Human GBM cell lines T98G (accession no. CVCL_0556) and U87 (glioblastoma of unknown origin; accession no. CVCL_0022) were purchased from American Type Culture Collection. GBM cell lines SHG-44, U251 and human normal glial HEB cells and human embryonic kidney 293T cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. T98G and U87 cells were maintained in modified Eagle's medium (MEM; Hyclone; Cytiva), SHG-44 cells were maintained in RPMI-1640 medium (Hyclone; Cytiva) and U251, HEB and 293T cells were maintained in DMEM (Hyclone; Cytiva). All culture mediums were supplemented with 10% FBS (Hyclone; Cytiva). Cells were maintained at 37˚C in 5% CO₂ incubators. To establish TMZ-resistant cell lines, SHG-44 and U251 cells were cultured and passaged over 8 weeks in the presence of increasing concentrations of TMZ (30 to 300 µM; Selleck Chemicals) to generate TMZ resistant lines at 37˚C in 5% CO₂ incubator, which were denoted as SHG-44TMZ+ and U251TMZ+ as per a previous study (28) and the parental cells were denoted ad SHG-44TMZ- and U251TMZ-.

overexpression plasmids (LGR6) were constructed by inserting the LGR6 coding sequence into a pcDNA3.1 plasmid (General Biosystems, Inc.). An empty pcDNA3.1 vector was used as the negative control (Vector). The small interfering (si)RNA targeting LGR6 (siRNA-LGR6) and the control (siRNA-Ctrl) were purchased from Shanghai GenePharma Co., Ltd. The microRNA (miR)-1236-3p mimic (miR-1236-3p) and scrambled oligonucleotides (miR-Ctrl) were purchased from Guangzhou RiboBio Co., Ltd. Sequences are presented in Table I. The day prior to transfection, ~2x10⁵ cells were plated in growth medium without antibiotics at a density of 30-50%. Both siRNAs and miRNAs were transfected into cells at a final concentration of 100 nM using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. DNA fragments containing the wild-type (WT) or mutated (Mut) miR-1236-3p 3'-untranslated region (3'-UTR) and their complementary fragments were cloned from SHG-44 cDNA. The annealed double-stranded DNA was then cloned into the dual-luciferase reporter gene vector psicheck-2 (Promega Corporation). The recombinant WT and Mut reporter gene vectors were named LGR6-3' UTR-WT and LGR6-3' UTR-Mut, respectively.

To investigate whether microRNAs regulated the expression of LGR6, the available complementary-based algorisms were predicted using TargetScan (www.targetscan.org/vert_72) and miRTarBase (mirtarbase.mbc.nctu.edu.tw/php/index.php). miR-1236-3p displayed a low mirSVR score (-2.69) and was selected as a prediction microRNA.

293T cells (~5x10³ cells/well) were plated in 96-well plates and co-transfected with 25 ng luciferase reporter gene vector and 50 nM miR-1236-3p or miR-Ctrl using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Following culturing at 37˚C for 48 h in a 5% CO₂ incubator, luciferase activity were detected using the Dual-Luciferase Reporter assay system (Promega Corporation). The results were normalized to Renilla luciferase and analyzed, according to the manufacturer's protocol.

Cells were plated in 96-well plates (~5x10³) and transfected with siRNA-LGR6, siRNA-Ctrl, LGR6 overexpression plasmids or empty pcDNA3.1 vector for 24 h at 37˚C in 5% CO₂ incubator, then TMZ was added to culture medium at final concentrations of 0, 100, 200, 300, 400 or 500 µM. At 0, 24, 48 and 72 h post-transfection, Cell Counting Kit-8 reagent (10 µl; Beyotime Institute of Biotechnology) was added to each well for 4 h at 37˚C. The absorbance of each well was measured at a wavelength of 450 nm using the Multiskan GO plate reader (Thermo Fisher Scientific, Inc.).

Selective inhibitors of Akt1/2/3 (MK-2206) were purchased from Selleck Chemicals. Frozen aliquots (-80˚C) were melted and dissolved in DMSO (Sigma-Aldrich; Merck KgaA) and diluted in growth medium (RPMI-1640 medium for SHG-44 cells; DMEM medium for U251 cells). A total of 5 µM MK-2206 was added to SHG-44 and U251 cells for 0, 24, 48 or 72 h following transfection with LGR6 overexpression plasmids. Cell Counting Kit-8 reagent (10 µl; Beyotime Institute of Biotechnology) was added to each well for 4 h at 37˚C. The absorbance of each well was measured at a wavelength of 450 nm using a Multiskan GO plate reader (Thermo Fisher Scientific, Inc.).

For cell invasion assays, Corning^® Transwell^® polycarbonate membrane cell culture inserts containing polycarbonate membranes with 8-µm pores (Corning, Inc) were precoated with Matrigel^® (BD Biosciences) for 30 min at 37˚C. Cells (~5x10⁴ cells/well) were suspended in culture medium supplemented with 5% FBS and plated into the upper chambers. The lower chambers were filled with culture medium supplemented with 20% FBS. Following incubation for 24 h at 37˚C in 5% CO₂ incubators, cells were washed with PBS and fixed with cold 99.9% methanol for 30 min at room temperature. After staining with 1% crystal violet for 30 min at room temperature, cells on the upper surface of the membrane were removed using cotton swabs. Stained cells were counted using a light microscope and analyzed using Image J software (v18.0; National Institutes of Health).

At 48 h post-transfection, cells were washed twice with cold PBS and harvested using trypsin. Cells were fixed with cold 75% (v/v) ethanol overnight at -20˚C. After washing twice with PBS, cells were suspended in staining buffer containing 5 µl PI and 5 µl RNAase A inhibitor for 30 min in the dark at room temperature using a Cell Cycle Analysis kit (Shanghai Yeasen Biotechnology Co., Ltd.), according to the manufacture's protocol. Stained cells were analyzed via ACEA NovoCyte flow cytometry instrument (ACEA Bioscience, Inc.) and cell cycle distribution was assessed using Novo Express software (https://www.aceabio.com.cn/support/software_download#edit-group-novocyte-software-download; ACEA Bioscience, Inc.).

Transfected cells were washed with cold PBS and total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with phosphatase inhibitors (Roche Applied Science). Total protein was quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Protein (30 µg per lane) was separated via 10% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.), which were blocked with 5% non-skimmed milk for 1 h at room temperature. The membranes were incubated overnight at 4˚C with primary antibodies targeted against: Phosphorylated (p)-Akt (Ser473; dilution, 1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.), Akt (dilution, 1:500; cat. no. OM238722; OmnimAbs), LGR6 (dilution, 1:1,000; cat. no. ab126747; Abcam) and β-actin (dilution, 1:8,000; cat. no. 60008-1; ProteinTech Group, Inc.). Following primary incubation, the membranes were incubated with goat anti-rabbit (dilution, 1:6,000, cat. no. SA00001-2; ProteinTech Group, Inc.) or donkey anti-mouse (dilution, 1:8,000, cat. no. 715-005-150; Jackson ImmunoResearch) IgG horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using a chemiluminescence kit (Thermo Fisher Scientific, Inc.). Protein expression was quantified using ImageJ software (National Institutes of Health) with β-actin as the loading control.

Total RNA was extracted from transfected cells using TRIzol^® (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using the Hifair^® II 1st Strand cDNA Synthesis kit (Shanghai Yeasen Biotechnology Co., Ltd.) or Hairpin-it^™ miRNA RT-PCR Quantitation kit (Shanghai GenePharma Co., Ltd.). Subsequently, qPCR was performed using SYBR Green Select Master Mix (Thermo Fisher Scientific, Inc.) and an ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95˚C for 5 min followed by 40 cycles at 95˚C for 10 sec, 58˚C for 20 sec, 72˚C for 20 sec, followed by melting curve detection at 95˚C for 15 sec, 60˚C for 1 min and 95˚C for 15 sec. The sequences of the primers used for qPCR are listed in Table II. miRNA and mRNA expression levels were quantified using the 2^-∆∆Cq method (29) and normalized to the internal reference genes U6 and β-actin.

Data are presented as the mean ± SD. Experiments were performed in triplicate. One-way ANOVA followed by Tukey's post hoc test was used to analyze comparisons among multiple groups. Comparisons between two groups were analyzed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The expression of LGR6 was detected in GBM cells and normal glial HEB cells. The results indicated that LGR6 mRNA levels were significantly increased in U251 and SHG-44 cells compared with HEB cells (Fig. 1A) and that LGR6 protein expression was increased in SHG-44, U251 and T98G cells compared with HEB cells (Fig. 1B). Additionally, SHG-44 and U251 cells exhibited higher LGR6 mRNA and protein expression levels compared with U87 and T98G cells (Fig. 1A, C and D); therefore, SHG-44 and U251 cells were selected for further experiments. In addition, siRNA-LGR6 significantly reduced LGR6 mRNA and protein expression levels compared with siRNA-Ctrl (Fig. 1B, E and F).

The effect of LGR6 knockdown on SHG-44 and U251 cell viability was investigated. The results indicated that LGR6 knockdown significantly reduced SHG-44 and U251 cell viability at 48 h compared with the siRNA-Ctrl group (Fig. 2A and B). Additionally, due to the invasive capability of glioma cells that induce malignancy or intracranial metastasis (30), the effect of LGR6 on cell invasion was assessed. The results suggested that LGR6 knockdown significantly reduced the number of invasive cells compared with the siRNA-Ctrl group (Fig. 2C-E).

Conversely, LGR6 overexpression significantly increased the expression levels of LGR6 in SHG-44 and U251 cells compared with the vector group (Fig. 3A-C). Furthermore, LGR6 overexpression significantly increased cell viability compared with the vector group (Fig. 3H and I) and promoted cell cycle progression. By contrast, LGR6 knockdown arrested the cell cycle at the S phase (Fig. 3D-G). The results suggested that LGR6 served a vital role in regulating GBM cell viability and invasion.

A TMZ-resistant GBM cell model was successfully established and used to investigate TMZ sensitization. A total of 2 TMZ-resistant human glioma cell sublines, SHG-44TMZ+ and U251TMZ+, were generated by increasing TMZ concentrations for 6 months. The IC₅₀ of SHG-44TMZ+ and U251TMZ+ exhibited a >2-fold increase compared with parental TMZ-sensitive cell lines (SHG-44TMZ- and U251TMZ- cells; Fig. 4A and B). Moreover, TMZ-resistant SHG-44 and U251 GBM cells displayed increased expression levels of LGR6 compared with TMZ-sensitive SHG-44 and U251 GBM cells (Fig. 4C and D). TMZ-resistant GBM cells displayed higher viability rates compared with TMZ-sensitive cells following treatment with a series of TMZ concentrations (0, 100, 200, 300, 400 and 500 µM; Fig. 4C and D). U251 and SHG-44 cell viability decreased in a time-dependent manner, whereas LGR6 knockdown decreased U251 and SHG-44 cell viability compared with the siRNA-Ctrl group (Fig. 4E-H). Based on the results, it was hypothesized that LGR6 participated in the failure of TMZ chemotherapy in GBM, which might indicate a new therapeutic target for the disease.

The Akt signaling pathway is involved in numerous types of cancer, including GBM (31,32); therefore, the levels of p- and total Akt in transfected GBM cells were measured. The results indicated that LGR6 overexpression significantly increased the levels of p-Akt compared with the vector group, but did not alter the total levels of Akt (Fig. 5A and B), which suggested that LGR6 might activate Akt signaling to mediate GBM malignancy. Therefore, it was hypothesized that as LGR6 induced the activation of Akt signaling during GBM progression, the loss of Akt activity may abolish the regulatory ability of LGR6.

Further experiments were conducted to investigate whether MK-2206, a specific inhibitor of Akt signaling, reversed LGR6-induced cell viability and reduced cell viability in response to TMZ treatment in SHG-44 and U251 cells. The results were consistent with the hypothesis (Fig. 5C-F), which suggested that LGR6 promoted GBM viability and chemoresistance by activating Akt signaling.

miR-1236-3p serves as a tumor suppressor in various types of cancer (33-35). To investigate whether LGR6 was a potential target of miR-1236-3p, the available complementary-based algorisms were predicted using TargetScan and miRTarBase. The results indicated that miR-1236-3p expression levels were significantly decreased in U251 and SHG-44 cells compared with HEB cells and SHG-44 and U251 cells displayed lower miR-1236-3p levels compared with U87 and T98G cells (Fig. 6A). Additionally, miR-1236-3p mimic significantly increased the expression of miR-1236-3p and significantly decreased LGR6 expression levels at the mRNA and protein level compared with miR-Ctrl (Fig. 6B-D). Based on the predicted targeting sites of miR-1236-3p, LGR6 3'-UTR WT and Mut luciferase reporter plasmids were constructed. The results indicated that miR-1236-3p mimic significantly decreased the luciferase activity of LGR6 WT 3'-UTR compared with miR-Ctrl, but did not alter the luciferase activity of LGR6 Mut 3'-UTR (Fig. 6E and F). The results suggested that LGR6 was an miR-1236-3p target, which may mediate its effects during cancer development.