MiR‐4524b‐5p‐targeting ALDH1A3 attenuates the proliferation and radioresistance of glioblastoma via PI3K/AKT/mTOR signaling

Abstract Increasing evidence has revealed a strong connection between the aldehyde dehydrogenase family member ALDH1A3 and tumorigenesis, therapy resistance, and prognosis in diverse types of cancer. However, the specific miRNA involved in the pathways that regulate ALDH1A3‐mediated glioblastoma (GBM) radioresistance remains to be elucidated. In this study, we demonstrated a high expression of ALDH1A3 in GBM cells, which plays a critical role in their proliferation and radioresistance. We also identified miR‐4524b‐5p, which is downregulated in GBM, as the ALDH1A3 upstream regulator. Overexpression of miR‐4524b‐5p reduced proliferation and radioresistance in GBM cells. Moreover, silencing ALDH1A3 reduced PI3K/AKT/mTOR signaling and glycolytic activity in GBM cells, whereas inhibiting mTOR reversed the radioresistance effects of ALDH1A3 on these cells. In vivo experiments have evidenced that ALDH1A3 silencing and miR‐4524b‐5p overexpression significantly reduced tumor growth and GBM cells radioresistance. In summary, targeting the miR‐4524b‐5p and ALDH1A3 axis is a promising therapeutic strategy for treating GBM.

aldehydes into carboxylic acids. 7Furthermore, ALDH1A3, a significant member of the ALDH family, has been associated with tumorigenesis, progression, and radioresistance in various types of cancer. 8cent studies have shown that miR-7 targets ALDH1A3, which leads to a reduction of breast cancer growth and stem cell subpopulations. 9Inhibition of ALDH1A3 results in a decrease in invasion and metastasis in colorectal cancer cells through the miR-200-ZEB1/ SANI2 pathway. 10However, the role of upstream miRNA in regulating radioresistance through ALDH1A3 signaling pathways in GBM remains to be elucidated.
MicroRNAs (miRNAs) are small, endogenous, non-coding nucleotides that bind to mRNA strands to degrade or prevent them from being translated into proteins. 113][14] A recent study has revealed that overexpressing miR-195-3p inhibits nasopharyngeal carcinoma growth, progression, and radioresistance by suppressing cyclin-dependent kinase 1. 15 Moreover, miR-153-3p enhances cell radiosensitivity by targeting BCL2. 16It has been also reported that miR-187 promotes the proliferation of prostate cancer cells by inhibiting the expression of ALDH1A3. 17wever, the potential of miRNA to interact with ALDH1A3 in the regulation of tumorigenesis and radioresistance in GBM remains to be investigated.
In the present study, we showed that increased expression of ALDH1A3 promotes tumorigenesis and radioresistance in GBM.
Moreover, we have demonstrated that miR-4524b-5p targets ALDH1A3 to reduce proliferation and radioresistance in GBM by regulating the PI3K/AKT/mTOR signaling pathway and glycolytic activity.Therefore, targeting the miR-4524b-5p-ALDH1A3 axis may be a promising therapeutic approach for treating GBM.

| Cell culture
The GBM cell lines U251, A172, U87, and LN229, and normal human astrocytes (NHA) were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences.Cells were cultured in DMEM (Gibco), containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin solution (Thermo Fisher Scientific, Inc.) and maintained in a humidified incubator at 37°C with 5% CO 2 and 95% air.

| Clinical samples
We obtained 13 pairs of GBM and adjacent normal tissues from glioma patients, undergoing brain surgery at The First Affiliated Hospital of Xi'an Jiaotong University, between June and July 2021.

The use of these tissues was approved by the Hospital's Ethics
Committee (Xi'an, Shaanxi, China 710,061; Approval No. 2020-G13) and consent forms were signed for the use of clinical samples.

| X-ray irradiation
U251 GBM cells and BALB/c nude female mice were exposed to Xray irradiation at 0, 2, 4, 6, and 8 Gy for 0, 24, 48, and 72 h, using a small animal radiation research platform (SARRP; Xstrahl Ltd.).Mice were anesthetized using a portable machine (VetTech Solutions Ltd), before being placed in the irradiation device.Following irradiation, they were kept in cages and monitored, until they regained full consciousness.

| RNA isolation and quantitative PCR
We used the TRIzol® reagent (AccuRef Scientific) to extract RNA, following the supplier's instructions.RNA concentration was measured in a NanoDrop 2000 device (Thermo Fisher Scientific, Inc.) and cDNA was synthesized with the PrimeScript® RT Master Mix Perfect Real-Time Reagent kit (Takara Biotechnology Co., Ltd.).GGG ACTAAA; GAPDH forward, GGAGC GAG ATC CCT CCA AAAT and reverse GGCTG TTG TCA TAC TTC TCATGG; U6 forward, CTCGC TTC GGC AGC ACA and reverse AACGC TTC ACG AAT TTGCGT.

| Cell counting kit-8 assay
We used the CCK-8 kit (AccuRef Scientific) to assess cell proliferation.U251 cells (2000 cells/well) were cultured in 96-well plates with different treatments.Ten microliters of the CCK-8 reagent were added to all wells and plates incubated for 2 h at 37°C.Absorbances were then read at 450 nm in an ELX800 microplate reader (BioTek Instruments, Inc.) to evaluate cell proliferation.

| Colony forming assay
U251 cells were incubated in 6-well plates at a density of 500 or 1000 cells per well for 14 days, refreshing the culture medium every other day.Next, cell colonies were fixed with 100% methanol at 4°C for 30 min and stained with 1% crystal violet in 20% methanol at room temperature for 30 min.Cell clones were observed using an inverted microscope.

| Flow cytometry
The Annexin V FITC Apoptosis Detection kit was purchased from AccuRef Scientific and apoptosis analysis was performed following the manufacturer's instructions.The level of apoptosis was determined by calculating the percentage of cells in the early and late stages of apoptosis.
The plasmid containing the reporter gene was co-transfected with phRL-TK into cells that were cultured to a confluence of 70% to 80% in 24-well plates.U251 cells were co-transfected with 100 ng of pGL3-WT, 20 ng of the transfection control Renilla vector (pRL-TK; Promega Corporation), and 100 nM of miR-4524b-5p mimic or negative control (NC) mimic (RIBOBIO Co., Ltd.), using the Lipofectamine® 3000 kit (Thermo Fisher Scientific, Inc.) and following supplier's instructions.

| Measurement of glucose uptake and lactate and ATP production
The impact of silencing ALDH1A3 on glucose uptake and lactate and ATP production in U251 cells was measured using the colorimetric glucose uptake assay kit (Abcam; cat.no.ab136955), the colorimetric lactate assay kit (Abcam; cat.No. ab65331), and the luminescent ATP detection assay kit (Abcam; cat.No. ab113849), following manufacturer's protocols.

| Xenograft mouse model
Female BALB/c nude mice, aged 4-6 weeks, were purchased from Jiangsu Aniphe Biolaboratory Inc.They were housed in a specific-pathogen-free (SPF) environment at the Experimental Animal Center of The First Affiliated Hospital of Xi'an Jiaotong University.Stable negative control lentivirus-infected U251 cells (LV) and stable lentivirus-infected U251 cells expressing miR-4524b-5p (LV-miR-4524b-5p) were suspended in PBS and mixed with an isopycnic Matrigel (BD Biosciences) at a final concentration of 5 × 10 6 cells/mL.Next, 1 × 10 6 cells were subcutaneously injected into nude mice, and tumor size was measured every 3 days, using Vernier calipers.Mice were then euthanized with an intraperitoneal injection of 180 mg/kg ketamine (Sigma-Aldrich) and 20 mg/kg xylazine (Sigma-Aldrich) at day 27, after which subcutaneous tumors were weighed, imaged, and analyzed.
All animal experiments were conducted following the guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" (National Academies Press).The animal experimental protocol was approved by the Ethical Review Committee for Animal Experiments of the First Affiliated Hospital of Xi'an Jiaotong University.

| Immunohistochemistry staining
Mouse tumor slices were incubated overnight with primary antibodies (1:200 dilution) at 4°C.After washing with PBS, slices were incubated with HRP-conjugated secondary antibodies (1:1000 dilution) for 1 h at room temperature.Immune signals were then detected using the DAB substrate kit (Vector Laboratories) and nuclei were counterstained with hematoxylin and Hoechst.Negative controls consisted of slices that were not exposed to primary antibodies.

| TUNEL assay
Apoptosis detection was performed using the in situ cell death detection kit POD (Roche Diagnostics).The procedure involved fixing coverslips with 4% paraformaldehyde, blocking with 3% H 2 O 2 , and washing twice with PBS.Coverslips were then permeated with 0.1% Triton X-100 on ice for 2 min, after which they were exposed to the TUNEL reagent in darkness at 37°C for 1 h in a humid chamber.After three washes, slides were observed by fluorescence microscopy (Olympus).

| Statistical analysis
Statistical analysis was determined using the SPSS 22.0 software (IBM).Data represented the mean ± standard deviation of triplicate determinations from three independent experiments.The normality of data distribution was assessed using the Shapiro-Wilk test.
We used the two-tailed Student t-test (normal distribution) or the Mann-Whitney test (nonnormal distribution) for the comparison between the two groups.Comparisons among multiple groups were estimated by one-way or two-way ANOVA, followed by Bonferroni's post hoc multiple comparison tests (normal distribution) or the Kruskal-Wallis's test (nonnormal distribution).

| Increased expression of ALDH1A3 promotes proliferation and radioresistance of GBM cells
Thirteen sets of GBM tissues were examined to determine the expression of ALDH1A3, using qRT-PCR.Results showed that ALDH1A3 expression levels were significantly higher in GBM tissues, compared with adjacent normal tissues (Figure 1A).Bioinformatics analysis of the CGGA database revealed that glioma patients exhibiting high levels of ALDH1A3 expression had a poor clinical prognosis (Figure 1B).Moreover, qRT-PCR and Western blot analyses were conducted to evaluate mRNA and protein expression levels of ALDH1A3.As shown in Figure 1C,D, U251 cells exhibited higher expression of ALDH1A3, compared with normal human astrocytes (NHA) and other GBM cell lines.[20][21] However, the role of ALDH1A3 on GBM radioresistance remains to be elucidated.To explore the potential correlation between ALDH1A3 and radioresistance, we assessed the mRNA expression of ALDH1A3 at different radiation doses and exposure times.As shown in Figure 1E, ALDH1A3 expression increased in U251 cells, which correlated with increased exposure to radiation doses and duration.Furthermore, three siRNAs were designed to silence the expression of ALDH1A3 in GBM cells.Among them, si-ALDH1A3-2 and si-ALDH1A3-3 were found to be the most effective in targeting ALDH1A3 (Figure 1F).Growth and colony-forming potential of U251 cells were significantly reduced after transfection with si-ALDH1A3-2 and si-ALDH1A3-3, compared with the si-negative control (si-NC) (Figure 1G).As shown in Figure 1H, si-NC-transfected U251 cells had a higher colony formation rate than cells treated with si-ALDH1A3-2 or si-ALDH1A3-3, after radiation exposure.Our results also indicated that silencing ALDH1A3 significantly increased U251 cells apoptosis rates, particularly those exposed to 6 Gy radiation, thus suggesting that suppressing ALDH1A3 may enhance the radiosensitivity of GBM cell lines (Figure 1I).

| Decreased expression of miR-4524b-5p in GBM cells
Bioinformatics analysis was conducted to identify the miRNA that binds to the ALDH1A3 consensus binding sites.As a result, miR-4524b-5p was identified as a potential miRNA that regulates the expression of ALDH1A3.To confirm the impact of miR-4524b-5p on ALDH1A3, a mutated version of ALDH1A3 was constructed (Figure 2A).Luciferase activities were significantly reduced in U251 cells that were co-transfected with the miR-4524b-5p mimic and ALDH1A3 wild type (WT), compared with the control.In contrast, an increase in luciferase activity was observed in U251 cells that were co-transfected with si-miR-4524b-5p and ALDH1A3 WT (Figure 2A).However, the ALDH1A3 mutant (MUT) evidenced no effect on luciferase activity in U251 cells, during co-transfection with miR-4524b-5p (Figure 2A).Moreover, overactivation of miR-4524b-5p resulted in a reduction of ALDH1A3 expression in U251 cells (Figure 2B).In contrast, inhibition of miR-4524b-5p increased ALDH1A3 expression in U251 cells (Figure 2C).Adjacent normal tissues exhibited a higher level of miR-4524b-5p expression, compared with GBM tissues (Figure 2D).As shown in Figure 2E, GBM patients with higher levels of miR-4524b-5p had a longer survival rate based on the Kaplan-Meier analysis of the CGGA database.Furthermore, U251 cells exhibited the lowest level of miR-4524b-5p expression in comparison with other GBM cell lines (Figure 2F).Therefore, miR-4524b-5p was significantly decreased in GBM.

| Overexpression of miR-4524b-5p reduced proliferation and enhanced radiosensitivity in GBM cells
As shown in Figure 3A, overexpression of miR-4524b-5p inhibited U251 cells proliferation.In addition, the sensitivity of U251 cells to radiation increased following transfection of miR-4524b-5p mimic after exposure to varying doses of radiation (Figure 3B).Furthermore, a significant decrease of U251 cells colony-forming potential and overexpression of miR-4524b-5p were observed after exposure to 6 Gy radiation (Figure 3C).A significant increase in U251 cells apoptosis rates, following the combination of overexpression of miR-4524b-5p and 6 Gy radiation, indicates that miR-4524b-5p plays a crucial role in the response of GBM cells to radiation (Figure 3D).Taken together, overexpression of miR-4524b-5p not only inhibited GBM cells growth but also increased their radiosensitivity.and glycolysis. 22,23We performed multiple Western blot tests to investigate the impact of ALDH1A3 on the regulation of the PI3K/AKT/mTOR pathway and glycolysis.As demonstrated in Figure 4A, the activities in PI3K/AKT/mTOR signaling pathways were significantly inhibited in U251 cells, when ALDH1A3 was silenced.Furthermore, a reduction in glucose consumption, and lactate and ATP production were observed in U251 cells, after silencing ALDH1A3 (Figure 4B).HK2 and PKM2, the key enzymes of glycolysis, were examined by qRT-PCR and Western blot, after ALDH1A3 knockdown in U251 cells.Results showed that ALDH1A3 decrease was associated with a reduction of mRNA and protein levels of HK2 and PKM2 in U251 cells (Figure 4C,D).

| ALDH1A3 knockdown suppressed the activity of the PI3K/AKT/mTOR signaling pathway and glycolysis in GBM cells
These results indicated that suppression of ALDH1A3 impaired GBM cells activity in the PI3K/AKT/mTOR signal pathway and glycolysis.

| Inhibition of mTOR enhanced radiosensitivity and counteracted the effects of ALDH1A3 on radioresistance
To further investigate the potential role of ALDH1A3 and the PI3K/AKT/mTOR signaling pathway in radioresistance, U251 cells were treated with rapamycin, an mTOR inhibitor.In comparison with DMSO, the use of 10 nM rapamycin significantly decreased the expression of ALDH1A3 and PI3K/AKT/mTOR (Figure 5A).As shown in Figure 5B, U251 cells colony formation was significantly reduced after treatment with 10 nM rapamycin for 24 h, compared with the control group, at various radiation doses.Furthermore, the rate of apoptosis in U251 cells significantly increased when treated with 10 nM of rapamycin and exposed to varying levels of radiation (Figure 5C).Our previous data have revealed that si-ALDH1A3 enhances radiosensitivity in U251 cells (Figure 1H,I).However, as observed in Figure 5D, rapamycin partially blocked the impact of inhibiting ALDH1A3 on the promotion of radiosensitivity.Moreover, the impact of inhibiting ALDH1A3 on the apoptosis rate of U251 cells was abrogated by rapamycin at varying radiation doses (Figure 5E).
Overall, we demonstrated that inhibition of mTOR increased sensitivity to radiation and counteracted the effects of ALDH1A3 on radioresistance in GBM cells.

| ALDH1A3 silencing or miR-4524b-5p overexpression suppressed in vivo tumorigenesis and radioresistance of GBM cells
A series of in vivo experiments was conducted to evaluate the impact of silencing ALDH1A3 or overexpressing miR-4524b-5p on tumorigenesis and radioresistance in GBM.The tumor volume of the xenograft mice was measured every 3 days, using Vernier calipers.
The group that received a radiation dose of 6 Gy showed a significant decrease in tumor volume, compared with the control group (Figure 6A).Furthermore, immunocompromised mice implanted with si-ALDH1A3 U251 cells had smaller subcutaneous tumors than mice that received si-NC cells, when exposed to 6 Gy radiation (Figure 6A).Tumor growth was reduced in immunocompromised mice that were implanted with si-ALDH1A3 U251 cells and subjected to radiation therapy, as evidenced by a decrease in the levels of Ki-67 expression (Figure 6B).The TUNEL assay indicated that transfection with si-ALDH1A3 or exposure to radiation resulted in a higher level of cell death in the tumor area of immunocompromised mice (Figure 6C).Moreover, we observed a reduction in the expression of p-mTOR, HK2, and PKM2 in the subcutaneous tumors of immunocompromised mice that were implanted with si-ALDH1A3 U251 cells or exposed to radiation (Figure 6D,E).These results demonstrated that silencing ALDH1A3 inhibited in vivo tumorigenesis and radioresistance of U251 cells.As shown in Figure 6F, transfection with miR-4524b-5p mimic or radiation treatment reduced in vivo U251 proliferation.We also observed a decrease in Ki-67 expression levels, thus indicating a tumor growth reduction in nude mice that received radiation treatment, after being implanted with miR-4524b-5p mimic U251 cells (Figure 6G).TUNEL assay results showed that overexpression of miR-4524b-5p and exposure to radiation significantly increased cell death within the tumor region of nude mice (Figure 6H).Furthermore, we showed a decrease in p-mTOR, HK2, and PKM2 levels in the subcutaneous tumors of nude mice that were either treated with miR-4524b-5p mimic U251 cells or exposed to radiation (Figure 6I,J).Therefore, results revealed that overexpression of miR-4524b-5p reduced in vivo tumorigenesis and radioresistance of U251 cells.

| DISCUSS ION
Despite the use of radiotherapy and chemotherapy, following maximum tumor resection, the prognosis of GBM patients remains poor with 5-year survival rates of 5%. 24[28] A recent study has shown that increased levels of miR155HG act as a sponge for miR-185, thereby promoting annexin A2 expression, which in turn, contributes to GBM growth and progression. 29reover, miR-4524b-5p targets the WTX/β-catenin pathway to regulate cervical cancer migration and invasion. 30In the present study, miR-4524b-5p was identified as the upstream regulator of ALDH1A3 signaling, which affects tumor proliferation and radioresistance in GBM.Overexpression of miR-4524b-5p resulted in a reduction in ALDH1A3 expression in GBM cells and was associated with poor prognosis in GBM patients.These results suggest that miR-4524b-5p acts as a tumor suppressor in GBM.
Growing evidence has shown that ALDH1A3 participates in various biological processes, including development, progression, and therapy resistance in multiple types of cancer. 8,31We have previously demonstrated that ALDH1A3 serves as a functional marker for mesenchymal glioma stem cells, which are characterized by radioresistance and activated glycolytic metabolism. 4Furthermore, a recent study has shown that ALDH1A3 plays a key role in the lipid peroxidation of GBM cells, leading to resistance to temozolomide therapy. 32Our data suggest that ALDH1A3, which is highly expressed in GBM, is strongly associated with poor prognosis and radioresistance in GBM, which agrees with other studies.In addition, silencing ALDH1A3 not only suppressed glycolytic activity but also targeted the PI3K/AKT/mTOR signaling pathway in GBM.
Radiotherapy primarily kills tumor cells by inducing free radical damage to DNA. 33It is well known that the PI3K/AKT/mTOR pathway is one of the most critical mechanisms of radioresistance.Its activation may enhance DNA repair potential in cancer cells. 23,34r data showed that silencing ALDH1A3 significantly impaired the activities in the PI3K/AKT/mTOR signaling pathways.In addition, we found that inhibiting mTOR counteracted the effects of ALDH1A3 knockdown on GBM cell proliferation and radioresistance.It has been reported that ALDH1A3 activates the PI3K/AKT/ mTOR signaling pathway by cross-talking with the transcription factor PPARγ in pancreatic cancer. 35Furthermore, abnormal expression of ALDH1A3 promotes chemotherapy resistance in prostate cancer, via the PI3K/AKT/mTOR axis. 36Our study revealed a potential regulatory mechanism between ALDH1A3 and the PI3K/AKT/mTOR axis, which is consistent with previous studies.Reports have shown that malignant tumor cells utilize aerobic glycolysis to accumulate pyruvate, lactate, and glutathione, thus enhancing their antioxidant activity and resistance to the harmful effects of free radicals and oxidative stress resulting from radiotherapy. 37In addition, the metabolic by-products resulting from aerobic glycolysis pathways form a network that facilitates the scavenging of free radicals and reactive oxygen species (ROS) within tumor cells.This may reduce the effectiveness of radiotherapy treatment. 38In this study, we observed a significant decrease in glucose consumption, and lactate and ATP production in GBM cells, following ALDH1A3 suppression.
As expected, ALDH1A3 knockdown significantly decreased mRNA and HK2 and PKM2 protein levels, which are key enzymes involved in glycolysis.Taken together, we demonstrated that suppression of ALDH1A3 impaired the efficacy of radiotherapy in GBM by deactivating the PI3K/AKT/mTOR signaling pathway and glycolysis.
In conclusion, the results of the present study indicated that miR-4524b-5p directly targets ALDH1A3, which reduces GBM cells proliferation and radioresistance by regulating the PI3K/AKT/mTOR signaling pathways and glycolysis.Therefore, targeting the miR-4524b-5p-ALDH1A3 axis may be a promising novel approach for GBM treatment.

Furthermore
, qRT-PCR was performed on an ABI 7500 Real-Time PCR instrument (Applied Biosystems), using cDNA and SYBR Green Reagent (Takara Biotechnology Co., Ltd.), under the following conditions: 95°C for 5 min, then 40 cycles of 95°C for 15 s, 58°C for 20 s, and 72°C for 10 s.The internal control was either GAPDH (mRNA) or U6 (miRNA).Relative quantification was calculated as 2 −ΔCT .ΔCt values were calculated by subtracting the mean Ct value of the control gene from the mean Ct value of the target gene.The following primers were used in this study: ALDH1A3 forward, GATAA GCC CGA CGT GGACAA and reverse, ATACA GCC CTC CAG GTCGAT; miR-4524b-5p forward, CGCGA TAG CAG CAT AAGCC and reverse, AGTGC AGG GTC CGA GGTATT; HK2 forward, GTGAA TCG GAG AGG TCCCAC and reverse, CAAGC AGA TGC GAG GCAATC; PKM2 forward, GCCGA GAG CCA AGA AAAGAC and reverse, GCCCT TTC GGT | 3 of 11 YU et al.
Recent studies have demonstrated that tumor radioresistance is linked to aberrant activation of the PI3K/AKT/mTOR pathway F I G U R E 1 Increased expression of ALDH1A3 promotes proliferation and radioresistance in GBM cells.(A) ALDH1A3 was highly expressed in GBM tissues, compared with adjacent normal tissues.(B) The Kaplan-Meier analysis of the CGGA database revealed that glioma patients exhibiting elevated levels of ALDH1A3 expression had a poor clinical prognosis.(C) mRNA expression levels of ALDH1A3 were elevated in GBM cell lines, compared with that of normal human astrocytes (NHA).(D) The amount of ALDH1A3 protein was enriched in GBM cell lines, compared with that of NHA.(E) mRNA expression levels of ALDH1A3 were measured in U251 cells exposed to 0, 2, 4, and 6 Gy doses of radiation for 0, 6, 12, and 24 h.(F) Levels of ALDH1A3 mRNA decreased in U251 cells, after siRNA transfection.(G) Silencing ALDH1A reduced U251 cells growth rate.(H) U251 cells colony-forming potential was significantly reduced, after transfection with si-ALDH1A3-2 and si-ALDH1A3-3 and upon exposure to 0 Gy and 6 Gy radiation.(I) Silencing ALDH1A3 and exposing GBM cells to 6 Gy radiation significantly increased U251 cells apoptosis rates.NC represents the negative control.β-actin served as a control.*p < 0.05, **p < 0.01.

F I G U R E 2
Decreased expression of miR-4524b-5p in GBM cells.(A) Luciferase activity significantly decreased, after co-transfecting miR-4524b-5p mimics and ALDH1A3 WT.In contrast, luciferase activity increased in U251 cells that were co-transfected with si-miR-4524b-5p and ALDH1A3 WT. (B) Overexpression of miR-4524b-5p inhibited ALDH1A3 expression in U251 cells.(C) Inhibition of miR-4524b-5p increased ALDH1A3 expression in U251 cells.(D) Adjacent normal tissues exhibited higher expression of miR-4524b-5p, compared with GBM tissues.(E) The Kaplan-Meier analysis of the CGGA database revealed that GBM patients with higher levels of miR-4524b-5p experienced longer survival times.(F) GBM cell lines exhibited a decrease in the expression of miR-4524b-5p in comparison with normal human astrocytes (NHA).NC represents the negative control.**p < 0.01.

F I G U R E 5
Inhibition of mTOR enhanced radiosensitivity and counteracted the effects of ALDH1A3 on radioresistance in U251 cells.(A) Treatment with 10 nM rapamycin, an mTOR inhibitor, significantly suppressed the expression of ALDH1A3 and PI3K/ AKT/mTOR in U251 cells, compared with DMSO treatment.(B) After 24 h of treatment with 10 nM rapamycin, a significant decrease in colony-forming potential was observed in U251 cells, when exposed to various radiation doses.(C) Combination of 10 nM rapamycin and 6 Gy radiation resulted in a higher rate of apoptosis in U251 cells.(D) Promotion of radiosensitivity through ALDH1A3 silencing was compromised in U251 cells, following pretreatment with 10 nM of rapamycin.(E) Rapamycin counteracted the impact of inhibiting ALDH1A3 on U251 cells apoptosis rate, after 6 Gy radiation.NC represents the negative control.**p < 0.01.

F
I G U R E 6 ALDH1A3 silencing or miR-4524b-5p overexpression suppressed in vivo tumorigenesis and radioresistance of GBM cells.(A) Xenograft nude mice that were either exposed to 6 Gy radiation or had si-ALDH1A3 GBM cells implanted, experienced a significant decrease in tumor size, compared with the control group.(B) Ki-67 expression was suppressed in xenograft nude mice that received either si-ALDH1A3 U251 cells or 6 Gy radiation, compared with the control group.(C) According to the TUNEL assay, transfection of si-ALDH1A3 or exposure to 6 Gy radiation increased levels of cell death within the cancerous region of nude mice.(D) Expression of p-mTOR decreased in the subcutaneous tumors of nude mice that were implanted with si-ALDH1A3 U251 cells or exposed to 6 Gy radiation.(E) Decrease HK2 and PKM2 levels were observed in the subcutaneous tumors of nude mice that were implanted with si-ALDH1A3 U251 cells or exposed to 6 Gy radiation.(F) U251 cells tumorigenesis was reduced by miR-4524b-5p mimic or 6 Gy radiation.(G) Decrease Ki-67 expression was observed in xenograft nude mice that had either received 6 Gy radiation or were implanted with miR-4524b-5p mimic U251 cells.(H) Overexpression of miR-4524b-5p and exposure to 6 Gy radiation increased cell death in the tumor region of nude mice.(I) P-mTOR amount decreased in the subcutaneous tumors of nude mice, after exposure to 6 Gy radiation or following implantation of miR-4524b-5p mimic U251 cells.(J) Overexpression of miR-4524b-5p and exposure to 6 Gy radiation decreased mRNA expression of HK2 and PKM2 in xenograft nude mice.NC represents the negative control.*p < 0.05, **p < 0.01.