Overexpression of miR-124 enhances the therapeutic benefit of TMZ treatment in the orthotopic GBM mice model by inhibition of DNA damage repair

Given previous studies used only one independent cohort to analyze the differential expression genes in GBM tissues in comparison with normal tissues, it may not rule out the biased sample selection and data analysis. We wondered to globally analyze the dysregulated miRNAs and deep explore their biological roles in advanced progression and therapeutic response in GBM patients. Accordingly, we employed the “limma” R package to construct differential expression genes analysis from three public cohorts. We found that 1657, 333 and 602 miRNAs are downregulated in these three GBM cohorts, respectively (Fig. 1A). Among them, we selected the top-50 ranking downregulated miRNAs from each cohort to analyze the overlapped subjects. And then, 8 miRNA candidates, including miR-873, miR-124, miR-7, miR-338, miR-129, miR-137, miR-132 and miR-490 were identified to all downregulated in three tested cohorts (Fig. 1B). To further estimate the prognostic value of these miRNA candidates in the clinic, we used the Kaplan–Meier plotter to analyze the contribution of these miRNAs for survival rate in the Chinese GBM Genome Atlas (CGGA) dataset. Interestingly, we found that miR-873, the most significant fold change and false discover rate miRNA, has no significance for survival analysis, whereas miR-124, ranking the second miRNA was significantly associated with poor prognosis in a low expression level (Fig. 1D). Accordingly, we focused on the function and regulatory mechanism of miR-124 in GBM. As shown in the tested datasets (GSE90603↗, GSE138746↗ and GSE63319↗), the miR-124 expression is in a significant low level in the tumors compared with normal tissues (Fig. 1C). Taken together, all these results indicated that miR-124 is a bona fide potential indicator for prognosis in GBM patients.

To explore the function of miR-124 in GBM cells, we increased exogenous expression of miR-124 by transfecting miR-124 mimics in U87 and U251 GBM cells lines. Although miR-124 has been documented as a tumor-suppressive miRNA in cancers, strikingly, we found that overexpression of miR-124 has no significant effect on cell proliferation in two tested GBM cell lines using the CCK-8 assay (Fig. S1A). Then, we asked whether miR-124 could have an influence on the activity of the first-line agent TMZ (Fig. 2A, B). GBM cells were treated with a range of concentrations of TMZ with or without the miR-124 mimic transfection, and the ability of TMZ to inhibit cell viability was assessed using the CCK-8 assay. As shown in the Fig. 2A, overexpression of miR-124 enhances TMZ-mediated inhibition on GBM cell proliferation. Vice versa, when we used miR-124 antagomiR to decrease the endogenous miR-124 in GBM cell lines, TMZ-mediated inhibition on GBM cell proliferation was significantly attenuated using CCK-8 assay (Fig. 2B). And the similar phenotypes were also observed with high dose TMZ treatment (Fig. S1B). These results indicated that miR-124 may sensitize the GBM cells to TMZ treatment rather than suppress tumor growth directly.

Furthermore, we established a co-culture cell competition assay to more accurately estimate the effect of miR-124 on responsiveness of GBM cells to the TMZ. Parental cells stably expressed green fluorescent protein EGFP or red fluorescent protein mCherry, and then fluorescent GBM cell lines were transfected with miR-124 or control mimics, respectively. Next, 1:1 mixed cells were treated with TMZ in the indicated dosage. As expected, the number of red cell was reduced and the variation of mCherry/GFP ratio was increased (Figs. 2C, D, S2A, B). The similar phenotypes were also observed with high-dose TMZ treatment (Fig. S1C, D). Overall, these data supported that miR-124 enhances TMZ sensitivity in GBM cells.

To further confirm the effect of miR-124 on the TMZ treatment in GBM cells, we utilized Annexin V staining to test its influence on TMZ-induced cell death. It was shown that the treatment of miR-124 plus TMZ significantly increases percentage of Annexin V positive cells compared with TMZ alone (Figs. 2E and S2E). Additionally, analysis of Western blot showed that overexpression of miR-124 increases cleavage of Caspase-3 in TMZ-treated GBM cells (Fig. S1E). Taken together, our data revealed that overexpression of miR-124 enhances TMZ-mediated cell death in GBM cells.

Although it has been reported that miR-124 enhances TMZ-involved chemosensitivity by targeting Ras family members, Ras-driven intracellular signaling cannot rationally explain miR-124-involved regulation on TMZ responsiveness [23]. Given that the major mechanism of TMZ-induced cell death is DNA damage and known mechanisms of TMZ resistance are closely associated with the increasing activation of DNA damage repair [24], we wondered to known whether miR-124-mediated high responsiveness of GBM cells to TMZ is in a DNA damage repair dependent behavior. We measured the persistence of double-strand breaks after TMZ treatment as an indicator of unrepaired damaged DNA. Single-cell gel electrophoresis (alkaline comet assay) was carried out to measure DNA damage. GBM cells with ectopic overexpression of miR-124 had lower levels of RAD51 protein and statistically significantly higher residual DNA damage than control cells (Fig. 3A–D). Moreover, we examined the expression of γ-H2AX, a canonical biomarker for DNA damage by immunofluorescence staining. The results showed that a much higher level of γ-H2AX foci appears in GBM cells treated with overexpression of miR-124 plus TMZ in comparison with miR-ctrl plus TMZ (Fig. 4E, F). Taken together, these data indicated that miR-124 augments DNA damage in GBM cells with TMZ treatment. More importantly, among the reported mechanistic studies, there is no any report on how miR-124 affects TMZ responsiveness by modulating the DNA damage repair pathway.

To deep explore the underlying regulatory mechanism of miR-124 in GBM, four miRNA-mRNA binding prediction software (miRcode, miRDB, miRTarBase and TarBase) were used to predict the potential target genes regulated by miR-124. And 269 candidates were overlapped in all four-prediction software (Fig. 4A). We wondered to know whether the novel target(s) of miR-124 is upregulated in GBM tissues. Accordingly, in the TCGA GBM dataset, we set the parameters that fold change is more than 3 and p-value less than 0.05, and 2688 differential upregulated genes were screened. And then, we performed intersection analysis on the bioinformatic predicted target genes and the differential genes analyzed in the TCGA GBM database. RAD51 and PRDM13 were finally identified in the overlapped section (Fig. 4B). To further confirm the downstream gene, we used Pearson analysis to determine the relationship between miR-124 with RAD51 or PRDM13, respectively. Notably, the level of RAD51 expression in GBM was negatively correlated with the miR-124 expression, whereas the correlation between miR-124 and PRDM13 had no obvious significance (Fig. 4C).

To confirm the regulatory relationship between miR-124 and RAD51, we overexpressed miR-124 in a series of GBM cell lines and examined the expression of RAD51 using RT-qPCR and Western blot. As shown in Fig. 4D–F, when GBM cells were treated with miR-124 mimic, the mRNA and protein levels of RAD51 reduced significantly. Conversely, cancer cells transfected with miR-124 antagomiR expressed a much higher level of RAD51 than those transfected with the control mimics (Fig. 4G), indicating that RAD51 is negatively regulated by miR-124 in GBM cells. Next, we wondered to confirm the direct interaction between miR-124 and the RAD51 mRNA. The conserved targeting sequences for miR-124 were found in the 3′UTR of RAD51 mRNAs (Fig. 4H). This observation was further substantiated by the luciferase assay. 293 cells were transfected with the luciferase reporter plasmid containing the WT 3′UTR of RAD51 mRNA and miR-124 mimics, resulting in the inhibition of luciferase activity, whereas the luciferase activity of mutant 3′UTR was not suppressed by miR-124 (Fig. 4I). Collectively, these results suggested that RAD51 is a novel and bona fide target post-transcriptionally silenced by miR-124 in GBM cells.

Given that we identified that RAD51 is a novel target of miR-124 in GBM cells, we wondered to identify the biological function of RAD51 in GBM. TCGA and CGGA datasets were applied to evaluate the expression of RAD51, and it was shown that the RAD51 level is significantly higher in GBM tissues compared with the normal ones (Fig. 5A). More importantly, it was found that a higher level of RAD51 is significantly associated with poor prognosis in GBM patients (Fig. 5B). To evaluate the biological contribution of RAD51 on TMZ responsiveness in GBM, we used two independent shRNAs to knock down RAD51 expression in U87 and U251 cell lines, respectively. The efficiency of RAD51 knockdown in GBM cell lines was validated by Western blot and RT-qPCR (Fig. 5C, D). Also, we performed qRT-PCR assay and identified that RAD51 knockdown has no significant effect on the miR-124 expression level in GBM cell lines (Fig. S2C, D). Using CCK-8 assay, we found that RAD51 knockdown obviously increased TMZ-mediated inhibition on GBM cell proliferation, emphasizing the importance of the regulatory role of RAD51 in TMZ-treated GBM cells (Fig. 5E, F). Furthermore, with the treatment of TMZ, we also used immunofluorescence staining of γ-H2AX to determine the influence of RAD51 on TMZ-induced DNA damage repair. As shown in Fig. 5G, H, RAD51 knockdown increased γ-H2AX foci in GBM cells. Finally, we determined the role of RAD51 inhibition on TMZ-induced GBM cell death. Accordingly, we used flow cytometry to evaluate the percentage of Annexin V positive cells in RAD51 knockdown GBM cells with the TMZ treatment (Fig. 5I, J). Our data revealed that RAD51 knockdown sensitizes TMZ-induced DNA damage and consequently results in more cell death in GBM cells.

Given that we identified a miR-124-RAD51 regulatory axis in GBM cells, we wondered to know whether RAD51 is essential for mir-124 mediated regulation of GBM to TMZ treatment. We performed a rescue experiment to overexpress RAD51 in the miR-124 overexpressing GBM cells. The exogenous expression of RAD51 was validated by Western blot and RT-qPCR (Fig. 6A, B). Strikingly, using CCK-8 assay, we found that restoration of RAD51 significantly attenuates overexpression of miR-124 induced high responsiveness of GBM cells to TMZ treatment (Fig. 6C, D). Additionally, we used the immunofluorescence staining to test the effect of RAD51 on miR-124 sensitized DNA damage. The result showed that restoration of RAD51 inhibits γ-H2AX foci in miR-124 overexpressing GBM cells with the TMZ treatment (Fig. 6E, 6). Finally, we used FACS to test the influence of RAD51 on miR-124 promoted high responsiveness of GBM cells to TMZ. We found that rescue of RAD51 decreases percentage of Annexin V positive GBM cells treated with TMZ (Fig. 6G, H).

To further verify the role of RAD51 in miR-124 involved regulation on TMZ treatment in vivo, we performed the rescue experiment by restoring RAD51 expression in vivo with TMZ treatment. Notably, HE staining showed that enforced expression of RAD51 in miR-124 overexpressing tumor tissues obviously attenuates TMZ-induced suppression on tumor growth, indicating the potential role of a miR-124-RAD51 axis in TMZ resistance in vivo (Fig. 7A–C). Moreover, the result of immunofluorescence staining of γ-H2AX, Ki67 and TUNEL showed that restoring RAD51 expression decreases DNA damage and cell apoptosis, and concurrently increases the tumor growth index (Fig. 7D–F). Together, these results demonstrated that RAD51 is essential for miR-124 mediated regulation of GBM to TMZ treatment in vitro and in vivo.

To verify the effect of miR-124 in vivo, we applied stereotaxic apparatus to establish an intracranial orthotopic GBM mice model. U87 tumor-bearing mice were divided into four groups, including “saline plus Vector”, “saline plus miR-124”, “TMZ plus Vector” and “TMZ plus miR-124”. As shown in the HE staining, data of the tumor size and tumor occupancy rate showed that tumorigenicity is most significantly delayed in the TMZ plus miR-124 treated group (Fig. 8A, B). More importantly, Kaplan–Meier plotter analysis showed that the combinational utilization of miR-124 and TMZ potentially prolongs the survival time of tumor-bearing mice among four groups (Fig. 8C). Furthermore, data of the immunofluorescence staining showed that the expression of RAD51 obviously decreases in the miR-124 overexpressing xenografts. In addition, the result of immunofluorescence staining on γ-H2AX signaling showed that combinational treatment group leads to more DNA damage phenotype than TMZ or miR-124 treatment alone (Fig. 8D–F). Additionally, Ki67 and TUNEL staining in tumor tissue showed that the TMZ plus miR-124 treated group has a more effect on inhibition of cancer cell proliferation and induction of cancer cell death in vivo (Fig. 8G–J). Finally, we used a co-culture fluorescence competition assay in vivo to validate therapeutic benefit of the combinational utilization of mR-124 and TMZ in the orthotopic GBM model. The result showed that xenografts expressing miR-124 and mCherry fluorescent protein was almost eliminated, compared with the tumor tissues only expressing EGFP fluorescent protein (Figs. 8K, L, S2F). Together, these results demonstrated that miR-124 potently augments TMZ treatment efficiency in vivo.

The miRNA expression data (GSE90603↗, GSE138764↗, GSE63319↗) based on none-coding RNA profiling array in GBM were obtained from the open-access Gene Expression Omnibus (GEO) database [47–49], including microRNA sequencing data from both GBM and adjacent tissues. The mRNA expression data based on mRNA microarray or mRNA sequencing and clinical information in GBM were downloaded from The Cancer Genome Atlas (TCGA) and CGGA databases [50, 51]. miRcode, TarBase, miRDB, miRTarBase were used to predict target genes of miR-124 [52–55]. The differential expression genes analysis, clinical survival analysis and correlation analysis were respectively processed by “limma” package, “survival” package or “psych” package of R studio. All the Venn plots, butterfly plot and correlation scatter plot were painted by “ggplot” package of R project.

Human glioblastoma cell lines (U87, U251, A172) and human embryonic kidney cell lines (HEK 293T) were obtained from the American Type Culture Collection (ATCC). All cell lines were routinely authenticated by STR and tested negative for mycoplasma. U-87 MG, U251, A172 and HEK 293T cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum. All the cells were incubated with a humidified atmosphere of 5% CO₂ at 37 °C.

The mimics of miR-124, negative control, miR-124 inhibitor and microRNA inhibitor negative control were obtained from the GenePharma (Shanghai, China). Cells were seeded in 6-well-plates at 50% density before transfection. Using Lipofectamine 2000 (Invitrogen) delivered 20 μM miR-124 mimics or miR-124 inhibitor to cell interior. Total RNA and protein were extracted 48 h after transfection.

To allow perform the luciferase reporter assay, the PGL3-enhancer-RAD51 3′UTR plasmid was established. The 3′UTR of RAD51 containing the predicted binding site of miR-124 was amplified from whole genome by polymerase chain reaction. The primers used in PCR were showed in Supplementary Table. Then the clone fragments were inserted into PGL3-enhancer vector. Before transfection, HEK-293T cells were seeded in 48-well plates at 50% confluence. Successively, the cells were co-transfected with 100 ng pGL3-RAD51-3′UTR, 5 ng PRL-TK and 20 μM miR-124 mimics or negative control by using Lipofectamine 2000 reagent. 48 h after transfection, cells were subjected to lysis and luciferase activity were determined as for a dual-luciferase reporter system, according to the manufacture instructions. 2

Stable expressing green fluorescent protein (GFP) or red fluorescent protein (mCherry) GBM cell lines (U87-GFP, U87-mCherry, U251-GFP, U251-mCherry) were established. GFP cells were transfected with miR-Ctrl, meanwhile, miR-124 was overexpressed in mCherry cells. After 24 h of transfection, cells were collected, successively seeded in 6-well plates with 1 × 10⁵ GFP cells and 1 × 10⁵ mCherry cells and cocultured. Before treated with Temozolomide (Selleck, S1237), flow cytometry or fluorescence microscopy imaging were used to detect the number of GFP and Cherry cells as initial control group. Treated with TMZ for 72 h, samples were collected for flow cytometry and fluorescence microscopy imaging. The variation of Cherry/GFP cells ratio was calculated and regarded as the sensitivity of cells to TMZ.

Primary β-actin antibody was obtained from Sigma-Aldrich (Merck, A5441). Mouse RAD51 monoclinal antibody (67024-1-lg), rabbit RAD51 polyclonal antibody (14961-1-AP) and rabbit Caspase3/p17/p19 polyclonal antibody (19677-1-AP) were purchased from Poteintech (China, Wuhan). Detailed antibody information was included in Supplementary Table. Total proteins were separated from cells by using RIPA buffer (Solarbio, R0010) supplemented with PMSF (Solarbio, P0100). Protein concentration was quantified using BCA kit (Solarbio, PC0020). In brief, 15 μg of whole-cell lysate from each sample was loaded on a 10% polyacrylamide gel for electrophoresis and transferred onto nitrocellulose filter (NC) membranes. Then, the membranes were blocked in 5% non-fat milk in Tris-buffered saline solution (PH 7.4) containing 0.1% Tween-20 and incubated with primary antibody at a concentration of 1:2500 (for β-actin), 1:5000 (for RAD51) or 1:3000 (for Caspase3) overnight at 4 °C. The secondary antibodies were used at a concentration of 1:10,000 for 1 h at room temperature. Enhanced chemiluminescence reagent was used to visualize the protein. 1

Comet assay was performed as previously described [56]. Proliferating U-87 MG and A172 cells were transfected with miR-Ctrl or miR-124 mimics. After 24 h, transfected cells were treated or not with 500 μM TMZ for 4 h and repaired for 15 h to prepare single cell samples. Briefly, treated and untreated cells were collected and resuspended in ice-cold PBS (without Mg²⁺ and Ca²⁺) at 1 × 10⁵ cells per mL. Combined the cells with low-melt agarose at 1/10 ratio at 37 °C, mixed well by pipetting, and immediately transferred 75 μL suspension onto frosted glass slides. The slides were successively placed in pre-chilled Lysis Buffer and Alkaline Solution (Beyotime, C2041M) at 4 °C in the dark, after the agarose solidified. Slides were then subjected to alkaline electrophoresis (1 V per cm of distance between electrodes) for 20 min in alkaline solutions and stained with propidium iodide. DNA damage was quantified for the average Tail DNA% of 6 views, a fraction of tail DNA intensity with total DNA intensity, analyzed by the OpenComet in ImageJ software.

Total RNA was isolated from cells using TRIzol reagent (Thermo Fisher Scientific, 15596018CN). Reverse transcription for mRNA was performed using PrimeScript RT reagent Kit (Perfect Real Time) (Takara, RR047A) according to the manufacturer’s protocol. Mir-X miRNA First-Strand Synthesis Kit (Takara, 638313) for miR-124 reverse transcription was purchased from Takara. β-actin and U6 snRNA (sn-RNU6) were used as normalization controls. All the primer of qPCR were acquired from Tsingke (Supplementary Table). Bio-Rad C1000 Thermal Cycler (Bio-Rad, California, CA, USA) was used to detect the relative expression of mRNA or microRNA and data were analyzed by using the 2^-(ΔΔCt) method. 2

Cells transfected with miR-Ctrl or miR-124 were plated on coverslips and treated with 200 μM TMZ for 24 h. After treatment, 4% paraformaldehyde were used to fix cells for 15 min. Before primary antibody incubation, cells were blocked with 3% bovine serum albumin containing 0.3% Triton-x-100 for 1 h at room temperature. Then cells were incubated with primary antibody against RAD51 (Abcam, ab133534, 1:1000) or γ-H2AX (ABclonal, AP0099, 1:300) in 4 °C overnight. Coralite594-conjugated secondary antibody (Proteintech, SA00013-4, 1:500) or coralite488-conjugated secondary antibody (Proteintech, SA00013-2, 1:1000) was used to combine primary antibody for 1 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Phase images were captured by confocal laser scanning microscope FV3000 series (Olympus) at a magnification of 100× and processed by ImageJ.

After transfected with mimics miR-Ctrl or miR-124, cells were treated with 200 μM TMZ for 24 h. Cells were then collected and resuspended in PBS. FITC Annexin-V Apoptosis Detection Kit I (BD Bioscience, 556547) was used to stain the valgus phosphatidylserine and the FITC positive cells were analyzed by flow cytometry. In xenograft orthotopic tumor tissues, apoptosis of tumor cells was detected by TUNEL staining using an In Situ Cell Death Detection Kit, Fluorescein (Servicebio, G1504) according to the manufacturer’s protocol. The nuclei were then counterstained with DAPI. Phase images were captured by confocal laser scanning microscope FV3000 series (Olympus) at a magnification of 100× and processed by ImageJ.

Cells were transfected with mimics or microRNA inhibitor and then collected to seed at 3.0 × 10³ cells per well onto 96-well plates. Cells were treated with TMZ and counted by Cell Counting Kit-8 assay (CCK-8) (Yeasen, 40203ES80) at 0, 24, 48 h. Cell survival was calculated by normalizing the absorbance to that of untreated controls.

Five-week-old male BALB/c nude mice were purchased from the GemPharmatech LLC (Chengdu, China). All animal procedures were approved by the Laboratory Animal Welfare and Ethics Committee of Air Force Medical University (AFMU). To establish an intracranial tumor model, U87 (5 × 10⁵ in 5 μL PBS) cells with stable expression miR-Ctrl or miR-124, were stereo-tactically implanted into the nude mouse brain by 69100 Rotational Digital Stereotaxic Frame (RWD Life Science). During the surgery, a Hamilton syringe was inserted at lateral 2 mm and ventral 1 mm to the bregma to a depth of 3.5 mm and then pulled back 0.5 mm to allow space for tumor cells. Injection process was sustained 10 min at a speed of 0.5 μL/min and the Hamilton syringe was held for 10 min to avoid the tumor cells overflowed before syringe extraction [57]. After 5 days of intracranial injection, nude mice were treated with normal saline or TMZ (20 mg/kg) by intraperitoneal injection for 4 weeks (once every other day) [58]. The number of surviving nude mice was used to plot Kaplan–Meier survival curve. The brains of nude mice were obtained and embedded with Optimal cutting temperature compound (OCT) for further analysis.

Each experiment was repeated at least three times and data analyses were performed using GraphPad Prism version 8.0.2 (GraphPad, San Diego, USA) and R project (version 4.3.0). All data were expressed as mean values ± SD. The student t-test, one-way analysis of variance (ANOVA), log-rank text and Person correlation test were used to determine significant differences between the groups with p values of less than 0.05 considered statistically significant.

The data supporting the finding of this study are available from the corresponding authors upon reasonable request.