Lnc-TALC promotes O6-methylguanine-DNA methyltransferase expression via regulating the c-Met pathway by competitively binding with miR-20b-3p

We next investigated the underlying mechanism of lnc-TALC upregulation in TMZ-resistant GBM cells. We did not observe copy number aberrance of the lnc-TALC gene in the TMZ-resistant cell genomes (Supplementary Fig. 3a). Inhibition of DNA methyltransferase also had no effect on the lnc-TALC expression in GBM cells (Supplementary Fig. 3b).

Based on the DIANA, RegRNA2.0, TargetScan, miRWalk, and miRNA.org databases (Supplementary Table 2), two candidate miRNAs (miR-20b-3p and miR-335-3p) were predicted to target both the lnc-TALC and MET 3′UTR regions (Supplementary Fig. 5c). Furthermore, the dual luciferase reporter assays (Fig. 5c and Supplementary Fig. 5d) revealed that only miR-20b-3p directly bound to the lnc-TALC and MET 3′UTR regions (Fig. 5d and Supplementary Fig. 5e). The MS2-RIP assay was conducted to pull down endogenous miRNAs associated with lnc-TALC and showed that miR-20b-3p was significantly enriched in RNAs retrieved from MS2bs-lnc-TALC, further confirming that miR-20b-3p specifically targeted lnc-TALC (Fig. 5e and Supplementary Fig. 5f). Then, qRT-PCR was used to quantify the molecular numbers of lnc-TALC, MET, and miR-20b-3p per cell (Supplementary Fig. 5g). The dual luciferase reporter assays showed that miR-20b-3p bound the MET mRNA 3′UTR sequence in parental or TMZ-resistant GBM cells (Fig. 5f and Supplementary Fig. 5h, i). Moreover, c-Met and its downstream activity were restored after miR-20b-3p was inhibited in lnc-TALC knockdown cells. In contrast, the overexpression of miR-20b-3p inhibited c-Met and its downstream expression (Fig. 5g, h and Supplementary Fig. 5j–l). The CCK-8 and colony formation assays showed that miR-20b-3p was involved in lnc-TALC-mediated TMZ resistance in GBM cells (Supplementary Fig. 5m–p).

It was necessary to further investigate how the c-Met signaling pathway regulates MGMT expression in TMZ-resistant GBM cells. The pyrosequencing assay showed no significant change in the methylation levels of the MGMT gene promoter region between parental cells and TMZ-resistant cells or between the lnc-TALC knockdown and control groups (Supplementary Fig. 7a). In public databases, multiple miRNAs that were reported to posttranscriptionally regulate MGMT expression did not exhibit a significant differential expression between recurrent GBMs and primary GBMs (Supplementary Fig. 7b). We also did not observe obvious differential expression of these miRNAs between parental cells and TMZ-resistant cells using qRT-PCR (Supplementary Fig. 7c). Since Stat3-mediated regulation of MGMT does not depend on its transcriptional activity³², we investigated whether Stat3 could specifically modify histones in the promoter region of the MGMT gene. We found that histone acetyltransferase p300 (p300) was markedly enriched in the MGMT promoter region of TMZ-resistant cells compared with that of parental cells (Fig. 6d). Protein coimmunoprecipitation indicated that p-Stat3 could bind to p300 at a higher level in TMZ-resistant GBM cells (Fig. 6e). Moreover, there was an increased enrichment of H3K9ac, H3K27ac, and H3K36ac, but not H3K4ac, levels in the MGMT promoter region in TMZ-resistant GBM cells (Fig. 6f and Supplementary Fig. 7d). Furthermore, suppression of lnc-TALC or Stat3 in TMZ-resistant GBM cells impaired the enrichment of p300 in the MGMT promoter region (Fig. 6g), the interactivity between p300 and p-Stat3 (Fig. 6h), and the enrichment of H3K9ac, H3K27ac and H3K36ac, but not H3K4ac, in the MGMT promoter region (Fig. 6i and Supplementary Fig. 7e). Collectively, these findings demonstrate that the lnc-TALC/c-Met axis regulates MGMT expression by modulating the acetylation of H3K9/27/36 through the Stat3/p300 complex.

Human GBM LN229 and U251 cells were purchased from the Chinese Academy of Sciences Cell Bank. These cells were authenticated using STR assay (Genetic Testing Biotechnology, Jiangsu, China). Patient-derived GBM cells 551 W and HG7 were isolated from discarded GBM specimens. Briefly, mechanically minced tissues were digested with 0.1% trypsin (Invitrogen, USA) and 10 U ml⁻¹ of DNase I (Promega, USA) at 37 °C for 45 min. ACK lysis buffer (Beyotime, Shanghai, China) was used to lyse the red blood cells. After being washed, the tissues were triturated by pipetting and passed through a 100 μm cell strainer. TMZ-resistant GBM 229R, 251R, 551WR, and HG7R cells were induced from LN229, U251, 551 W and HG7 cells, respectively. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM/F12 with 10% fetal bovine serum (Gibco, USA) at 37 °C in a humidified atmosphere with 5% CO₂ and were tested negative for mycoplasma contamination.

LN229, U251, 551 W, and HG7 cells were seeded into 96-well plates at 6000 cells per well, and IC50 of TMZ was determined. TMZ was then added to the cell culture medium at an IC50 1/50 concentration (LN229/0.83 ± 0.26 μM, U251/1.1 ± 0.19 μM, 551 W/1.0 ± 0.22 μM, HG7/1.48 ± 0.14 μM) to culture LN229, U251, 551W, and HG7 cells in six-well plates. After the cells grew stably, the drug dose was increased in multiples. Each dose was maintained for 15 days, to the end of the fifth month. The induced TMZ-resistant cells were named 229R, 251R, 551WR, and HG7R.

The RNA expression profiling was performed using Agilent custom human lncRNA and mRNA microarrays (SHBIO Biotechnology Corporation, Shanghai, China). The raw data were normalized using the quantile algorithm from the limma package in R. Heatmaps representing differentially regulated genes were generated using Cluster 3.0 and Gene Tree View. The microarray data have been deposited in NCBI’s Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo↗) under accession number GSE113510↗. The top 10 upregulated lncRNAs of TMZ-resistant GBM cells are presented in Supplementary Table 6.

Total RNA was isolated with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. 5′RACE and 3′RACE analyses were performed with 1 μg of total RNA. The SMARTer™ RACE cDNA kit (Clontech, CA) was used according to the manufacturer’s instructions. RACE PCR products were separated on a 1% agarose gel. Gel extraction products were subcloned into pGH-T vectors and sequenced bi-directionally using indicated primers (Sagene, China). The gene specific primers used for PCR are presented in Supplementary Table. 7

Four different methods including Open reading frame finder from NCBI⁵⁹, phyloCSF⁶⁰, coding probability from Coding potential assessment tool (CPAT)⁶¹, and coding potential score from coding potential calculator (CPC)⁶² were performed to calculate the coding potential of lnc-TALC. Prediction of putative proteins encoded by lnc-TALC using ORF Finder. Nuclear Enriched Abundant Transcript 1 (NEAT1) served as a control non-coding gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) served as control coding genes. We defined phyloCSF = 0⁶³, coding probability = 36.4%⁶¹ and coding potential score = 0⁶² as thresholds.

Cells were transfected with siRNAs, miRNA mimics or plasmids using Lipofectamine 2000 (Invitrogen, USA). The sequences of siRNAs against specific targets are listed in Supplementary Table 8. The MET and FOXO3 plasmids were purchased from GeneChem (Shanghai, China). For knocking down lnc-TALC, LV-Cas9 lentiviruses were transfected into cells for 48 h at an MOI of 10 and selected for 7 days with puromycin at a final concentration of 3 μg ml⁻¹. Cells were subsequently infected with lentiviruses carrying sgRNAs designed for lnc-TALC. Twenty-four hours later, expression level of lnc-TALC was confirmed by qPCR. Lentiviruses expressing lnc-TALC, Cas9, sgRNAs targeting lnc-TALC or the negative control (LV-NC) were prepared by GeneChem. The details of the sgRNA sequences are shown in Supplementary Table 9.

A total of 1 × 10⁶ cells were resuspended in a single cell suspension and washed two times with PBS solution. The cell apoptosis analysis was performed with the Annexin V-FITC or Annexin V-APC Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer’s instructions. The rates of apoptosis were detected by flow cytometry (BD FACSCanto II, USA).

GBM cell viability was evaluated with the Cell Counting Kit 8 (Dojindo, Japan) and was measured at OD 450 nm with the BioTek Gen5 system (BioTek, USA).

Cells were seeded (0.3 × 10³ cells per well) in a six-well plate and cultured for 11 days. The resulting colonies were then washed twice with PBS and fixed with 4% formaldehyde for 10 min and stained for 30 min with 0.1% crystal violet. The number of colonies was then captured by an Olympus camera (Tokyo, Japan) and counted by ImageJ.

Cell proliferation was detected by an EdU Cell Proliferation Assay Kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. The proportion of cells that incorporated EdU was determined with a fluorescence microscope (Nikon C2, Tokyo, Japan).

Molecule inhibitors, including SGX-523, SAHA/NaB, decitabine, and azacitidine were used in this study. The cells were treated with SGX-523, a small-molecule inhibitor of c-Met, with a final concentration of 200 nM. SAHA and NaB, inhibitors of histone deacetylase, were added to the cells at concentrations of 2 μM and 10 mM, respectively. Decitabine and azacitidine are inhibitors of DNA methyltransferase and were added to the cells at concentrations of 0.5 μM and 10 μM, respectively.

The CGGA microarray database was obtained from the website http://www.cgga.org.cn↗. TCGA microarray and protein databases were downloaded from the TCGA data portal (http://cancergenome.nih.gov/↗). The GSE32466↗ database was downloaded from the website https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE32466↗.

In this study, fresh GBM tissues were collected from 79 patients by surgical resection (Department of Neurosurgery, The Second Affiliated Hospital of Harbin Medical University) from 2013 to 2018. The pathological diagnoses for GBM were determined by pathologists. Recurrent GBMs were defined as an increase in residual lesions after treatment or the emergence of new lesions. The clinical characteristics of the GBM patients are presented in Supplementary Table–. Written informed consent for use of the tissues and data for this research was obtained from the patients or from immediate family members or guardians. This research was approved by the Clinical Research Ethics Committee of Harbin Medical University. 3 4

Genomic DNA was isolated from the LN229, 229R, 229R Scra, and 229R KD_lnc groups using a QIAamp DNA Mini Kit (Qiagen, China). The pyrosequencing analysis was carried out by Gene Tech Company Limited (Shanghai, China). Methylation values >10% in GBM cells were considered to be methylated.

In the coimmunoprecipitation assay, LN229, 229R, 229R Scra, and 229R KD_lnc cells were first lysed with IP lysis buffer. The lysates were incubated with 5 µg of anti-p-Stat3 or anti-p300 overnight at 4 °C. Ten microliters of protein A agarose beads were then added to the samples and incubated at 4 °C with gentle shaking for 3 h. After incubation, the bead–protein mixtures were centrifuged and washed three times with lysis buffer. The immunoprecipitated samples were further analyzed by western blot.

Total proteins were prepared from GBM cells or clinical GBM tissues using prechilled RIPA buffer with proteinase and phosphatase inhibitor cocktails (Selleck.cn, Shanghai, China). The PVDF membranes were incubated overnight at 4 °C with primary antibodies (Supplementary Table) and were then incubated with an HRP-labeled secondary antibody (Zsbio Store-bio, Beijing, China) at room temperature for 1 h. The protein bands were visualized using a chemiluminescence reagent (ECL) kit (Boster, Wuhan, China). Uncropped scans of these blots are reported in Supplementary Fig.. 10 9

The total RNA of the GBM cells or clinical GBM tissues was extracted using TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. The nuclear and cytoplasmic components were separated using 0.5% NP-40 (Solarbio, Beijing, China) with an RNAase inhibitor (Promega, USA), followed by extraction using TRIzol reagent (Sigma, USA). One microgram of total RNA was used as a template for cDNA synthesis using a PrimeScript RT Reagent Kit (Takara, Japan). Real-time quantitative PCR was performed on triplicate samples in a reaction mix of SYBR Green (Takara, Japan) with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). Quantification of the miRNA was performed with a stem-loop real-time PCR miRNA kit (RiboBio, Guangzhou, China). The levels of total RNA were normalized to β-Actin. The levels of miRNA RNA were normalized to U6 snRNA (small nuclear RNA). The expression of the indicated genes was normalized to the endogenous or exogenous reference control by using the 2^−ΔΔCt method. Sequences of the primers used for qRT-PCR in this study are listed in Supplementary Table 11. The molecular numbers of lnc-TALC, miR-20b-3p and MET were measured using the standard curve method and were calculated by relating the Ct value to the standard curve. Plasmids containing lnc-TALC or MET were purchased from GeneChem (Shanghai, China) and synthetic miR-20b-3p polynucleotide was purchased from RiboBio (Guangzhou, China).

Cells were seeded into 96-well plates and were transfected with the GV272 luciferase vector (GeneChem, Shanghai, China). Lnc-TALC and MET wild type with potential miR-20b-3p/miR-335-3p binding sites or mutants of each binding site (Supplementary Table) were generated and fused to the luciferase reporter vector GV272. The firefly luciferase activity in each well was measured with a Dual Luciferase Reporter Assay System (Promega, USA) and normalized to Renilla luciferase activity, according to the manufacturer’s protocol. 12

Cells were plated on a cell smear (WHB-24-CS, Shanghai, China) in 24-well tissue culture plates. The cells were stained using standard procedures. Primary antibodies, including FOXO3, MGMT, and c-Met, were diluted in 1% BSA in PBS. After overnight incubation at 4 °C, the cells were washed three times with PBS and incubated with FITC-labeled anti-IgG antibodies (Alexa Fluor 488 and 594, Thermo Fisher) for 1 h at room temperature. The DNA was stained with DAPI (Sigma, USA) and visualized with a fluorescence microscope (Nikon C2, Tokyo, Japan) and a laser scanning confocal microscope (LSCM) (ZEISS LSM700, Germany).

ISH was performed with the RNA ISH Kit (BersinBio, Guangzhou, China), according to the manufacturer’s instructions. Briefly, the samples were treated with pepsin for 10 min at 37 °C and incubated with 500 nM of probe at 55 °C for 4 h. The cells were subsequently incubated with 3% hydrogen peroxide to block potential endogenous peroxidase, and the probes were then detected with peroxidase-conjugated anti-DIG-Ab. Finally, the sections were stained with diaminobenzidine (DAB) for detection. The probe sequences are listed in Supplementary Table. 13

RIP was performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Massachusetts, USA), according to the manufacturer’s instructions. The antibodies used in the RIP assays of Ago2 were purchased from Abcam. MS2-based RIP assay with anti-GFP antibody in LN229 cells 48 h after transfection with MS2bp-YFP plasmid along with MS2bs-lnc, MS2bs-lnc Mut, or MS2bs-Rluc (GenScript, Nanjing, China). The RNA fraction precipitated by RIP was analyzed via qPCR.

ChIP assays were performed with an EZ-ChIP kit (Millipore, USA), according to the manufacturer’s instructions. The ChIP-PCR products were detected with 4.8% agarose gel electrophoresis. Sequences of the primers used for ChIP-PCR in this study are listed in Supplementary Table. Uncropped scans of these blots are reported in Supplementary Fig.. 14 9

Immunohistochemistry was carried out in 4 μm paraffin sections with a three-step process and a DAB staining kit (ZSGB-BIO, Beijing, China). In brief, formalin-fixed, paraffin-embedded tissue sections were incubated at 80 °C for 15 min, dewaxed in xylene, rinsed in graded ethanol, and rehydrated in double-distilled water. For antigen retrieval, the slides were pretreated by steaming them in sodium citrate buffer for 15 min at 95 °C. After washing with PBS for 3 min, the sections were immunostained with primary antibodies to c-Met, p-MAPK, p-Stat3, p-AKT, and MGMT, and incubated at 4 °C overnight. After being washed in PBS buffer, the tissues were covered by an antimouse/rabbit polymer HRP-label for 30 min. The staining reactions were performed by covering the tissue samples with the prepared DAB chromogen solution and incubating them for ~1 min to allow for proper brown color development.

Four-week-old female athymic BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). A total of 3 × 10⁵ GBM cells (LN229/229R and 229R Scra/229R KD_lnc) per mouse were stereotactically injected into the brain. After the surgery, the mice were treated for 2 weeks with DMSO or TMZ (60 mg kg⁻¹ day⁻¹). The intracranial tumors were measured with bioluminescence imaging. The mice were sacrificed, and the brain tissues were removed. The mouse brain tissue was embedded in paraffin and sectioned at a thickness of 4 μm for immunohistochemistry assays. All procedures were approved by the Committee on the Ethics of Animal Experiments of Harbin Medical University.

Significant differences between the groups were estimated by Student’s t-test. One-way analysis of variance (one-way ANOVA) was used for at least three groups. The overall survival curves were used to describe the survival distributions, and the log-rank test was applied for assessing statistical significance between different groups. The survival data were further processed by using univariate and multivariate Cox regression analysis. The Pearson correlation coefficient was used to analyze the correlations between variables. GO and KEGG Pathway analysis was performed using the DAVID website (http://david.abcc.ncifcrf.gov/home.jsp↗). Statistically significant gene sets were visualized by Cytoscape, and GSEA was used to analyze biological processes. SsGSEA was used to calculate the enrichment score of every gene set for every sample⁶⁴. Heatmaps were constructed and produced using Gene Cluster 3.0 and Gene Tree View software. All results are expressed as the mean ± SD. A value of p < 0.05 was considered to be statistically significant. All statistical analyses were performed using GraphPad software version 7.0 (GraphPad Software, CA, USA) or IBM SPSS Statistics 23.0 (SPSS, Chicago, USA).

We have complied with all relevant ethical regulations for animal testing and research. All research performed was approved by the Institutional Review Board at the Second Affiliated Hospital of Harbin Medical University and was in accordance with the principles expressed at the Declaration at Helsinki. Written informed consent was received from all participants. All animal experiments were performed according to Health guidelines of Harbin Medical University Institutional Animal Use and Care Committee.

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