Hypoxia-inducible miR-196a modulates glioblastoma cell proliferation and migration through complex regulation of NRAS

Sonam Takkar; Vikas Sharma; Sourabh Ghosh; Ashish Suri; Chitra Sarkar; Ritu Kulshreshtha

doi:10.1007/s13402-020-00580-y

. 2021 Jan 19;44(2):433–451. doi: 10.1007/s13402-020-00580-y

Hypoxia-inducible miR-196a modulates glioblastoma cell proliferation and migration through complex regulation of NRAS

Sonam Takkar ¹, Vikas Sharma ¹, Sourabh Ghosh ², Ashish Suri ³, Chitra Sarkar ⁴, Ritu Kulshreshtha ^1,^✉

PMCID: PMC12980754 PMID: 33469841

Abstract

Background

Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in humans. Hypoxia has been correlated with the aggressive form of glial tumors, poor prognosis, recurrence and resistance to various therapies. MicroRNAs (miRNAs) have emerged as critical mediators of hypoxic responses and have shown great potential for cancer diagnostics and therapeutics. Here, we focus on the regulatory and functional characterization of miR-196a, a hypoxia-inducible miRNA, in GBM.

Methods

Hypoxia/HIF regulation of miR-196a was assessed by RT-qPCR, promoter-luciferase and ChIP assays in GBM cell lines. miR-196a levels were analyzed in The Cancer Genome Atlas (TCGA)-GBM, Chinese Glioma Genome Atlas (CGGA) and Indian GBM patient cohorts. miR-target interactions were studied using RNA/protein quantification and 3’UTR luciferase assays. The effect of miR-196a overexpression/inhibition was assessed on cellular viability, migration and apoptosis under hypoxia and normoxia. Microarray-based gene expression profiling studies were performrd to study the effect of miR-196a on the GBM cellular transcriptome under hypoxia.

Results

We identified miR-196a as a hypoxia-inducible and hypoxia-inducible factor (HIF)-regulated miRNA that plays an oncogenic role in GBM. miR-196a was found to be significantly up-regulated in TCGA-GBM, CGGA glioma as well as Indian GBM patient cohorts. miR-196a overexpression was found to induce cellular proliferation, migration, spheroid formation and colony formation and to inhibit apoptosis, while miR-196a inhibition using anti-miR-196a yielded opposite results, suggesting an oncogenic role of miR-196a in GBM. We further unveiled NRAS, AJAP1, TAOK1 and COL24A1 as direct targets of miR-196a. We also report a complex competitive regulation of oncogenic NRAS by miR-196a, miR-146a and let-7 in GBM. Analysis of microarray-based gene expression data obtained by miR-196a inhibition under hypoxia revealed a role of miR-196a in HIF, calcium adhesion, Wnt and cell adhesion pathways. Interestingly, miR-196a was found to positively regulate the expression of various genes involved in the induction or stabilization of HIFs and in maintenance of hypoxic conditions, thereby suggesting the existence of an indirect miR-196a/HIF positive feedback loop under hypoxia.

Conclusions

Overall, our work identifies a novel association between hypoxia/HIF signalling and miR-196a in GBM and suggests its therapeutic significance.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-020-00580-y.

Keywords: Hypoxia, HIF-1, miR-196a, NRAS, Glioblastoma

Introduction

Glioblastoma (GBM) is the most pervasive and aggressive malignant primary brain tumor in humans and one of the most lethal types of all cancers [1, 2]. Despite advancements in medical treatments that have been made, the outcome of the currently available therapies remains poor leading to overall low survival rates and, thus, urging for the development of novel therapeutic options. Hypoxia is a common feature of solid tumors and has been clinically well correlated with aggressiveness, metastasis and poor prognosis [3, 4]. Recent studies have shown that hypoxic microenvironments promote tumor aggressiveness, cellular reprogramming and maintenance of stem cell characteristics in GBM [5–7]. Also, high levels of hypoxic markers such as VEGF (Vascular Endothelial Growth Factor), OPN (Osteopontin) and CA9 (Carbonic Anhydrase 9) have been correlated with a poor survival of GBM patients [8].

MicroRNAs (miRNAs) constitute a class of small non-coding RNA molecules (∼22 nt in length) that have been reported to act as critical players in tumorigenesis and to serve as promising candidates for cancer diagnosis, prognosis and therapy [9–11]. Altered expression of miRNAs has been reported to be associated with the development and progression of several human cancers, including GBM, by regulating the expression of various oncogenes and tumor suppressor genes [12–14]. Tumoral hypoxia has been found to lead to a dynamic regulation of protein coding genes and, more recently, miRNAs (hypoxia regulated miRNAs, HRMs) to exert various effects on biological processes such as cell cycle progression, apoptosis and angiogenesis, thereby acting as molecular mediators of hypoxic responses [15–18].

The present study is based on the analyses of our previously published data of a hypoxia regulated small RNA transcriptome of GBM in which 141 miRNAs were found to be differentially regulated under severe hypoxic conditions (0.2% O₂) in U87MG cells, i.e., 102 up-regulated and 39 down-regulated [19]. miR-210 was found to be the most induced miRNA by hypoxia and to play an oncogenic role in GBM [19, 20]. Here, we focused on a miRNA, miR-196a, that was found to be among the top hypoxia induced miRNAs based on our RNA sequencing studies. We observed a novel association of miR-196a with the hypoxia and HIF-1 (Hypoxia Inducible Factor-1) pathway in GBM and identified its direct target genes and pathways using a combination of bioinformatics and biochemical approaches. We show that miR-196a induces downregulation of AJAP1 (Adherens Junctions Associated Protein 1), TAOK1 (Thousand and One Amino Acid Protein Kinase 1) and COL24A1 (Collagen type XXIV alpha 1 chain) and induces NRAS upregulation (NRAS proto-oncogene) involving a complex interplay with tumor suppressive miRNAs (miR-146a and let-7). We also found that miR-196a was upregulated in The Cancer Genome Atlas (TCGA) dataset and in Indian GBM patients and that its high levels were correlated with a poor prognosis. Overall, our work identifies miR-196a as an important mediator of hypoxia signalling in GBM and as a putative novel therapeutic target.

Materials and methods

Cell culture

The current study was carried out using U87MG and A172 glioma cell lines. U87MG was obtained from the cell repository of the National Centre for Cell Sciences (NCCS), Pune, and A172 was a kind gift from Dr. Kunzang Chosdol (All India Institute of Medical Sciences (AIIMS), Delhi, India). Both cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% Fetal Bovine Serum (FBS, GIBCO), 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were incubated at 37 ºC, 5% CO₂ in a humidified incubator (Memmert).

Glioblastoma patient samples

Glioblastoma (GBM) samples collected during surgery at the All India Institute of Medical Sciences (AIIMS) were used in this study. The protocol of the study was approved by the AIIMS ethics committee (Ref. No. IEC-130/07.04.2017, RP-24/2017). One part of the resected tumors was snap-frozen in liquid nitrogen and stored at -80 ºC, and the remaining tissue was formalin-fixed and paraffin-embedded for routine histopathology and other studies. Hematoxylin and eosin stained slides of all cases were reviewed independently by 3 neuropathologists to confirm their diagnosis as per World Health Organization (WHO) 2007 classification [21]. Samples containing excessively large amounts of necrotic material and/or significant regions of normal cell contamination (> 10%) were excluded. Based on these criteria, 18 GBM samples were selected. Additionally, three epileptic brain samples were collected and used as controls for real-time PCR-based quantitative studies.

Transfections and cellular assays

Detailed information on oligos and plasmids used in the study and detailed protocols of transient transfection, hypoxic treatment, cloning of miRNAs, cell viability, caspase, PE (Phycoerythrin) Annexin V apoptosis detection, 3D-spheroid formation, clonogenicity, migration and luciferase assays are provided in Supplementary materials and methods.

DNA, RNA and protein analyses

Detailed protocols of RNA isolation, cDNA synthesis, stem loop RT-PCR, quantitative real-time RT-PCR (qRT-PCR), Western blotting and chromatin immunoprecipitation (ChIP) are provided in Supplementary materials and methods.

Prediction of HREs in the miR-196a promoter

To check whether hypoxic-modulation of miR-196a is caused by the presence of a transcriptional target of HIF (Hypoxia Inducible Factor), Hypoxia Response Elements (HREs) were searched for within its promoter region (10 kb upstream of 5’ end of the precursor miRNA). The upstream region of miR-196a was extracted from the Ensembl genome browser 97 [22], after which HREs were predicted using the online PROMO program which employs version 8.3 of the TRANSFAC database [23, 24]. Potential HREs were subsequently cloned in a promoter luciferase reporter vector and binding of HIF on HREs was confirmed using a dual luciferase assay (Supplementary materials and methods).

miRNA target prediction

To search for probable miR-196a targets, the in silico target prediction program TargetScan [25] was used. The resulting list was integrated with a list of genes known to play significant roles in various cellular processes (migration, invasion and apoptosis) obtained from the UniProt database [26]. The target list was also analysed using DAVID Bioinformatics Resources v6.8 to check the prevalence of the probable target genes in various signalling pathways [27]. Genes that were found to be involved in cellular processes (i.e., migration, invasion and apoptosis) and signalling pathways were selected for further analyses. Predicted targets were validated using qRT-PCR and 3’UTR (Untranslated Region) luciferase reporter assays as described in Supplementary materials and methods.

Microarray analysis

A172 cells were transfected with anti-miRNA oligos or its respective controls and incubated for 48 hours in a hypoxic workstation maintained at 0.2% O₂, 5% CO₂, 37 ºC. RNA lysates were prepared inside the hypoxia workstation using a lysis kit buffer, after which total RNA was isolated as described above. Microarray-based gene expression analysis was performed using an Affymetrix platform (GeneChip Human PrimeView Array) by StellarGene Technologies Pvt. Ltd, India. Differentially regulated genes with p-values < 0.05 were considered to be significant and analyzed using DAVID Bioinformatics Resources v6.8 to assess their prevalence in various signalling pathways [27].

Statistical analysis

All the experiments carried out in vitro were performed thrice in triplicates unless otherwise specified in the figure legends. Statistical analyses were carried out with Microsoft Excel using a paired two-tailed Student’s t-test. Data with p-values < 0.05 were considered to be statistically significant.

Results

Hypoxia induces miR-196a in a HIF-1 dependent manner

Previously, we reported a miRNA signature of hypoxia in GBM using small RNA deep sequencing (sRNA-seq) [19]. Based on the sRNA-seq data, miR-196a was found to be highly induced under hypoxia in U87MG cells. To validate and further study hypoxic regulation of miR-196a, we incubated U87MG and A172 cells at varying oxygen concentrations (21 − 0.2% O₂) for 48 hours in a hypoxia workstation and quantified miR-196a levels. We found a time-dependent increase in the levels of mature miR-196a with a maximal increase at 0.2% O₂ (Fig. 1a, b). To further study the mechanism underlying the hypoxic induction of miR-196a, we modulated the levels of HIFs in U87MG cells by transient transfection of HIF-1 and HIF-2 overexpression plasmids or their specific shRNAs. Interestingly, we found that miR-196a expression levels were induced by HIF-1 but not by HIF-2 (Fig. 1c). To further understand the HIF-1 mediated regulation of miR-196a, we scanned the miR-196a upstream region (10 kb) for the presence of HIF binding sites/Hypoxia response elements (HREs) using PROMO software and, by doing so, identified four potential HREs (Fig. 1d). These HREs were cloned in a pGL3-tk luciferase reporter vector as three clones (HRE-1, 2 and 3) and their functionality was assessed by dual luciferase reporter assay. All three HRE clones were found to be responsive to HIF-1 stimulation, with HRE-1 showing the maximum induction (Fig. 1e). These findings were further validated by a ChIP assay using an anti-HIF-1 antibody in U87MG cells grown in a hypoxic (0.2% O₂) environment. The HIF-1 immunoprecipitated DNA was checked for enrichment of these HREs in the miR-196a promoter along with a negative control. In concordance with the luciferase data, HRE-1 showed a maximal enrichment upon immunoprecipitation with the anti-HIF-1 antibody (Fig. 1f). We also checked for correlation of miR-196a levels with those of a known hypoxia miRNA biomarker, miR-210, in GBM patient samples [19]. Interestingly, we found that the miR-196a levels correlated positively with miR-210 levels in the TCGA-GBM dataset (Betastasis analyses software) and in CGGA glioma patient samples (CGGA data portal) (Fig. 1g). Overall, we found that hypoxia significantly induces miR-196a levels through binding of HIF-1 to HREs present within the miR-196a upstream region.

Fig. 1 — miR-196a is regulated by HIF-1 under hypoxia. qRT-PCR data showing miR-196a expression at different oxygen concentrations (0.2%-21% O₂ for 48 hours) in a U87MG and b A172 cells (n = 2). c qRT-PCR data showing the effect of HIF overexpression (HIF-1α or HIF-2α) or its inhibition (shHIF1α or shHIF2α) under normoxic or hypoxic conditions, respectively, on miR-196a expression levels in U87MG cells (n = 3). d Schematic diagram showing the position of four potential hypoxia response elements (HREs) upstream of miR-196a. e Bar graph representing dynamic binding of HIF-1 to the HREs in the promoter region of miR-196a in terms of relative luciferase units obtained by Dual Luciferase Reporter Assay (n = 3). f ChIP analysis validating the binding of HIF-1 to the HREs in the promoter region of miR-196a. Bar graph representing the fold enrichment of bound chromatin compared with input. g miRNA expression scatterplot showing a positive correlation of miR-196a levels and miR-210 levels in TCGA-GBM and CGGA glioma patient samples. Error bars denote ± S.D (** p < 0.01; * p > 0.01 and < 0.05)

miR-196a is upregulated in GBM patients and correlates with a poor prognosis

We next assessed miR-196a levels in GBM patients through the analysis of TCGA-GBM patients using the starBase v2.0 online data portal (n = 566), the TCGA GBM data portal (n = 195), the CGGA glioma data portal (n = 198) and an Indian GBM (n = 18) patient cohort, and found that miR-196a was indeed up-regulated in GBM patients compared to controls (Fig. 2a-d). We also found that the miR-196a levels were higher in high-grade versus low-grade glioma patients in the CGGA patient cohort and correlated with a poor overall survival (Fig. 2e, f). Further, we assessed correlations between miR-196a expression levels and overall survival rates in TCGA-GBM patients using Betastasis analysis software. Interestingly, we found that patients with high miR-196a levels exhibited a significantly poorer overall survival compared to the low expressing ones (Fig. 2g). We also noted that especially in GBM patients belonging to the classical subtype or GBM patients who had not received chemotherapy, the high miR-196a expressors exhibited a significantly poorer survival compared to the low expressors (Fig. 2g, h).

Fig. 2 — miR-196a is up-regulated in GBM tissues and cell lines and its high levels are associated with a poor survival. a-c Analysis of miR-196a expression levels in global GBM patient samples (starBase v2.0, TCGA cohort and CGGA cohort). d Analysis of miR-196a expression levels in Indian GBM patient samples (qRT-PCR analysis). e Analysis of miR-196a expression levels in different grades of glioma patient samples obtained from the CGGA data portal. f Patient survival data obtained from the CGGA data portal showing correlation of miR-196a levels with survival probability in primary glioma. g Kaplan-Meier plot showing survival association of miR-196a levels in TCGA-GBM or TCGA-GBM (classical subtype) patient cohorts (25% expression threshold). h Kaplan-Meier plot showing survival association with miR-196a levels in GBM patients who have or have not undergone chemotherapy (25% expression threshold) in the TCGA-GBM patient cohort

miR-196a promotes GBM cell growth

To study the role of miR-196a in glioma cell growth, we transiently transfected U87MG and A172 cells with a miR-196a overexpression plasmid or anti-miR-196a oligos along with their respective controls under normoxic and hypoxic conditions. Using a MTT assay we found that under both normoxia and hypoxia, miR-196a overexpressing GBM cells exhibited an increased viability compared to the respective controls, while GBM cells treated with anti-miR-196a oligos showed the opposite (Fig. 3a-d). We also found that miR-196a significantly increased the colony forming capacities of both U87MG and A172 cells (Fig. 3e, f). Additionally, we assessed the effect of miR-196a on 3D-spheroid formation. We found that miR-196a significantly increased the numbers as well as the sizes of the spheroids formed compared to controls (Fig. 3g, h).

Fig. 3 — miR-196a promotes GBM cell viability, clonogenic potential and 3D tumor spheroid formation. Bar graphs showing relative increases in cell viability using a MTT assay upon transient transfection with a miR-196a over-expression plasmid or its control in a U87MG and b A172 cells under normoxic and hypoxic conditions (n = 3). Bar graphs showing relative decreases in cell viability using a MTT assay upon transient transfection with anti-miR-196a oligos or its Negative Control in c U87MG and d A172 cells under normoxic and hypoxic conditions (n = 3). e Bar graph showing the effect of miR-196a expression modulation on colony forming abilities in U87MG and A172 cells (n = 3). f Image representing colonies formed by A172 cells upon overexpression or inhibition of miR-196a levels in a 6-well plate. g Microscopic image (4x) showing increases in size or number of tumor spheroids of U87MG cells overexpressing miR-196a compared to its control in a 6-well plate (multiple wells) (n = 3). h Bar graph representing average number of spheroids formed upon miR-196a overexpression compared to its control in U87MG cells (n = 3). Error bars denote ± S.D (** p < 0.01; * p > 0.01 and < 0.05)

miR-196a promotes GBM cell migration

We also studied the effect induced by miR-196a overexpression on GBM cell migration. Both wound-healing and transwell chamber migration assays clearly revealed that miR-196a overexpression increased the migratory potential of GBM cells incubated under both normoxic and hypoxic conditions (Fig. 4a-c). Since cell migration and epithelial-to-mesenchymal transition (EMT) have been reported to be interconnected, we also analyzed changes in expression levels of EMT-related genes upon modulating the level of miR-196a. We found that upon miR-196a induction most of the EMT makers tested were up-regulated [including MMPs (Matrix Metalloproteases)-MMP2, MMP9, ZEB (Zinc finger E-box-binding homeobox)-ZEB1, ZEB2, TWIST2 and Vimentin] (Fig. 4d, e). To validate our results, Vimentin protein levels were analyzed by Western blotting (Fig. 4f). Interestingly, we found that high Vimentin protein levels were correlated with high miR-196a levels. Thus, our data indicate that miR-196a is involved in modulating mesenchymal-specific properties in GBM cells.

Fig. 4 — miR-196a promotes GBM cell migration. U87MG and A172 cells were transfected with a miR-196a overexpression plasmid or inhibitor oligos along with their respective controls after which the migratory potentials were assessed by a and b wound-healing and c transwell chamber migration assays under normoxia and hypoxia (n = 3). qRT-PCR data showing changes in transcript levels of various EMT-related genes upon miR-196a d overexpression and e inhibition in U87MG cells (n = 3). f Analysis of Vimentin expression in U87MG cells with modulated miR-196a levels by Western blotting (n = 2). The numbers below the lanes represent relative protein levels quantified using ImageJ software and normalized to the respective controls. Error bars denote ± S.D (** p < 0.01; * p > 0.01 and < 0.05)

miR-196a inhibits GBM cell apoptosis

We also assessed the role of miR-196a in GBM cell apoptosis by flow cytometry using a 7-AAD PE Annexin V Apoptosis Detection Kit and by scoring Caspase 3/7 activity. In cells overexpressing miR-196a, we observed a considerable decrease in the percentage of apoptotic cells in both cell lines tested (Fig. 5a-c). Also, there was a significant decrease in Caspase 3/7 activity in cells overexpressing miR-196a (Fig. 5d). We verified our findings by Western blotting using an anti-cleaved PARP (poly-ADP ribose polymerase) antibody. Opposite results were obtained when miR-196a levels were inhibited using anti-miR-196a oligos, underscoring the anti-apoptotic role of miR-196a in GBM cells (Fig. 5e).

Fig. 5 — miR-196a inhibits GBM cell apoptosis. FACS analysis showing reductions in apoptotic cell populations upon miR-196a overexpression, while miR-196a inhibition shows the opposite in a A172 and b U87MG. c Bar graph showing percentages of Annexin-V positive cells obtained by FACS analysis upon modulation of miR-196a levels in A172 and U87MG (n = 2). d Bar graph showing caspase 3/7 activity in U87MG cells transfected with a miR-196a overexpression plasmid or its inhibitor along with their respective controls under normoxia and hypoxia (n = 3). e Analysis of cleaved-PARP expression in U87MG cells with modulated miR-196a levels by Western blotting (n = 2). The numbers below the lanes represent relative protein levels quantified using ImageJ software and normalized to the respective controls. The bar graph represents mean ± S.D of at least three independent experiments performed in triplicates. Error bars denote ± S.D (** p < 0.01; * p > 0.01 and < 0.05)

miR-196a targets NRAS, AJAP1, TAOK1 and COL24A1 in GBM cells

To identify target genes of miR-196a, we obtained a putative target list from TargetScan and used two approaches to identify potential target genes. First, we analyzed the target list using DAVID microarray software and shortlisted those genes that were enriched in the ECM (Extracellular matrix) receptor family and ERbB pathway [27]. Secondly, we integrated our target list with the list of known genes involved in regulating cellular proliferation, migration and apoptotic signalling (obtained from the UniProt database), as our functional analyses indicated that miR-196a may actively regulate these processes (Supplementary Fig. 1) [26]. We finally selected nine target genes [AJAP1, BIRC6 (Baculoviral IAP repeat-containing protein 6), COL3A1 (Collagen Type III Alpha 1 Chain), COL24A1, ERBB2 (Erythroblastic oncogene B), NRAS, RUFY3 (RUN And FYVE Domain Containing 3), TAOK1 and TEAD3 (TEA domain transcription factor 3)] that could potentially be regulated by miR-196a in GBM cells. To next determine whether miR-196a indeed targets the selected genes, we first validated the transcript levels of these genes by qRT-PCR upon modulation of miR-196a levels in GBM cells. We used NFκBIa (NF-kappa-B-Inhibitor Alpha; a known miR-196a target in GBM) as a positive control [28]. We observed a significant down-regulation of four of the selected target genes (AJAP1, COL24A1, COL3A1 and TAOK1) and, interestingly, a significant overexpression of NRAS upon miR-196a overexpression, while an opposite trend was seen upon miR-196a inhibition using anti-miR-196a oligo treatment (Fig. 6a, b). To further confirm the role of miR-196a in regulating these four genes, miR-196a binding sites in the 3’UTRs of these genes were cloned in a pMIR-REPORT luciferase reporter vector and dual luciferase assays were performed (Fig. 6c). We found a significant down-regulation of AJAP1, COL24A1 and TAOK1 upon miR-196a overexpression, whereas NRAS up-regulation was seen in both 3’UTR clones (N1 and N2) (Fig. 5d, e). The luciferase results were in accordance with the qRT-PCR results and, thus, we infer that AJAP1, COL24A1, TAOK1 and NRAS act as targets of miR-196a in GBM cells.

Fig. 6 — miR-196a targets AJAP1, COL24A1, TAOK1 and NRAS in GBM. Validation of predicted miR-196a target genes by qRT-PCR analysis in U87MG cells transfected with a miR-196a overexpressing construct and b anti-miR196a oligos along with their respective controls (n = 2). c Schematic diagram showing the position of miR-196a binding sites within the 3’UTRs of AJAP1, COL24A1, TAOK1 and NRAS. d Luciferase activity of 3’UTR luciferase constructs of AJAP1, COL24A1 and TAOK1 bearing wild-type miR-196a binding sites (n = 3). e Luciferase activity of NRAS 3’UTR luciferase constructs bearing a wild-type miR-196a binding site (n = 3). f Diagram showing a mutated miR-196a binding site in the NRAS 3’UTR. g Luciferase activity of NRAS 3’UTR luciferase construct bearing a mutated miR-196a binding site (n = 3). h Analysis of NRAS expression by Western blotting upon modulation of miR-196a in U87MG (n = 3) and A172 (n = 2) cells. The numbers below the lanes represent relative protein levels quantified using ImageJ software and normalized to the respective controls. i Analysis of expression level of NRAS in Indian GBM, TCGA-GBM and CGGA glioma patient cohorts. Error bars denote ± S.D (** p < 0.01; * p > 0.01 and < 0.05)

Since NRAS, belongs to the RAS family of proteins that play salient roles in various cancer types, we extended our study on this target and further validated it as a direct target by mutating one of the miR-196a binding site in its 3’UTR by site-directed mutagenesis (Fig. 6f). When we performed dual luciferase reporter assays with this mutated 3’UTR, we observed a significant decrease in luciferase activity, suggesting a critical role of this binding site in NRAS expression (Fig. 6g). To further validate NRAS as a direct target of miR-196a, Western blotting was performed, and we found that the NRAS protein levels increased upon miR-196a overexpression and decreased upon miR-196a inhibition (Fig. 6h). We also assessed NRAS levels in Indian GBM patients, TCGA-GBM patients and CGGA glioma patients. Similar to miR-196a, NRAS was found to be highly expressed in GBM patients and to show a grade-dependent increase in the CGGA glioma cohort (Fig. 6i). This is the first report showing that NRAS activation may be associated with overexpression of a miRNA. We also checked the levels of other miR-196a targets and found that, similar to their regulation by miR-196a, significant down-regulation of AJAP1, COL24A1 and TAOK1 was seen in the TCGA-GBM patients (Supplementary Fig. 2).

Complex regulation of NRAS by miR-196a, miR-146a and let-7

To understand the role of miRNAs in regulating the expression of NRAS, we looked for all predictive miRNA binding sites in the NRAS 3’UTR region and identified 20 other miRNA binding sites. Interestingly, we noted that let-7-5p and miR-196a have overlapping binding sites while miR-146a-5p was found to be binding in close proximity to the miR196a binding site (Fig. 7a). Next, we checked the levels of miR-146a and let-7g in GBM patients and found let-7g to be significantly down-regulated in the TCGA-GBM and CGGA glioma patients, while the miR-146a levels were minimally affected (Fig. 7b). We also assessed the potential role of these miRNAs in GBM biology using literature data and found that both of them may act as tumor suppressors in GBM [29–31]. We hypothesized that these three miRNAs could be competitively binding and, thus, be instrumental in regulating NRAS expression in cancer. To test this hypothesis, we first cloned their precursors in a pcDNA3.1 mammalian expression vector and checked the functionality of clones in U87MG cells by qRT-PCR (Supplementary Fig. 3a, b). We next transiently overexpressed these miRNAs individually and in combinations with miR-196a in U87MG cells to check their effect on NRAS transcript levels. We found that the NRAS levels were down-regulated after miR-146a-5p and let-7g-5p overexpression (Fig. 7c). Notably, the effect of NRAS up-regulation by miR-196a was strongly inhibited by miR-146a and let-7g (Fig. 7c). We also checked the effect of miR-146a and let-7g on the NRAS 3’UTR using a dual luciferase reporter assay and found that overexpression of these miRNAs significantly decreased the luciferase activity (Fig. 7d). The varied expression pattern of NRAS after miR-196a, miR-146a and let-7g overexpression was confirmed at the protein level using Western blotting (Fig. 7e). These data indicate a complex regulation of NRAS expression through the binding of various miRNAs, including miR-196a, miR-146a and let-7g-5p, to its 3ʹUTR.

Fig. 7 — Complex regulation of NRAS in GBM. a Diagram showing the presence of proximal binding sites of miR-196a, miR-146a and let-7g in the NRAS 3’UTR obtained from TargetScan. b Expression analysis of let-7g in TCGA-GBM and CGGA glioma patients. c qRT-PCR data showing changes in relative NRAS transcript levels in U87MG cells transiently overexpressing miR-196a, miR-146a and let-7g individually or in combination with miR-196a (n = 3). d Luciferase activity of NRAS 3’UTR luciferase construct bearing wild-type miR-146a and let-7g binding sites in close proximity to the miR-196a binding site (n = 3). e Analysis of NRAS expression by Western blotting upon transient overexpression of miR-196a individually or in combination with miR-146a and let-7g in U87MG cells (n = 3). The numbers below the lanes represent relative protein levels quantified using ImageJ software and normalized to the respective controls. Error bars denote ± S.D (** p < 0.01; * p > 0.01 and < 0.05)

NRAS promotes GBM cell proliferation and migration

Since we found that miR-196a may directly induce NRAS overexpression, we next set out to assess the effect of NRAS overexpression on key cellular processes, including proliferation, migration and apoptosis. For this, we cloned the CDS of NRAS in a pcDNA3.1 mammalian expression vector and confirmed its overexpression by qRT-PCR (Supplementary Fig. 3c). We next checked the effect of NRAS overexpression on GBM cell proliferation using MTT and colony formation assays in both U87MG and A172 cells. We found that NRAS overexpression led to increases in proliferation and colony formation in both U87MG and A172 cells (Fig. 8a-c). Since we additionally found that miR-196a affected cellular migration, we also decided to look for the effect of NRAS overexpression on cell migration using wound-healing and transwell chamber migration assays. We found that NRAS overexpression induced GBM cell migration (Fig. 8d,e). To confirm our findings, we subsequently assessed the expression of EMT markers in U87MG cells upon NRAS overexpression by qRT-PCR and found that NRAS overexpression was positively associated with increases in the expression of Vimentin, TWIST1 (Twist-related protein 1), MMP2 and MMP9 (Fig. 8f). We also observed an increase in Vimentin protein expression by Western blotting in U87MG cells upon NRAS overexpression (Fig. 8g). In addition, we assessed the effect of NRAS overexpression on GBM cell apoptosis using a Caspase3/7 assay, but no significant effects were noted (Supplementary Fig. 4). Overall, our results indicate that NRAS acts as an oncogene in GBM cells by promoting their proliferation and migration.

Fig. 8 — NRAS functions as an oncogene in GBM. a Bar graph showing relative increases in cell viability using a MTT assay upon transient transfection with a NRAS overexpression plasmid or its control in U87MG andA172 cells (n = 3). b Bar graph showing the effect of NRAS overexpression on colony formation by U87MG and A172 cells (n = 3). Migratory potential of NRAS assessed by **c, d** wound-healing and e transwell chamber migration assays in A172 cells transfected with a NRAS overexpression plasmid or its control (n = 3). f qRT-PCR data showing changes in transcript levels of EMT marker genes upon NRAS overexpression in U87MG cells (n = 2). g Analysis of Vimentin expression in U87MG cells upon NRAS overexpression by Western blotting (n = 2). The numbers below the lanes represent relative protein levels quantified using ImageJ software and normalized to the respective controls. Error bars denote ± S.D (** p < 0.01; * p > 0.01 and < 0.05)

Identification of miR-196a regulated genes and pathways in GBM

To study the impact of miR-196a inhibition under hypoxia on the GBM cell transcriptome, we performed gene expression profiling using an Affymetrix microarray platform on A172 cells treated with anti-miR-196a or its control under hypoxic conditions. In total 692 genes were found to be differentially regulated (307 up-regulated and 385 down-regulated) (Supplementary Table 1). Subsequent gene enrichment analysis using DAVID Bioinformatics Resources v6.8 led to the identification of HIF, calcium adhesion, Wnt and cell adhesion pathways as being most enriched (Fig. 9a) [27]. Since we found that miR-196a is hypoxia regulated in a HIF-1 dependent manner, we selected 8 genes enriched in the HIF pathway for further validation by qRT-PCR in both U87MG and A172 cells. We found that the transcript levels of only three genes, i.e., Bcl-2 (B-cell lymphoma 2), CYBB (NADPH oxidase 2) and EP300 (E1A Binding Protein P300) were significantly reduced upon miR-196a inhibition under hypoxia, thereby suggesting a positive correlation (Fig. 9b, c). Although the observed modulation in EP300 levels was contradictory to our microarray-based data, our conclusions are based on experiments conducted in technical and biological replicates in two different GBM cell lines. Since previous studies have shown that Bcl-2, CYBB and EP300 can promote HIF stability and activation, we next wanted to test whether miR-196a indirectly affects the HIF pathway (Fig. 9d) [32–35]. To this end, we assessed the effect of miR-196a inhibition under hypoxia on the HIF target genes VEGF and CA9. We found that miR-196a inhibition led to significant decreases in VEGF and CA9 levels in U87MG and A172 cells (Fig. 9e). We next assessed the effect of miR-196a on a HIF reporter plasmid under hypoxia. To this end, we co-transfected a HIF reporter plasmid containing tandem HRE repeats of the erythropoietin (EPO) gene along with anti-miR-196a oligos and its respective controls under hypoxic conditions and scored luciferase activity using a dual luciferase assay. We found that miR-196a inhibition under hypoxia significantly reduced the luciferase activity, suggesting that miR-196a is involved in the promotion of HIF signalling (Fig. 9f). Overall, our results establish miR-196a as a hypoxia regulated oncogenic miRNA (‘oncomir’) and indicate a role of this miRNA in the regulation of NRAS expression levels and the establishment of a positive regulatory loop with the HIF pathway (Fig. 9g).

Fig. 9 — miR-196a promotes HIF signalling. a Pathway analysis of the miR-196a gene signature under hypoxia by DAVID Bioinformatics Resources v6.8. qRT-PCR data showing downregulation of various genes upon inhibition of miR-196a under hypoxia in b U87MG and c A172 cells. d Diagram showing the biological functions of genes modulated by miR-196a inhibition under hypoxia (n = 2). e qRT-PCR data showing the effect of miR-196a inhibition under hypoxia on the expression of hypoxic markers VEGF and CA9 in U87MG and A172 cells (n = 3). f Luciferase activity of an EPO-HRE construct upon co-transfection with anti-miR-196a oligos or its control under hypoxic conditions by dual luciferase reporter assay (n = 3). g Scheme summarizing miR-196a function, regulation and targets as part of hypoxia signalling in GBM. Error bars denote ± S.D (** p < 0.01; * p > 0.01 and < 0.05)

Discussion

GBM is characterized by extensive regions of hypoxia that directly correlate with tumor aggressiveness, treatment failure and poor prognosis. Thus, hypoxia represents a major clinical concern. However, incomplete understanding of hypoxia signalling hampers the design of novel and effective therapies for GBM. Here, we identify miR-196a as a novel target of hypoxia/HIF signalling in GBM. We report hypoxic stress-induced upregulation of miR-196a in GBM and its direct regulation via HIF-1. HIF-1 has amply been correlated with aggressiveness in GBM and, recently, been reported as an important switch that affects GBM responsiveness to temozolomide sensitivity [36, 37]. It is interesting to note that only HIF-1, but not HIF-2, was able to induce miR-196a levels, reiterating the growing body of data showing that HIF-1 and HIF-2 have specific alternate targets next to shared ones [38]. A recent study also identified miR-196a as being hypoxia regulated in hepatocellular carcinoma cells (Huh7 cells exposed to 1% O₂) based on microarray data, although no further validation or functional studies were performed [39].

We noted high miR-196a levels in TCGA-GBM, CGGA glioma and Indian GBM patient cohorts. Previous studies have also shown higher miR-196a levels in GBM patient cohorts [28, 40, 41]. Guan et al. [41] additionally reported a higher miR-196 expression in GBM patients (Grade IV) versus anaplastic astrocytoma patients (Grade III), suggesting a grade-dependent increase in miR-196a levels signifying its prognostic importance. We also found that GBM patients (TCGA cohort) with high miR-196a levels exhibited poor overall survival rates compared to the low expressing ones, especially in the GBM patients belonging to the classical subtype. Similarly, GBM patients with high miR-196a levels exhibited significantly poorer survival rates than the low expressing ones among patients who had not received chemotherapy. Higher miR-196a expression levels have also been reported in other human malignancies including head and neck cancer, lung cancer, esophageal cancer, pancreatic cancer, oral cancer, cervical cancer, breast cancer and acute myeloid leukaemia, but their upstream regulatory mechanisms have only been explored by limited studies [42–44]. These studies have shown that miR-196a may be regulated by epigenetic modification (methylation) of CpG islands present in its promoter and by binding of TGFβ1 and MYC to the promoter. Whether, miR-196a is regulated by these factors in GBM remains to be established. Since hypoxic environments are a common feature of various solid tumors, high miR-196a levels in these tumors can at least partly be attributed to hypoxia, although this needs to be validated in individual tumor types. Overall, miR-196a may serve as a diagnostic biomarker for GBM patients.

Several reports have shown that miR-196a functions as an oncogene ('oncomir') in various cancers. There are, however, only few studies that have addressed the functional characterization of miR-196a in GBM, with none under hypoxia. Yang et al.[28] were the first to report oncogenic properties of miR-196a (promotion of cell proliferation and inhibition of apoptosis) in GBM. This was followed by another study in which it was shown that miR-196a promotes proliferation, migration and invasion, in addition to reducing apoptosis of glioma stem cells [45]. Here, we found that miR-196a was able to induce GBM cell proliferation and migration and inhibit apoptosis under both normoxic and hypoxic conditions, suggesting that the oncogenic effects of miR-196a are maintained under hypoxic conditions as well. There are several reports showing distinct cellular effects under different oxygen concentrations, stressing the need to perform experiments under conditions mimicking the tumor microenvironment. Many of these studies have also suggested downstream effectors regulated by miR-196a. In one study by Sun et al. miR-196a was found to directly target the CDK inhibitor p27kip1 by interacting with its 3′-UTR in gastric cancer [46]. Similarly, Han et al. identified UBE2C as a key downstream target of miR-196a in breast cancer [47]. As yet, in GBM only three direct targets of miR-196a, i.e., ZMYND11 (Zinc Finger MYND-Type Containing 11), FOXO1 (Forkhead Box O1) and NFκBIA have been reported to induce proliferation [28, 45, 48]. Here, we have identified additional downstream targets of miR-196a that may play a role in the oncogenic effects of miR-196a under normoxia as well as hypoxia. We found that miR-196a downregulates AJAP1, TAOK1 and COL24A1 and upregulates NRAS levels through binding sites in their 3’UTRs. AJAP1, TAOK1 and COL24A1 were found to be downregulated in primary GBM samples. AJAP1 is known to function as a tumor suppressor in various cancers including breast cancer, hepatocellular carcinoma and GBM [49–51]. In GBM, AJAP1 has been found to be deregulated during early stages of tumor development and to promote temozolomide-induced apoptosis. Its low levels have been correlated with increased tumor progression and poor survival [49, 52, 53]. Thus, it will be important to further study the role of the miR-196a/AJAP1 axis in GBM. TAOK1 is a serine/threonine protein kinase while COL24A1 is a collagen type protein. Their putative roles in GBM have not yet been reported.

We also found that miR-196a positively regulates NRAS. This may be important as NRAS has been reported to act as an oncogene and to be deregulated in various cancers including gliomas [54–56]. Interestingly, based on the Intogen somatic mutation data analysis tool, NRAS was found to be a cancer driver gene in 21 different types of cancer including GBM and lower-grade glioma (LGG) (Supplementary Fig. 5) [57]. NRAS is an important member of the RAS oncogene family, which codes for small GTPases that are involved in signal transduction. RAS family-induced oncogenic signalling triggers many important downstream signalling pathways including the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT (Protein kinase B) pathways that lead to modulation of cell growth and survival [55]. Interestingly, two other studies have reported aberrant NRAS activation and mutation in GBM [54, 58]. Specific miRNAs (such as miR-181a, miR-340, let-7 and miR-145) have been shown to suppress RAS expression, and thus to act as tumor suppressors [54, 55, 59, 60]. We here report miR-196a to play a key role in promoting NRAS oncogene expression in GBM cells. We further show that overexpression of NRAS stimulates the viability/proliferation and migration in GBM cells, thereby suggesting a role in establishing oncogenic effects of miR-196a in GBM. Interestingly, we found that miR-146a and let-7 binding sites lie in close proximity of the conserved miR-196a binding site within the NRAS 3’UTR. Both of these miRNAs have been shown to function as tumor suppressors in GBM [29–31]. Our current results indicate competitive binding between these three miRNAs at the 3’UTR of NRAS and, thereby, NRAS (de)regulation in GBM cells. These results suggest the existence of a complex network of miRNAs that interact co-operatively or competitively to establish specific gene signatures affecting GBM aggressiveness and survival.

Additionally, to decipher the role of miR-196a in hypoxia, we inhibited its level under hypoxia and, by subsequent microarray analysis, we found that various important cellular pathways were affected including the HIF, calcium adhesion, Wnt and cell adhesion pathways. We found that under hypoxia miR-196a induces the levels of Bcl-2, CYBB and EP300 in GBM cells. While Bcl-2 and CYBB have been shown to promote HIF-1 stability, EP300 is known to act as a crucial HIF-1 co-activator [32–35]. Thus, by modulating the levels of oncogenes, i.e., Bcl-2, CYBB and EP300, under hypoxia miR-196a may promote the establishment of a hypoxia-induced gene signature in GBM cells. This feedback loop of HIF and miR-196a needs to be further deciphered and may serve as a novel target for GBM therapy.

Our work adds to the clinical significance of the genetic loci bearing miR-196a in GBM, i.e., chromosome 17 (miR-196a-1) and chromosome 12 (miR-196-a-2), since they lie within HOX (Homeobox) gene clusters that are deregulated in many human cancers including GBM [61]. Also, HOXB9, HOXC9 and HOXC10, which are located in the vicinity of miR-196a-1 and miR-196a-2, respectively, have been reported to promote oncogenic activities in GBM [62–65]. Interestingly, several HOX cluster gene members such as HOXB8, HOXC8, HOXD8, HOXA7 and HOXB7, have been shown to be targeted by miR-196a in various cancers, suggesting the existence of feedback loops [66–68]. It was also reported that HOTAIR, a lincRNA (long intergenic noncoding RNA) located upstream of miR-196a-2, is upregulated in gastrointestinal tumors and correlated with their malignancy [69].

In comprehension, our work identifies miR-196a as a novel mediator of hypoxia signalling in GBM and establishes it as a potential therapeutic target in GBM. Inhibiting miR-196a may hamper GBM progression by targeting NFκB, NRAS and HIF-mediated oncogenic pathways. Since several studies identified miR-196a as a negative prognostic biomarker for GBM, further studies to unveil miR-196a interactions and pathways for their therapeutic potential are warranted.

Electronic supplementary material

ESM 1^{(27.1KB, docx)}

Detailed materials and methods are provided in this file. (DOCX 27.1 kb)

Supplementary Fig. 1^{(369.4KB, png)}

Analysis of miR-196a target genes. Schematic diagram representing integration of various datasets to identify potential target genes of miR-196a (PNG 369 kb)

High Resolution Image (TIF 841 kb)^{(841.9KB, tif)}

Supplementary Fig. 2^{(426KB, png)}

Expression pattern of AJAP1, COL24A1, TAOK1 and NRAS in different subtypes of GBM. Figures showing expression of AJAP1, COL24A1 and TAOK1 in GBM tissue versus non-tumor in GBM patients obtained from Gliovis web portal (Agilent platform). (***P<0.001; **P<0.01; *P<0.05) (PNG 426 kb)

High Resolution Image (TIF 0.98 mb)^{(1,006KB, tif)}

Supplementary Fig. 3^{(137KB, png)}

qPCR analysis of cloned miRNAs and gene. qPCR data showing relative increase in a miR-146a, b let-7g and c NRAS transcript levels as compared to their control in U87MG cells transfected with their respective overexpression plasmid or its control (n=3). Error bars denote ± S.D (**P < 0.01; *P>0.01 and < 0.05) (PNG 137 kb)

High Resolution Image (TIF 294 kb)^{(294.5KB, tif)}

Supplementary Fig. 4^{(58.8KB, png)}

Effect of NRAS on apoptosis in GBM. Bar graph showing caspase 3/7 activity in U87MG cells transfected with NRAS overexpression plasmid or its control (n=3). Error bars denote ± S.D (**P < 0.01; *P>0.01 and < 0.05) (PNG 58.8 kb)

High Resolution Image (TIF 161 kb)^{(161.9KB, tif)}

Supplementary Fig. 5^{(1.5MB, png)}

Figure showing data for driver mutations in NRAS across various cancer types obtained using Intogen data analyses tool. The data for GBM and LGG is highlighted in the table shown below. (PNG 1.45 mb)

High Resolution Image (TIF 7.26 mb)^{(7.3MB, tif)}

Supplementary Table 1^{(41.6KB, xlsx)}

List of differentially regulated mRNAs (p-value <0.05) obtained by gene expression profiling in A172 cells upon inhibition of miR-196a-5p levels under hypoxia. (XLSX 41 kb)

Supplementary Table 2^{(19.9KB, docx)}

List of primers used for cloning (miRNAs, mRNA, 3’UTR, 5’UTR and site-directed mutagenesis) and qRT-PCR detection of various transcripts (miRNAs and mRNAs). (DOCX 19 kb)

Acknowledgements

RK and CS thank Department of Biotechnology (DBT), Government of India for the financial support (BT/PR16851/MED/122/45/2016). ST thanks Department of Science and Technology (DST), Government of India, for the DST-INSPIRE fellowship and Indian Council of Medical Research (ICMR), Government of India for ICMR-SRF fellowship. VS thanks Department of Biotechnology, Research Associate fellowship. SG thanks Department of Biotechnology (DBT), Government of India for the financial support (BT/IN/Swiss/54/SG/2018-19, BT/PR15451/MED/32/539/2016).

Abbreviations

AJAP1: Adherens junctions associated protein 1
Bcl-2: B-cell lymphoma 2
BIRC6: Baculoviral IAP repeat-containing protein 6
CA9: Carbonic anhydrase 9
CGGA: Chinese Glioma Genome Atlas
ChIP: Chromatin immunoprecipitation
COL3A1: Collagen type III alpha 1 chain
COL24A1: Collagen type XXIV alpha 1 chain
CYBB: NADPH oxidase 2
ECM: Extracellular matrix
EMT: Epithelial to mesenchymal transition
EP300: E1A binding protein P300
EPO: Erythropoietin
ERBB2: Erythroblastic oncogene B
GBM: Glioblastoma
HIF: Hypoxia inducible factor
HOX: Homeobox.
HRE: Hypoxia response element
HRM: Hypoxia regulated microRNAs.
LGG: Lower-grade glioma
miRNA/miR: MicroRNA
MMP: Matrix metalloproteases
NFĸBIA: NF-kappa-B-inhibitor alpha
NRAS: NRAS proto-oncogene
OPN: Osteopontin
PARP: Poly-ADP ribose polymerase
qRT-PCR: Quantitative reverse transcription-PCR
RUFY3: RUN and FYVE domain containing 3
sRNA-Seq: Small RNA sequencing (deep sequencing)
TAOK1: Thousand and one amino acid protein kinase 1
TCGA: The Cancer Genome Atlas
TEAD3: TEA domain transcription factor 3
TWIST1: Twist-related protein 1
UTR: Untranslated region
VEGF: Vascular endothelial growth factor
ZEB: Zinc finger E-box-binding homeobox

Funding

The work was funded by Department of Biotechnology (DBT), Government of India; project number: BT/PR16851/MED/122/45/2016.

Data availability

The microarray-based gene expression data have been submitted to the Gene Expression Omnibus (GEO) repository with accession number GSE152575.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ESM 1^{(27.1KB, docx)}

Detailed materials and methods are provided in this file. (DOCX 27.1 kb)

Supplementary Fig. 1^{(369.4KB, png)}

Analysis of miR-196a target genes. Schematic diagram representing integration of various datasets to identify potential target genes of miR-196a (PNG 369 kb)

High Resolution Image (TIF 841 kb)^{(841.9KB, tif)}

Supplementary Fig. 2^{(426KB, png)}

High Resolution Image (TIF 0.98 mb)^{(1,006KB, tif)}

Supplementary Fig. 3^{(137KB, png)}

High Resolution Image (TIF 294 kb)^{(294.5KB, tif)}

Supplementary Fig. 4^{(58.8KB, png)}

High Resolution Image (TIF 161 kb)^{(161.9KB, tif)}

Supplementary Fig. 5^{(1.5MB, png)}

Figure showing data for driver mutations in NRAS across various cancer types obtained using Intogen data analyses tool. The data for GBM and LGG is highlighted in the table shown below. (PNG 1.45 mb)

High Resolution Image (TIF 7.26 mb)^{(7.3MB, tif)}

Supplementary Table 1^{(41.6KB, xlsx)}

List of differentially regulated mRNAs (p-value <0.05) obtained by gene expression profiling in A172 cells upon inhibition of miR-196a-5p levels under hypoxia. (XLSX 41 kb)

Supplementary Table 2^{(19.9KB, docx)}

List of primers used for cloning (miRNAs, mRNA, 3’UTR, 5’UTR and site-directed mutagenesis) and qRT-PCR detection of various transcripts (miRNAs and mRNAs). (DOCX 19 kb)

Data Availability Statement

The microarray-based gene expression data have been submitted to the Gene Expression Omnibus (GEO) repository with accession number GSE152575.

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PERMALINK

Hypoxia-inducible miR-196a modulates glioblastoma cell proliferation and migration through complex regulation of NRAS

Sonam Takkar

Vikas Sharma

Sourabh Ghosh

Ashish Suri

Chitra Sarkar

Ritu Kulshreshtha

Abstract

Background

Methods

Results

Conclusions

Supplementary Information

Introduction

Materials and methods

Cell culture

Glioblastoma patient samples

Transfections and cellular assays

DNA, RNA and protein analyses

Prediction of HREs in the miR-196a promoter

miRNA target prediction

Microarray analysis

Statistical analysis

Results

Hypoxia induces miR-196a in a HIF-1 dependent manner

Fig. 1.

miR-196a is upregulated in GBM patients and correlates with a poor prognosis

Fig. 2.

miR-196a promotes GBM cell growth

Fig. 3.

miR-196a promotes GBM cell migration

Fig. 4.

miR-196a inhibits GBM cell apoptosis

Fig. 5.

miR-196a targets NRAS, AJAP1, TAOK1 and COL24A1 in GBM cells

Fig. 6.

Complex regulation of NRAS by miR-196a, miR-146a and let-7

Fig. 7.

NRAS promotes GBM cell proliferation and migration

Fig. 8.

Identification of miR-196a regulated genes and pathways in GBM

Fig. 9.

Discussion

Electronic supplementary material

Acknowledgements

Abbreviations

Funding

Data availability

Compliance with ethical standards

Conflict of interest

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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