1
Identification of transcription factor binding sites in the pre-miR-124 promoter was performed using Cisgenome 2.0 software to identify putative transcription factors that could potentially bind and regulate the expression of miR-124. The motif resembling the known Gli binding site was CTGGGTGGTC (
A chromatin immunoprecipitation (CHIP) analysis was performed to detect the occupation of the Gli2 transcription factor on the putative regions of miR-124. In brief, H4 cells were cultured in a 10-cm dish until reaching approximately 90% confluence. After discarding the original medium, H4 cells were crosslinked with 5 ml of PBS containing 1% formaldehyde at room temperature for 10 min with gentle shaking. Then, DNA was sonicated into a range of 200–1000 base pairs in size using a Bioruptor Sonicator (Diagenode) for five cycles of 3 sec on/3 sec off. The extracts were pre-cleared in BSA-blocked protein A beads and incubated with anti-Gli2 or IgG control overnight at 4°C. After being washed, DNA was eluted and reverse cross-linked overnight at 65°C and then purified and amplified by PCR. The primers for PCR are shown in
The wild-type (WT) AURKA-3′-UTR was amplified by PCR from human cDNA using the primers (forward) 5′-CAA GCT TCA CAT CAG GTG GAT GGA GAG AC-3′ and (reverse) 5′-GAG CTC GGC AGG GGA AAG CTG TAG GAA T-3′. The mutant-type (Mut) AURKA-3′ UTR was amplified using the primers (forward) 5′-CAA GCT TCA CAT CAG GTG GAT GGA GAG AC-3′ and (reverse) 5′-GAG CTC GGC AGG GGT ATG GTC TAG GAA T-3′. Then, the cDNA fragments were inserted into a pGL3 Vector using the
HEK293T cells were cultured in 24-well plates and co-transfected with pGL3.0 vectors containing either the WT or mutated AURKA-3′-UTR vectors and miR-124 expression plasmid or control plasmid using Lipofectamine 2000. After 48 h, cells were lysed, and luciferase assays were performed using a dual luciferase reporter assay kit (Promega, Madison, WI, USA).
Cells were washed with chilled PBS and harvested by trypsinization. Then, cells were lysed in lysis buffer at 4°C for 30 min and centrifuged (12,000 rpm, 15 min at 4°C) to collect the supernatant. Protein concentrations were determined by the BCA method using Pierce™ BCA Protein Assay kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. Subsequently, the lysates were separated on SDS-PAGE gels and immunoblotted using standard procedures. The primary antibodies used were anti-AURKA (Abcam, 1:1000) and anti-GAPDH (Millipore, 1:2000). Finally, immunostaining was visualized using Kodak X-ray film, which was subsequently scanned with an Epson 1680 scanner. Quantitative analysis was performed on scanned images of blots using ImageJ software.
Cell viability assays were performed as previously described (
The statistical significance between two groups was calculated by unpaired Student's t-test using SPSS 16.0 software. For experiments involving more than one group for comparison, ANOVA was used with a suitable post hoc test. All data are expressed as the mean ± SD for experiments performed at least three times. Differences were considered significant at P<0.05 or P<0.01.
To identify miRNAs potentially regulated by Hh signaling, we treated H4 cells with GANT61, a specific inhibitor of Gli1 and Gli2 (
In vertebrates, the Gli family of transcription factors, specifically Gli1 and Gli2, mediates the Hh signaling pathway by regulating the transcription of target genes. Their cooperative roles are vital in Hh signaling, while their specific roles have only been partially defined. To interrogate which one of the Glis influences miR-124 biogenesis, we transfected H4 cells with either a Gli1-shRNA or Gli2-shRNA plasmid. We found that sh-Gli1-2855 and sh-Gli2-228 were more efficient in knocking down endogenous Gli1 and Gli2 expression (
To investigate the molecular mechanism by which Gli2 orchestrates miR-124 expression, we measured the expression level of pri-miR-124 and pre-miR-124 following Gli2 knockdown in H4 cells. As shown in
Next, in order to further illustrate the mechanism, a search for putative Gli2 binding sites, using Cisgenome 2.0, identified seven putative Gli2-binding DNA elements (BS1: +9789 ~ +9798, BS2: +9255 ~ +9234, BS3: +6235 ~ +6244, BS4: +6025 ~ +6034, BS5: +5538 ~ +5547, BS6: +2935 ~ +2944, and BS2: +551 ~ +560) located upstream of the transcriptional start site of miR-124 (
To elaborate the functional consequences of the Gli2-mediated inhibition of miR-124 expression, we analyzed the target genes of miR-124. Noteworthy, based on the bioinformatic analysis of potential miR-124 targets (
In addition, overexpression of miR-124 significantly decreased the expression of AURKA protein in H4 cells (
The results described above show that Hh signaling inhibits the expression of miR-124, and miR-124 downregulates the expression level of AURKA (
A real-time PCR assay showed that the AURKA mRNA level was downregulated in the GANT61 group, while the miR-124-inhibitor rescued the expression of AURKA mRNA (
To illustrate the molecular mechanisms by which Hh signaling regulates the proliferation process in glioma cells, we inhibited the expression of Gli2 in H4 cells. Strikingly, the number of colonies formed by H4 cells was significantly decreased following Gli2 knockdown, while transfection with the miR-124 inhibitor rescued the proliferative ability of the cells (
In this study, we determined that miR-124 acts as a downstream effector of the Hh signaling pathway. Noteworthy, we found that miR-124 potentially interacts with the 3′-UTR region of AURKA. Further experiments showed that the Hh signaling pathway regulated the expression of AURKA through miR-124, and overexpression of miR-124 significantly decreased the expression of AURKA and the proliferation of glioma cells. Our results suggest that the Gli2/miR-124/AURKA axis is essential for the proliferation and growth of human glioma cells.
The Gli transcription factors constitute the final effectors of the Hh signaling pathway, which is frequently hyperactivated in human cancers through multiple mechanisms. Hence, targeting Gli may offer a highly effective therapeutic strategy for the treatment of lethal tumors. Currently, there are multiple studies aimed at assessing the efficacy of Gli inhibitors in cancers. However, the downstream mechanisms initiated by Gli are poorly understood, in part because relatively little is known about the multiple specific genes directly regulated by Gli. Before the discovery of noncoding RNAs, searches for transcription factor-targeted genes were focused on protein-coding genes. Intriguingly, it is worth noting that recently several transcription factors have been discovered to regulate the expression of miRNAs.
The central tumor suppressor p53 enhances the transcriptional activity of the miR-34 family by binding to the promoter of miR-34a (
In this study, we demonstrated that Gli2 can directly modulate the expression of miR-124 by binding to one binding site in the upstream region of the transcriptional start site, thereby fine tuning the function of miR-124. Notably, most studies have focused attention on protein-coding genes that can be regulated by Hh signaling. Our findings suggest that, in addition to many protein-coding genes, miRNAs can also be regulated by Gli2. Our study raises the possibility that Gli2 functions as a global modifier of gene expression through the regulation of miRNA transcription. However, it is not known whether there are other miRNAs that might be directly modulated by Gli2. Further investigations will provide insight into how great a portion of the pri-miRNAs are regulated by Gli2 to fully understand the regulatory mechanism of Gli2 and miRNAs.
The biogenesis of miRNAs in mammalian systems is composed of multiple steps, including transcription of primary miRNA (pri-miRNA), cleavage of pri-miRNA to precursor miRNA (pre-miRNA), nucleocytoplasmic transport of pre-miRNA and cleavage of pre-miRNA to an miRNA duplex (
Consistent with Gli2 function, many signature miRNAs, especially miR-124, are considered tumor-associated molecules, and miR-124 expression is lost in diverse types of tumors (
AURKA, also referred to as Aurora-2, BTAK, ARK1, and STK15, maintains cell division by regulating centrosome separation, bipolar spindle assembly, and chromosome segregation (
In summary, our data indicate that aberrant expression of miR-124 through Gli2 inhibition in glioma cells can lead to the repression of AURKA, which can repress cell proliferation in glioma cells. Our results highlight an additional mechanism by which the Hh signaling pathway controls gene expression and influences cancer progression, and they elucidate a new mechanism through which the Hh signaling pathway regulates glioma development.
This work was supported in part by grants from the National Natural Science Foundation of China (31560314 to Q.L.) and the Natural Science Foundation of Jiangxi Province (2016BAB204168 to Q.L.).
Hedgehog (Hh) signaling is involved in the regulation of miRNA expression. (A) Gli was downregulated in H4 cells by treatment with GANT61 after 48 h. Real-time PCR was performed to analyze Gli mRNA in H4 cells after treatment with DMSO (control) or GANT61 (20
Gli2 binds directly to the miR-124 genomic locus. (A) H4 cells and U87 cells were transfected with control plasmid or sh-Gli2-228 plasmid and were subjected to real-time PCR analysis to determine pri-miR-124 expression. (B) H4 cells and U87 cells were transfected with either control plasmid or sh-Gli2-228 plasmid (48 h). The expression of pre-miR-124 was quantified by real-time PCR. (C) Schematic diagrams of Gli2 regions indicated the putative Gli2-binding sites in the upstream region of the miR-124 transcription start site. (D) Chromatin was isolated from H4 cells, and ChIP assays were performed with control (IgG) and anti-Gli2 antibodies. Specific primers for each putative binding element were used for PCR analyses (
AURKA is a direct target of miR-124. (A) miR-124 sequences and the predicted miR-124 binding sites in the 3′-UTR of human AURKA. (B) Overexpression of miR-124 in H4 cells. H4 cells were transfected with control plasmid or miR-124 expression plasmid for 48 h, and then, miR-124 levels were detected by real-time PCR. (C) miR-124 expression downregulates the mRNA level of AURKA in H4 cells. H4 cells were transfected with control plasmid or miR-124 expression plasmid for 48 h, and then, AURKA mRNA levels were detected with real-time PCR. (D) miR-124 expression downregulates the protein level of AURKA in H4 cells. Cells were transfected with control plasmid or miR-124 expression plasmid for 48 h, and then, AURKA protein levels were detected by western blotting. (E) Downregulation of the expression of miR-124 in H4 cells. Cells were treated with control oligonucleotides or miR-124 inhibitor for 48 h, and then, miR-124 levels were detected with real-time PCR. (F) Downregulation of the expression of miR-124 in H4 cells. Cells were treated with control oligonucleotides or miR-124 inhibitor for 48 h, and then, AURKA mRNA levels were detected with real-time PCR. (G) Downregulation of the expression of miR-124 in H4 cells. Cells were treated with control oligonucleotides or miR-124 inhibitor for 48 h, and then, AURKA protein levels were detected by western blotting. (H,I,J) The miR-124 binding site in the human AURKA 3′-UTR mediates the repression of luciferase activity in HEK-293T cells. luciferase reporter constructs containing the wild-type or mutant human AURKA 3′-UTR were fused to the 3′-end of the firefly luciferase gene. Then, the AURKA 3′-UTR luciferase plasmid was transfected into HEK293T cells together with or without the miR-124 expression plasmid. The relative luciferase activity was measured 48 h after transfection with a dual luciferase assay. The values shown are the means ± SD for triplicate samples. *P<0.05; **P<0.01; NS, no significance.
Gli2 influences the expression of AURKA via miR-124. (A) H4 cells were transfected with miR-124 inhibitor for 12 h and subsequently treated with GANT61 (20
Gli2/miR-124/AURKA mediates the growth of glioma cells. (A and B) Inhibition of miR-124 influences the effect of Gli2 on proliferation. H4 cells were transfected with sh-Gli2-228 plasmid and miR-124 inhibitor as indicated. The proliferation of H4 cells was assessed by a colony formation assay (12 days). The quantitative analysis was performed using ImageJ software. (C and D) Overexpression of AURKA restores the inhibitory effect of miR-124 on proliferation. H4 cells were transfected with miR-124 and Flag-AURKA plasmid as indicated, and the proliferation of H4 cells was assessed by a colony formation assay (10 days). The quantitative analysis was performed using ImageJ software. (E) H4 cells transfected with sh-Gli2-228 plasmid were co-transfected with NC or miR-124 inhibitor for 72 h. An MTT assay was performed to determine the proliferation of H4 cells. (F) H4 cells transfected with miR-124 overexpression plasmid were co-transfected with NC or Flag-AURKA plasmid for 72 h. An MTT assay was performed to determine the proliferation of H4 cells. The bar graph shows the means ± SD, n=5; *P<0.05; **P<0.01.
Interference sequences.
Genes | Target site | Target sequence |
---|---|---|
sh-Gli1-720 | 720 to 740 | 5′-TTCATACACAGATTCAGGCTC-3′ |
sh-Gli1-1863 | 1863 to 1883 | 5′-TTCATACACAGATTCAGGCTC-3′ |
sh-Gli1-2255 | 2255 to 2275 | 5′-AAGACCTATCCGATCCAGCGG-3′ |
sh-Gli2-233 | 233 to 253 | 5′-AATGGTACCTTCCTTCCTGGT-3′ |
sh-Gli2-1127 | 1127 to 1147 | 5′-TGGCCTGAAACGATGTCATC-3′ |
sh-Gli2-2058 | 2058 to 2078 | 5′-TGTGAATGGCGACAGGGTTGA-3′ |
Primer sequences for real-time PCR.
Genes | Forward primer (5′ to 3′) | Reverse primer (5′ to 3′) |
---|---|---|
Gli1 | 5′-TCCTACCAGAGTCCCAAGTT-3′ | 5′-CCCTATGTGAAGCCCTATTT-3′ |
Gli2 | 5′-CTGTGGGTTAGGGATGGACTG-3′ | 5′-GTAAAGTGGGTGGACGTTGCA-3′ |
AURKA | 5′-GGAATATGCACCACTTGGAACA-3′ | 5′-TAAGACAGGGCATTTGCCAAT-3′ |
GAPDH | 5′-GAAGGTGAAGGTCGGAGTC-3′ | 5′-GAAGATGGTGATGGGATTTC-3′ |
miR-301b | 5′-GCAGTGCAATGATATTGTCAAAGC-3′ | Uni-miR PCR Primer |
miR-302d | 5′-GCTAAGTGCTTCCATGTTTGAGTGT-3′ | Uni-miR PCR Primer |
miR-519a | 5′-GCAAAGTGCATCCTTTTAGAGTGT-3′ | Uni-miR PCR Primer |
miR-335 | 5′-GCTCAAGAGCAATAACGAAAAATGT-3′ | Uni-miR PCR Primer |
miR-122 | 5′-GTGGAGTGTGACAATGGTGTTTG-3′ | Uni-miR PCR Primer |
Primer sequences for CHIP.
Genes | Forward primer (5′ to 3′) | Reverse primer (5′ to 3′) |
---|---|---|
BS1 | 5′-TACAGAGGGATCTGTTGGGAGT-3′ | 5′-TGGCCTTACCTACAAAATGGG-3′ |
BS2 | 5′-AGGCTGGTTTCAAACTCCTG-3′ | 5′-TAGTGTCTAGGCTGGGTGC-3′ |
BS3 | 5′-AGGGAAATGATTCCAAGCC-3′ | 5′-CTGGGAAGTTCTGAATGTTTG-3′ |
BS4 | 5′-GAACTTCCCAGTCTAAACAGC-3′ | 5′-GGCTTAGGGATTGCTACAAC-3′ |
BS5 | 5′-CGCTTCCAACCTCCTCTTG-3′ | 5′-GGGCTGGTCTTGAACTCCT-3′ |
BS6 | 5′-GCTGGGAACTGTAGTCTTGC-3′ | 5′-GCCACTGGAGGTAGTGATT-3′ |
BS7 | 5′-TTCTTCCCAGCAGAGTCAAG-3′ | 5′-TAATACCTCGCAAAGCATGG-3′ |