The full name of the cell line used is U87-MG human brain astroblastoma, which is a GBM of unknown origin. Cells were cultured in Minimum Essential Medium (MEM; Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) containing 10% fetal bovine serum (FBS; HyClone; Cytiva) at 37°C with 5% CO₂. The cell line was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

pLVX–IDH1-mCMV-ZsGreen-PGK-Puro and pLVX–IDH1(MUT)-mCMV-ZsGreen-PGK-Puro lentiviruses were purchased from Beijing Xibei Hongcheng Biotechnology Co., Ltd. (http://www.xbhcbio.com/). Trypsin (0.25%)-EDTA was purchased from Invitrogen (Thermo Fisher Scientific, Inc.) and 0.45-µm PVDF membranes and a chemiluminescence kit (Immobilon Western Chemiluminescent HRP Substrate, eCl@ss cat. no. 42029053) were purchased from EMD Millipore. A total protein extraction kit (Whole protein extraction kit, cat. no. KGP250), a BCA assay protein content detection kit (BCA Protein Quantitation Assay, cat. no. KGPBCA), an SDS-PAGE gel preparation kit (KGI SDS-PAGE Gel Preparation kit, cat. no. KGP113) and a flow cytometry cell cycle analysis kit (Cell Cycle Detection kit, cat. no. KGA511) were purchased from Jiangsu Kaiji Biotechnology Co., Ltd. (http://keygentec.com.cn/index.php?cid=1). The BSA (BSA-V, cat. no. A8020) used to dilute the antibody was purchased from Beijing Solarbio Science & Technology Co., Ltd. Mouse anti-IDH1 (R132H; cat. no. SAB4200548) was purchased from Sigma-Aldrich (Merck KGaA). Rabbit anti-IDH1 (cat. no. 8137S), rabbit anti-BMAL1 (cat. no. 14020S), rabbit anti-CLOCK (cat. no. 5157S), rabbit anti-phosphorylated (p)-Smad2 (cat. no. 18338S), rabbit anti-p-Smad3 (cat. no. 9520S), rabbit anti-Smad2 (cat. no. 5339S), rabbit anti-Smad2-3 (cat. no. 86855S), rabbit anti-Smad3 (cat. no. 9513S), rabbit anti-Smad4 (cat. no. 46535S), mouse anti-β-actin (cat. no. 3700S) and mouse anti-GAPDH (cat. no. 51332S) primary antibodies, as well as HRP-labelled goat anti-rabbit IgG (cat. no. 7074S) and goat anti-mouse IgG (cat. no. 7076S) secondary antibodies, were purchased from Cell Signaling Technology, Inc. Rabbit anti-Cyclin A (cat. no. AF0142), rabbit anti-CyclinB1 (cat. no. DF6786), rabbit anti-CyclinD3 (cat. no. DF6229), rabbit anti-CDK2 (cat. no. AF6237), rabbit anti-CDK4 (cat. no. DF6102), rabbit anti-P18 (cat. no. AF0620), rabbit anti-P21 (cat. no. AF6290), rabbit anti-P27 (cat. no. AF6324), rabbit anti-PER1 (cat. no. DF9080), rabbit anti-PER2 (cat. no. DF12304), rabbit anti-PER3 (cat. no. DF7349), rabbit anti-Cry1 (cat. no. DF8932) and rabbit anti-Cry2 (cat. no. DF12919) primary antibodies were purchased from Affinity Biosciences. An IX73 inverted fluorescence microscope was purchased from Olympus Corporation, and an Amersham Imager 600 gel imaging analysis system was purchased from General Electric.

BMAL1 and CLOCK expression data of all types of tumor analyzed in the present study were derived from TCGA database (https://portal.gdc.cancer.gov/) and the online analysis software GEPIA2 (http://gepia2.cancer-pku.cn/#index).

Construction of recombinant lentiviral vector: 1 µg Plasmid and pLVX/IDH1, 1 µl EcoRI, 1 µl BamHI, 2 µl pLVX, 6 µl target gene, 1 µl 10X DNA Ligase Buffer and 1 µl T4 DNA Ligase, were used to construct lentiviral vectors. The 239T cell line (Beijing Xibei Hongcheng Biotechnology Co., Ltd.) was used for virus purification. After culturing for 12 to 16 h, the medium was discarded (500 µl HET Buffer A + 10 µl recombinant lentiviral vector + 15 µl lentiviral packaging vector + HET Buffer B 50 µl + ddH₂O 425 µl), and replaced with 10 ml fresh complete medium solution (DMEM + 10% FBS + P/S). The cells were placed in a 37°C, 5% CO₂ incubator for 48 h, the supernatant was collected after centrifugation at 500 × g and room temperature for 5 min and the cell debris was discarded. The supernatant was filtered with a 45 µm PVDF filter into a 50 ml round bottom centrifuge tube. This was centrifugated at a high speed of 6,000 × g at 4°C for 2 h, and purified recombinant lentivirus was obtained after centrifugation. Lentivirus can be introduced into U87-MG cells after 48 h of infection with MOI value (20:1) (43). U87-MG cells were inoculated in MEM containing 10% FBS at a density of 2×10⁵ cells/100-mm culture dish, incubated at 37°C and then subjected to lentivirus infection 6–8 h after becoming adherent. The titre of the treatment and control lentiviruses was set to an MOI of 20:1, and MEM with 2% FBS was used to prepare viral titre gradient solutions for subsequent experiments. The experimental groups were the IDH1 wild-type (WT) and the IDH1 R132H mutant (MUT) groups, and the corresponding lentiviral vectors were pLVX–IDH1-mCMV-ZsGreen-PGK-Puro and pLVX–IDH1(MUT)-mCMV-ZsGreen-PGK-Puro, respectively. Subsequent experiments were performed 72 h after lentiviral infection.

Following lentiviral transfection, U87-MG cells in each group were collected for extraction of total protein using the aforementioned whole protein extraction kit. The BCA assay kit was used to quantify the protein, and equal amounts of protein (40 µg/10 µl) were separated via 8% SDS-PAGE. Subsequently, the proteins were transferred to PVDF membranes, which were blocked at room temperature in 5% skimmed milk for 1 h and then incubated at 4°C overnight with the following primary antibodies diluted with 3% BSA: Anti-IDH1 (R132H; 1:1,000), anti-IDH1 (1:1,000), anti-GAPDH (1:2,000), anti-β-actin (1:2,000), anti-BMAL1 (1:1,000), anti-CLOCK (1:1,000), anti-PER1/2/3 (1:1,000), anti-CRY1/2 (1:1,000), anti-CyclinA (1:1,000), anti-CyclinB1 (1:1,000), anti-Cyclin D3 (1:1,000), anti-CDK2 (1:1,000), anti-CDK4 (1:1,000), anti-P18 (1:1,000), anti-P21 (1:1,000), anti-P27 (1:1,000), anti-p-Smad2 (1:1,000), anti-p-Smad3 (1:1,000), anti-Smad2 (1:1,000), anti-Smad2-3 (1:1,000), anti-Smad3 (1:1,000) and anti-Smad4 (1:1,000). The membranes were incubated with secondary antibodies (HRP-conjugated goat anti-rabbit IgG and goat anti-rat IgG; both 1:2,000) at room temperature for 1 h. The chemiluminescence reagent was used for color development for 1 min, and the membranes were exposed for greyscale measurement. The protein expression level was semi-quantified using Image Pro Plus 6.0 (Media Cybernetics, Inc.).

After seeding 1,000 lentivirus-infected U87-MG cells into 100-mm culture dishes, the cells were divided into the WT and MUT groups. After 5 days of culture, the medium was changed once, and whether the cells formed clumps or sheets was observed under a 10× light microscope. The medium was then discarded. The cells were fixed in 4% paraformaldehyde at room temperature for 20 min. Methylene violet solution (4 ml) was added to each dish, and the cells were stained at room temperature for 30 min before rinsing with pure water. After drying at room temperature, images were captured, and the colonies, which are clumps or flakes formed by cells, were counted using Image-Pro Plus 6.0 (Media Cybernetics, Inc.) for statistical analysis.

A cell cycle analysis kit (Cell Cycle Detection kit, cat. no. KGA511) was used for cell cycle detection. The cells in each group were digested with trypsin at 37°C for 1 min, collected by centrifugation at 1,500 × g for 5 min at normal temperature and rinsed with PBS to form a cell suspension. The concentration was adjusted to 1×10⁶ cells/ml. After removing the supernatant by centrifugation at 1,500 × g for 5 min at normal temperature, 500 µl precooled 75% ethanol was used to fix the cells overnight at 4°C. The fixation solution was removed by centrifugation at 1,500 × g for 5 min at 4°C, 100 µl RNase A was added and the cells were incubated in a water bath at 37°C for 30 min. A 400 µm mesh screen was used for filtration. Then, 400 µl PI dye was added and the solution was gently mixed and incubated at 4°C for 1 h in the dark. Analysis was performed using a flow cytometer (FACSCalibur, BD Biosciences; FlowJo, Version 10, FlowJo LLC).

SPSS 21.0 (IBM Corp.) statistical software was used for statistical analysis. The data are expressed as the mean ± SD. Each independent experiment was repeated three times. Independent sample t-test was used to analyze differences between two groups of continuous data. One-way ANOVA was used for the comparison among multiple groups followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

BMAL1 and CLOCK expression was analyzed in TCGA database. The expression levels of BMAL1 and CLOCK appeared to differ among the 33 types of tumor. BMAL1 expression in acute myeloid leukemia was higher than that in normal tissues (Fig. 1A). By contrast, BMAL1 expression was lower in diffuse large adrenocortical carcinoma (ACC), cervical squamous cell carcinoma, endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), lung adenocarcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, prostate adenocarcinoma, rectum adenocarcinoma, skin cutaneous melanoma, testicular germ cell tumors (TGCT), uterine corpus endometrial carcinoma and uterine carcinosarcoma compared with that in normal tissues (Fig. 1A). Additionally, BMAL1 expression in brain lower grade glioma (LGG) tissues was lower than that in normal tissues (Fig. 1C). The expression of BMAL1 in GBM tissue was not significantly different from that in normal tissue (Fig. 1C). CLOCK expression in TGCT tissues was lower than that in normal tissues (Fig. 1B). By contrast, CLOCK expression was higher in diffuse large pancreatic adenocarcinoma (PAAD), stomach adenocarcinoma (STAD) and thymoma (THYM) tissues compared with that in normal tissues (Fig. 1B). Additionally, CLOCK expression in brain LGG was higher than that in normal tissues (Fig. 1D). The expression of CLOCK in GBM tissues was slightly higher compared with that in normal tissues (Fig. 1D). However, how the rhythm genes are expressed in IDH1 R132H mutated glioma is unclear. Therefore, further experiments were performed to investigate this.

U87-MG cells were transfected with lentivirus. Western blot analysis (Fig. 2A) indicated that after 72 h of lentiviral infection, IDH1 WT protein levels in WT U87-MG cells were slightly higher than those in the MUT U87-MG cells (Fig. 2B). The IDH1 MUT protein was highly expressed in the MUT group (Fig. 2C). The effect of IDH1 gene mutation on U87-MG cell proliferation was detected through colony formation experiments, and the results indicated that significantly fewer new colonies were formed in the MUT group than in the WT group (Fig. 2D and E). The current results indicated that a cell model of IDH1 lentivirus transfection was successfully established and that IDH1 R132H mutation influenced the colony formation of glioma cells.

Cell cycle distribution was detected by flow cytometry. In the WT group, 34.41% of cells were in S phase, while in the MUT group, 19.39% of cells were in S phase (Fig. 3A-C and G). The proportion of cells in S phase in the MUT group was markedly lower than those in the WT and Vector groups (Fig. 3A-C and G). The proportion of cells in G₁ phase in the MUT group was 72.83%, which was higher than that in the WT group (57.83%); additionally, there was no marked difference in the proportion of cells in G₂ phase among the groups (Fig. 3A-C and G), suggesting that the IDH1 R132H mutation may inhibit cells from entering S phase. To further describe the changes in the cell cycle, the expression levels of S phase-associated proteins (Cyclin A and CDK2), G₂/M phase-associated protein (Cyclin B1), G₁ phase-associated proteins (Cyclin D3 and CDK4), inhibition of early G₁ phase P18 protein via inhibiting Cyclin D-CDK4/6 (44,45) and inhibition of late G₁ phase P27 and P21 proteins via Cyclin E-CDK2 (46) were analyzed by western blot analysis (Fig. 3D). The results revealed that the protein expression levels of CyclinA and CDK2 were significantly lower in the MUT group than in the WT and vector groups (Fig. 3E and F). There were no significant differences in CyclinB1 expression among the groups (Fig. 3H). The expression levels of Cyclin D3 and CDK4 were significantly higher in the MUT group (Fig. 3I and J). The protein expression levels of P18 protein in the MUT group were significantly lower than in the WT group, and the expression levels of P27 and P21 protein in the MUT group were significantly higher than those in the WT and vector groups (Fig. 3K-M). Therefore, the flow cytometry results were verified by detecting the protein expression levels of relevant cyclins, indicating that the IDH1 R132H mutation may affect the regulation of the cell cycle.

Abnormal clock gene expression and circadian rhythm disorders are closely associated with the occurrence and development of tumors (47). Western blot analysis revealed that the expression levels of the biological rhythm genes BMAL1 and CLOCK in the IDH1 MUT group were significantly lower than those in the WT group (Fig. 4A-C). Transcriptionally, the clock is driven by positive regulators of the loop. Basic helix-loop-helix heterodimeric transcription factors (CLOCK/BMAL1 or BMAL1/NPAS2) regulate the expression levels of key circadian genes, including CRY genes (Cry1 and Cry2) and PER genes (PER1, PER2, and PER3), which are negative regulators of the circadian loop (48,49). CRY and PER form a transcriptional repressor complex that enters the nucleus to repress CLOCK/BMAL1 activity, thus creating a negative feedback loop to control the clock (50). Therefore, the expression levels of negative regulators of clock genes were also examined. The negative regulatory factors of biological rhythm PER1, PER2, PER3, Cry1 and Cry2 were significantly downregulated compared with the WT group (Fig. 4D-I). These results indicated that the IDH1 R132H mutation may change the expression levels of biological rhythm genes and may participate in the regulation of the cell biological rhythm.

IDH1 regulates the TGF-β signaling feedback loop through α-ketoglutarate (α-KG) and TGF-βR)-IDH1-Canine adenovirus 1 (Cav1) to enhance TGF-β signaling in a regulatory network between cellular signaling and cell metabolism (51). Additionally, TGF-β is an important regulator of the physiological clock (52,53). Therefore, the present study further investigated any changes in the TGF-β/Smad signaling pathway in IDH1 R132H mutated glioma cells. Western blot assays were used to detect the effects of MUT and WT IDH1 on the levels of p-Smad2, p-Smad3, Smad2, Smad2-3, Smad3 and Smad4 in U87-MG cells (Fig. 5A). Compared with the WT group, the MUT group exhibited significantly decreased protein expression levels of Smad2, Smad3 and Smad2-3 (Fig. 5B-D) and significantly increased levels of p-Smad2 and p-Smad3 (Fig. 5E and F). Furthermore, Smad4 expression was significantly upregulated in the MUT group compared with in the WT group (Fig. 5G). TGF-β initiates signaling pathways by phosphorylating Smad2 and Smad3, and phosphorylated Smad2 and Smad3 bind to Smad4 to form complexes that are transferred to the nucleus to interact with multiple transcription factors in order to induce cellular responses (54,55). Thus, it was hypothesized that the IDH1 R132H mutation may affect biological rhythm genes of glioma cells through the TGF-β/Smad signaling pathway.