Gene expression data and relevant clinicopathologic variable information were downloaded from TCGA database (http://cancergenome.nih.gov) (n=489). Additionally, miRNAs expression data and metadata containing survival information from the CGGA database (http://www.cgcg.org.cn) (n=198) were analyzed. Moreover, the ENCORI database (http://starbase.sysu.edu.cn/) was used to analyze the relationship between miR-301a and ZNRF3 expression in low-grade gliomas (LGG; n=525).

Furthermore, 39 clinical glioma specimens from 15 female and 24 male patients (age range, 19-76 years; mean age, 42.9±15.4 years) were obtained from the nerve tumor tissues bank at the Tianjin Institute of Neurosurgery, including 10 low grade (WHO I and II) and 29 GBMs (WHO IV), who were diagnosed by surgeons and pathologists at Tianjin Huan Hu Hospital (Tianjin, China) between January 2010 and July 2018. None of the patients had received any radiotherapy, chemotherapy or any other anticancer treatments prior to surgery. In total, five normal adult brain tissue samples were collected from 4 male and 1 female patient (age range, 58-74 years; mean age, 66±6.3 years) undergoing post-trauma surgery for severe traumatic brain injury. All the collected tissues were frozen immediately in liquid nitrogen and stored at −80°C. This study was approved by the Institutional Review Board of Tianjin Huan Hu Hospital, and written informed consent was obtained from all participants.

The human glioblastoma cell lines U87, U251 and A172 and the normal astrocyte cell line were obtained from the Peking Union Medical College Cell Library. Human LN308, LN229 and T98G glioblastoma cell lines were obtained from the China Academia Sinica Cell Repository. The U87 cell line was authenticated using short tandem repeat profiling analysis; the U87 cell line (glioblastoma of unknown origin) used in the present study was of the American Type Culture Collection type. Cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). All cell lines were incubated at 37°C with 5% CO₂.

The oligonucleotide sequencess of human miR-301a inhibitor (AS-miR; 5′- GCUUUGACAAUACUAUUGCACUG-3′), miR-301a mimics (5′-CAGUGCAAUAGUAUUGUCAA AGC-3′), miRNA-negative control (miR-NC; 5′-UUGUACU ACACAAAAGUACUG-3′), ZNRF3 small interfering (si)RNA (5′- CACUGGGCCUAUGUAAUAATT-3′) and transcription factor 4 (TCF4) overexpression plasmid were purchased from Shanghai GenePharma Co., Ltd. In brief, LN229 and U87 cells were incubated in a 6-well plate at a density of 2×10⁵ cells/well and then transfected with miRNA (2 µg) or siRNA (2 µg), or co-transfected both of the transfectants at room temperature using X-tremeGENE transfection reagent (Roche Diagnostics GmbH) according to the manufacturer's protocol. Subsequent experiments were performed after 72 h.

Lentivirus-transducing units carrying the ZNRF3 variant 3 (LV-ZNRF3) and Lentivirus vector control (LV-NC) were purchased from Shanghai Genechem Co., Ltd. For stable transfection, after seeding in 6-well plates (2×10⁵ cells/well) for 24 h, cells were infected with LV-ZNRF3 or LV-NC at a multiplicity of infection of 10 in the Opti-DMEM (Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37°C. In order to establish stable overexpressing cell lines, infected cells were treated with puromycin (2 µg/ml for the LN229 and U87 cells) for 7 days. The overexpression efficiency was evaluated via western blot analysis.

Total RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and was reverse transcribed to cDNA to evaluate the mRNA expression levels of ZNRF3, c-myc, cyclin D1 and TCF4 using the PrimeScript RT Master mix (Takara Bio, Inc.) according to the manufacturer's instructions. The protocol of RT was 37°C for 15 min, 85°C for 5 sec and held at 4°C. The PCR protocol was: Initital denaturation at 95°C for 5 sec, followed by 40 cycles at 60°C for 30 sec and by annealing and extension at 50°C for 30 sec. To evaluate miR-301a, the protocol of miRNA reverse transcription was 37°C for 60 min, 95°C for 5 min and held at 4°C, and stem-loop RT-PCR was performed with an RNA PCR kit (Qiagen GmbH) according to the manufacturer's instructions. qPCR was performed using the miScript SYBR Premix Green PCR kit (Qiagen GmbH) and the Roche LC480 quantitative Real-Time PCR system (Roche Diagnostics). The amplification conditions were: Initital denaturation at 95°C for 15 min, followed by 40 cycles at 94°C for 15 sec and by annealing and extension at 55°C for 30 sec. GAPDH or U6 levels were selected as internal controls, and fold changes were calculated using the relative quantification (2^-ΔΔCq) method (20). The primer sequences are presented in Table SI. Data were analyzed from three independent experiments and are presented as the mean ± SD.

StarBaseV2 (http://starbase.sysu.edu.cn/), TargetScan (http://www.targetscan.org/vert_72/) and Pictar (http://www.pictar.org/) algorithms were applied to identify targets of miR-301a, and the seed sequence of miR-301a was identified to match the 3′ untranslated region (3′UTR) of the ZNRF3 gene. The 3′UTR of ZNRF3 containing the miR-301a conserved binding sites and the corresponding mutant (MT) sites were inserted into the pMIR-REPORT vector (Promega Corporation). LN229 and U87 cells were cultured in 96-well plates, and co-transfected with the wild-type (WT) or MT luciferase reporters (0.15 µg) and the AS-miR-301a (0.15 µg) using the X-tremeGENE transfection reagent (Roche Diagnostics GmbH) at room temperature.

To analyze the transcriptional activity of β-catenin/TCF4, TOP-FLASH and FOP-FLASH luciferase reporter constructs were constructed. The TOP-FLASH (with repeats of the Tcf-binding site) or FOP-FLASH (with repeats of the MT Tcf-binding site) plasmids (EMD Millipore) (0.15 µg) were transfected into LN229 and U87 cells treated with the miR-301a inhibitor (0.15 µg) in 96-well plates at room temperature. After 72 h incubation, luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega Corporation), and Renilla luciferase activity was used as an internal control.

The PGL3-WT-miR-301a-promoter-Luc reporter or pGL3-MT-miR-301-promoter-Luc reporter constructs (0.15 µg; Promega Corporation) were transfected into LN229 and U87 cells or stable ZNRF3 overexpressing cells, following the co-transfection of the TCF4 overexpression plasmid or siTCF4 using the X-tremeGENE transfection reagent (Roche Diagnostics) at 37°C for 72 h. The luciferase activity was detected after 72 h as aforementioned.

The ExKine Total Protein Extraction kit was purchased from Abbkine Scientific Co., Ltd. and used to extract proteins. Protein concentrations were determined using a BCA Protein assay kit (Thermo Fisher Scientific, Inc.). Then, 40 µg protein lysate from each sample was resolved via 10% SDS-PAGE and subsequently transferred to PVDF membranes (EMD Millipore). After blocking in 5% skimmed milk for 1 h at room temperature, the membranes were incubated with diluted primary antibodies at 4°C overnight. After washing with TBS-Tween-20 (1% Tween-20), membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. nos. ab222772 and ab222759; Abcam) for 1 h at room temperature. The immune-reactive bands were visualized using ECL Western Blot Detection reagents (EMD Millipore) and semi-quantified using ImageJ software (version 1.47v; National Institutes of Health). The primary antibodies used were as follows: ZNRF3 (1:1,000; cat. no. A16026; ABclonal Biotech Co., Ltd.), cyclin D1 (1:10,000; cat. no. ab134175; Abcam), c-myc (1:1,000; cat. no. ab39688; Abcam), TCF4 (1:10,000; cat. no. ab76151; Abcam) and β-actin (1:5,000; cat. no. ab6276; Abcam), which was used as the control.

IHC assays were performed using antibodies against ZNRF3 (1:200; cat. no. A16026, ABclonal Biotech Co., Ltd.), β-catenin (1:500; cat. no. ab32572; Abcam), c-myc (1:100; cat. no. ab39688; Abcam), TCF4 (1:500; cat. no. ab76151; Abcam) and caspase-3 (1:100; cat. no. 9662; Cell Signaling Technology, Inc.). The protocols used were performed as described previously (21). Briefly, the animal tumor samples were fixed with 10% formalin for 48 h at room temperature, embedded with paraffin and sliced into 4-µm sections, followed by dewaxing. Subsequently, these sections were incubated with 3% hydrogen peroxide for 20 min at room temperature to block endogenous peroxidase. Following antigen retrieval, the slides were blocked with 10% normal goat serum (Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature and then incubated at 4°C overnight with appropriate primary antibody. After washing with PBS, the slides were incubated with biotinylated secondary antibody (cat. no. Sp-9001; 1:1,000; OriGene Technologies, Inc.) for 1 h at room temperature. A DAB Horseradish Peroxidase Color Development kit (Beyotime Institute of Biotechnology) was used for color development, and neutral gum (Sinopharm Chemical Reagent Co., Ltd.) was used for sealing. Finally, five randomly selected fields were imaged using the Olympus X71 inverted microscope (Olympus Corporation; magnification, x200), and semi-quantitatively analyzed in the form of mean optical density using Image-Pro Plus 6.0 (Media Cybernetics, Inc.). The signal density of the tissue areas from five randomly selected fields were counted in a blinded manner. The significance of the differences between two groups was determined using Student's t-test.

To examine the regulatory role of miR-301a on β-catenin/TCF4 activity, LN229 and U87 cells were transfected with AS-miR-301a or miR-scramble (miR-NC) or were co-transfected with AS-miR-301a and siZNRF3, followed by immunofluorescence analysis. The procedures were conducted as previously described (21). In brief, cells were fixed with 0.1% paraformaldehyde for 20 min at room temperature and were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) at room temperature for 2 h. Cells were incubated with anti-β-catenin primary antibody (1:200; cat. no. ab32572; Abcam) overnight at 4°C. After washing with PBS at room temperature, Goat anti-rabbit fluorescence conjugated second antibody (1:10,000; cat. no. A21245; Invitrogen; Thermo Fisher Scientific, Inc.) was added for 1 h at room temperature and imaged using a confocal microscope (magnification, ×50).

Cell viability was analyzed using a Cell-Counting Kit 8 (CCK-8; Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions. Following transfection with AS-miR-301a or co-transfection with AS-miR-301a and siZNRF3, LN229 and U87 cells were incubated at 37°C for 24, 48, 72 and 96 h. CCK-8 solution (10 µl) was added to each well, and the absorbance at 490 nm was measured after incubation at 37°C for 2 h to estimate the number of viable cells.

Apoptosis was quantified after transfection, using Annexin V labelling and caspase-3/7 activity. For the Annexin V assay, the Annexin V-FITC-labelled Apoptosis Detection kit (Abcam) was used according to the manufacturer's protocol. In brief, cells were harvested, washed and resuspended, and then 5 µl Annexin V-FITC and 5 µl PI were added and maintained for 15 min at room temperature in the dark. Within 1 h, the stained cells were analyzed with a flow cytometry system (BD FACSCalibur™ flow cytometer; BD Biosciences) equipped with CellQuest software (version-5.1; BD Biosciences). The apoptotic rate was calculated as the percentage of early + late apoptotic cells.

Caspase-3/7 activity was analyzed using the Caspase-Glo-3/7 reagent (cat. no. G8091; Promega Corporation). Caspase-Glo-3/7 reagent (100 µl) was added to a white-walled 96-well plate, which was gently mixed using a plate shaker at 50 × g for 30 sec at room temperature. Following incubation at room temperature for 1-2 h, the luminescence of each sample at 510 nm was measured in a plate-reading luminometer.

All animal protocols were performed following the approval of the Animal Care and Used Committee of Tianjin Huanhu Hospital. A total of 30 female BALB/c-A nude mice (Age, 5 weeks; weight,18-21 g) were purchased from the Animal Center of the Cancer Institute, Chinese Academy of Medical Science. Animal welfare was considered and all the mice were housed with filtered air, a 12-h light/dark cycle, regulated temperature (25±2°C) and humidity (50±10%), and with ad libitum access to sterilized food and water. Then, ~5×10⁶ U87 GBM cells in 100 µl PBS were subcutaneously injected into the left flank region of nude mice. When the tumor volume reached 100 mm³, the mice were randomly divided into two groups, namely NC group and AS-miR-301a group (5 mice per group). Each group was treated with oligonucleotides (10 µg AS-miR-301a or miR-NC) with a mixture of 10 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) via local injection into the xenograft tumor at multiple sites on all sides. The treatment was performed once every 3 days for 27 days, and the tumor volume was monitored with a caliper, using the formula: Volume = length × width²/2.

To evaluate the miR-301a-mediated role of ZNRF3 in tumor inhibition in vivo, 5×10⁶ U87 cells stably expressing LV-ZNRF3 or LV-NC in 150 µl PBS were injected into the left flank region of nude mice. When all the groups formed a tumor at ~4 weeks, the LV-NC and LV-ZNRF3 injected mice were separated into two groups (5 mice per group), respectively. One of the LV-NC groups was treated with miR-scramble (miR-NC), while one of LV-ZNRF3 groups was treated with miR-301a mimics and another one was treated with miR-NC every 3 days for 30 days. The tumor injection of oligonucleotides (10 µg AS-miR-301a or miR-NC) with a mixture of 10 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was also performed at multiple sites. At the end of the experiment, the mice were anesthetized by injecting 1% pentobarbital sodium (100 mg/kg body weight) and sacrificed via cervical vertebra dislocation. Finally, tumor weight was calculated. The removed tumor tissues were subjected to RNA extraction and immunohistochemistry assays.

All statistical analyses were performed using GraphPad software version 6.0 (GraphPad Software, Inc.) or IBM SPSS Statistics 23.0 (IBM Corp.). Data are presented as the mean ± SD of ≥3 independent experiments. The unpaired Student's t-test, and one-way ANOVA were performed to analyze statistically significant differences between two groups or multiple groups, respectively. Additionally, a Tukey's test was used for multiple comparisons following ANOVA. The Kaplan-Meier method was used to evaluate the differences in survival rates, which were analyzed using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the potential role of miR-301a in gliomagenesis, miR-301a expression was analyzed in gliomas with different grades, including 10 LGG and 29 GBM cases, and seven malignant glioma cell lines. miR-301a was significantly upregulated in the clinical samples and GBM cell lines (Fig. 1A and B), and its expression level was positively associated with the WHO grade (Fig. 1A). The clinicopathological characteristics of the 39 glioma specimens are summarized in Table I. There was a statistically significant statistically correlation between miR-301a expression and histology, but not for sex and age. Additionally, analysis of the data from the CGGA database indicated that miR-301a expression was positively associated with the WHO grade, which was in accordance with the data from the clinical specimens (Fig. 1C), and a higher level of miR-301a predicted a poor prognosis (Fig. 1D). Furthermore, the relationship between the miR-301a expression and prognosis from TCGA database was analyzed, and the results were consistent with the analysis of the CGGA data (Fig. S1). These findings indicated that miR-301a may act as a critical oncogene in gliomas.

miRNAs serve as oncogenes or tumor-suppressors by regulating gene expression by either mRNA degradation or translational inhibition (22). Thus, StarBaseV2, TargetScan and Pictar algorithms were used to verify the downstream effectors of miR-301a. Based on the database evaluation, the seed sequence of miR-301a that matched the 3′UTR of the ZNRF3 was identified (Fig. 2A).

To verify whether ZNRF3 expression was downregulated by miR-301a, the changes in ZNRF3 mRNA and protein expression levels were detected in LN229 and U87 cells after knocking down miR-301a expression. The results demonstrated that ZNRF3 expression was significantly increased at both the mRNA and protein levels after miR-301a knockdown (Fig. 2B and C). Additionally, to assess whether ZNRF3 was the direct functional target of miR-301a, the p-MIR-WT-ZNRF3-3′UTR and p-MIR-MT-ZNRF3-3′UTR reporter plasmids were constructed. Reporter assay results indicated that AS-miR-301a triggered a significantly increase of p-MIR-WT-ZNRF3-3′UTR luciferase activity, but no change in luciferase activity was observed for the MT reporter plasmid (Fig. 2D). Moreover, ZNRF3 expression was significantly downregulated in GBM tissues compared with healthy tissues (Fig. 2E and F).

The public database ENCORI was analyzed, and it was identified that miR-301a expression was weakly negatively associated with ZNRF3 expression in 525 LGG (r=-0.148; P<0.001; Fig. 2G). However, the correlation coefficient was very small, which may due to the LGG or the sample size. These data suggested that miR-301a directly modulated ZNRF3 expression at both the transcriptional and post-transcriptional levels via binding to the 3′UTR.

To further identify the functional relationship between miR-301a and its target ZNRF3, the ZNRF3-mediated role on proliferation and apoptosis exerted by miR-301a was investigated. The verification of miR-301a knockdown is presented in Fig. 3A. The successful establishment of lentivirus-mediated ZNRF3 overexpression is presented in Fig. 3B, and the successful establishment of ZNRF3 knockdown in LN2229 and U87 cells singly transfected with siZNRF3 is presented in Fig. S2.

When ZNRF3 expression was knocked down by siZNRF3 in LN229 and U87 cell lines co-transfected with AS-miR-301a, the ZNRF3 protein expression was significantly down-regulated compared with cells transfected with AS-miR-301a alone (Fig. 3B), indicating that siZNRF3 transfection could effectively abrogate the effect of AS-miR-301a-mediated ZNRF3 upregulation. CCK-8, Annexin V and caspase-3/7 activity analyses demonstrated that the inhibition of proliferation and the promotion of apoptosis induced by AS-miR-301a were partially abolished by siZNRF3 (Fig. 3C-E). Furthermore, ZNRF3 overexpression could significantly enhance caspase-3/7 activity compared with its NC (Fig. 3F), consistent with the effect mediated by AS-miR-301a. These results suggested that AS-miR-301a inhibited the GBM malignant phenotype, partially via ZNRF3.

Our previous studies have reported that ZNRF3 can repress the wnt/β-catenin signaling pathway (unpublished), and combined with the fact that ZNRF3 is a direct target of miR-301a, it was further examined whether miR-301a also regulates the wnt/β-catenin signaling pathway via ZNRF3. Firstly, a TOP/FOP luciferase assay was used to analyze the β-catenin/TCF4 transcriptional activity mediated by miR-301a, and the results indicated that AS-miR-301a suppressed TOP luciferase activity with no apparent change in FOP activity (Fig. 4A). Additionally, AS-miR-301a could decrease the mRNA and protein expression levels of TCF4, c-myc and cyclin D1, as demonstrated by RT-qPCR and western blotting, respectively (Fig. 4B and C). The immunofluorescence results indicated that AS-miR-301a repressed the expression of β-catenin both in the nucleus and cytoplasm (Fig. 4D). It was found that knockdown of ZNRF3 via siRNA following the transfection with AS-miR-301a could significantly abrogate the effects mediated by AS-miR-301a (Fig. 4A-D). Taken together, these data indicated that AS-miR-301a could attenuate the wnt/β-catenin signaling pathway, at least partially via ZNRF3, in LN229 and U87 cells.

Based on the in vitro experimental findings, the effects of AS-miR-301a on tumor growth and wnt/β-catenin signaling were assessed in vivo. U87 cells were implanted into the left flanks of nude mice via subcutaneous injection. AS-miR-301a and miR-NC were treated in a multisite manner every 3 days. After 27 days of treatment, tumors were excised and subjected to further analysis. The verification of miR-301a knockdown in tumor tissues is presented in Fig. 5A. The tumor growth curve indicated that AS-miR-301a significantly inhibited the tumorigenicity of GBM cells, as observed in AS-miR-301a treated mice (Fig. 5B). Additionally, a significant decrease in tumor weight was identified in AS-miR-301a-treated tumors (Fig. 5C). The IHC assay demonstrated that AS-miR-301a significantly increased ZNRF3 expression, and decreased β-catenin, c-myc and cyclin D1 expression levels (Fig. 5D), which was consistent with the in vitro results.

Based on the miR-301a-mediated regulatory effect on ZNRF3, ZNRF3 expression and its relationship with patient prognosis was evaluated in the CGGA dataset (CGGA Mseq325). The mRNA expression of ZNRF3 was significantly down-regulated in high-grade gliomas compared with the LGG, and ZNRF3 expression was negatively associated with the WHO grade (Fig. 6A). Analysis of ZNRF3 expression and its relevant clinical characteristics indicated that ZNRF3 expression was associated with IDH1 gene mutation status, 1p/19q codeletion status (Fig. 6B and C) and age (Fig. S3B). No statistical significance was observed for ZNRF3 expression and sex or progression status (Fig. S3A and C). Furthermore, Kaplan-Meier survival analysis identified that ZNRF3 upregulation conferred prolonged overall survival (Fig. 6D). In combination with the ZNRF3 expression data of the clinical specimens, it was concluded that ZNRF3 may serve as a key tumor suppressor and is involved in GBM progression.

A previous study revealed that miR-301a is activated by the wnt/β-catenin pathway by direct binding of TCF4 to the miR-301a promoter region (13). Thus, it was hypothesized that ZNRF3 restoration could, in turn, affect miR-301a expression via TCF4. Firstly, ZNRF3 could inhibit the wnt/β-catenin transcriptional activity, as verified by the TOP/FOP assay, and the ectopic restoration of miR-301a in stable ZNRF3-overexpressing LN229 and U87 cells could partially abrogate this effect mediated by ZNRF3 (Fig. 7A). The successful transfection efficiency of miR-301a overexpression via RT-qPCR is presented in Fig. S4A. ZNRF3 overexpression could significantly inhibit miR-301a expression, and the knockdown of TCF4 (siTCF4) decreased the miR-301a expression. Moreover, the TCF4 overexpression plasmid in stable ZNRF3-overexpressing LN229 and U87 cells could reverse the ZNRF3-mediated inhibitory effect (Fig. 7B). The successful transfection efficiency of TCF4 overexpression and siTCF4 detected via RT-qPCR is illustrated in Fig. S4B and C. Furthermore, the luciferase assay results with the constructed miR-301a promotor reporter plasmid indicated that ZNRF3 overexpression or the knockdown of TCF4 alone could significantly decreased the luciferase activities, while the TCF4 restoration in ZNRF3 stably overexpressing cells could abrogate this inhibitory effect mediated by ZNRF3 in WT groups as compared with the MT groups (Fig. 7C), indicating that TCF4 transcriptionally regulated miR-301a expression by binding to the promotor of miR-301a. These data demonstrated that ZNRF3 could repress TCF4-depedent miR-301a expression.

To further evaluate the miR-301a-mediated role of ZNRF3 on apoptosis, Annexin V-PI and caspase-3/7 activity assays were performed and the results demonstrated that the transfection of miR-301a mimics in stable ZNRF3-overexpressing LN229 and U87 cells reversed the ZNRF3-induced effect on apoptosis (Fig. 7D and E). Furthermore, in vivo experiments indicated that ZNRF3 overexpression significantly inhibited tumor growth and that the miR-301a mimics treatment partially reversed this effect mediated by ZNRF3 (Fig. 7F). The ZNRF3 expression from different treatment groups is presented in Fig. 7G, it was found that ZNRF3 expression in the ZNRF3 + miR-301a group was decreased compared with the ZNRF3 + miR-NC group (P<0.01; Fig. 7G). The IHC assay results for caspase 3 expression were in line with the in vitro results (Fig. 7H). In summary, the results indicated that the miR-301a/ZNRF3/wnt signal feedback loop may be involved in gliomagenesis.