Introduction
Gliomas, as common primary brain tumors, account for about 80% of all malignant brain and other central nervous system (CNS) tumors [
1]. Glioblastoma is a grade IV glioma, which is the most aggressive form and accounts for 50% of all gliomas. Although progress has been made in treatment, glioblastoma patients still have a very bad prognosis, with only 14–17-month median survival [
2]. The current standard treatment options include surgery, radiotherapy and chemotherapy, especially temozolomide (TMZ), which is the most commonly used clinical chemotherapy for Glioblastoma [
3,
4]. TMZ can efficiently cross the blood–brain barrier and induce glioma cell apoptosis; however, chemotherapy resistance often develops and represents a major treatment hurdle. Since glioma resistance to TMZ is associated with multiple factors, identifying more factors that enhance glioma cell sensitivity to TMZ may be an effective intervention to improve the prognosis of patients.
Adrenomedullin (ADM), an effective vasodilator peptide composed of 52 amino acids, is first found in human pheochromocytoma. ADM shows expression in human lung, breast, brain, prostate, colon, and other tumor cell lines [
5]. ADM mRNA expression in brain tumors is correlated with the type and grade of tumors [
6]. In an oncology environment, ADM influences tumor cell growth, angiogenesis and apoptosis [
5,
7,
8]. In gliomas, ADM expression levels have been found upregulated, and ADM acts as an effective inducer of the growth of glioblastoma cells [
6]. As previously reported, ADM exerts a significant effect on multiple critical pathways, for example, cAMP, PI3K/Akt-dependent, and Erk signaling. Through the PI3K/Akt-dependent signaling, ADM causes endothelium-dependent vasorelaxation [
9], and the infusion of ADM relieves myocardial ischemia or reperfusion injury [
10]. In addition, through MAPK/Erk activation, ADM signaling modulates additional downstream pathways which enhance the growth and survival of endothelial cells [
11]. ADM can also upregulate Bcl-2 to exert protective effects on cancer cells against hypoxia-induced apoptosis through autocrine or paracrine modes of action [
12]. Notably, blocking PI3K/Akt/mTOR signaling has been considered to be a complementary target to further overcome the resistance of glioblastoma under hypoxic conditions [
13]. Sato et al. [
14] revealed that TMZ in combination with the MEK inhibitor SL327 acts synergistically to sensitize glioblastoma stem-like cells to TMZ treatment. Thus, agents that could inhibit ADM expression might sensitize glioma cells to TMZ treatment.
MicroRNAs (miRNAs) have been found to act as critical posttranscriptional gene expression ‘fine-tuners’ within normal and tumor progression [
15‐
17]. miRNAs generally regulate gene expression via interacting with target mRNA 3ʹ-untranslated region (3ʹ-UTR) through mRNA destabilization and translation repression. Mounting evidence indicates that dysregulated miRNA expression signatures are tightly associated with various tumors, such as glioblastoma, and play context-dependent actions in tumorigenicity regulation and therapy response [
16,
18‐
21]. Interestingly, several miRNAs, including miR-27a-3p [
22], miR-93 [
23], miR-26a [
24], miR-1238 [
25], miR-129-5p [
26], miR-519a [
27] and so on, have been reported involved in glioma resistance to TMZ treatment. Thus, searching for miRNAs that might target ADM might be a promising strategy for improving glioma resistance to TMZ.
Herein, we verified ADM expression in TMZ-resistant and -sensitive glioma samples using bioinformatics and experimental analyses. ADM knockdown was achieved in glioma cells and the specific effects of ADM knockdown on glioma sensitivity to TMZ were investigated. We employed five online tools to predict miRNAs targeting ADM; miR-1297 was selected after confirming the expression profiles of predicted candidates. The predicted miR-1297 binding to ADM and miR-1297 regulation of ADM were verified. The specific effects of miR-1297 overexpression on glioma sensitivity to TMZ were investigated. Lastly, the dynamic effects of the miR-1297/ADM axis on glioma sensitivity to TMZ were investigated.
Materials and methods
Clinical samples collection
A total of 18 glioma tissue samples and 18 noncancerous adjacent tissue samples (peritumoral brain edema tissue) were obtained from patients undergoing surgical treatment at Sichuan Provincial People’s Hospital under the approval of the Ethics Committee of Sichuan Provincial People’s Hospital. All enrolled patients provided written informed consent. All the tissues were kept at − 80 °C or fixed in 4% paraformaldehyde before further experiments.
Expression data from Gene Expression Omnibus (GEO) datasets, The Cancer Genome Atlas Glioma (TCGA-GBMLGG) were downloaded. GSE68029 contains the expression profiling of 6 untreated glioblastoma stem cells (GSC) and 6 resistant GSC clones (survived from 500 μM TMZ treatment). GSE113510 contains the expression profiling of 3 LN-229 cells and 3 TMZ-resistant LN-229 cells. GSE46531 contains the expression profiling of 6 radiation-treated GSCs and 6 radiation-treated TMZ-resistant GSCs. The expression of ADM in different groups was analyzed by Student’s t-test (P-value < 0.05). For overall survival analysis of ADM, the expression matrix of TCGA-GBMLGG (n = 625) and Chinese Glioma Genome Atlas (CGGA) (n = 601) and correlated survival data were analyzed by R language packages survival and surviminer.
Cell treatment
Human non-cancerous glial cell line SVG p12 (CRL-8621) and human glioma cell lines [U87-MG (HTB-14) and T98G (CRL-1690)] were cultured (37 °C, 5% CO2) in Eagle's Minimum Essential Medium (EMEM; 30–2003) containing 10% FBS (Gibco, Waltham, MA, USA). Human glioma cell lines [A-172 (CRL-1620) and LN-229 (CRL-2611)] were kept (37 °C, 5% CO2 in 10% FBS (Gibco)-supplemented Dulbecco's Modified Eagle's Medium (DMEM; 30–2002). All cells and medium were obtained from ATCC (Manassas, VA, USA). For TMZ treatment, cells were exposed to 100 μM of TMZ (Sigma, USA) for 48 h and then collected for further investigation.
Cell transfection
Short hairpin RNA targeting ADM (sh1-ADM or sh2-ADM) was used to achieve the knockdown of ADM (GenePharma, Shanghai, China). miR-1297 expression was intervened through the transfection of miR-1297 mimics/inhibitor (GenePharma). All the transfection was performed using Lipofectamine 3000 reagent (Invitrogen). Cells were seeded in 6/96-well plates. After transfection, cells were subjected to 24/48-h incubation for subsequent analysis.
Cell counting kit-8 (CCK-8) for cell viability
Cell viability was measured using the CCK-8 (Beyotime, China). Cells (1.0 × 104 cells/well) were seeded into 96-well plates. Following an overnight incubation (37 °C), cells underwent 4-h incubation (37 °C) with 10 μL CCK-8 in 100 μL serum-free DMEM. A microplate reader (Bio-Rad, model 680; Hercules, CA, USA) was utilized to determine the optical density at 490 nm.
qRT-PCR
Total RNA of tissues and cells was extracted by TRIzol (Invitrogen, USA). miRNA and mRNA expressions were determined utilizing the SYBR Green PCR kit (Qiagen, Hilden, Germany). RNU6B (for miRNA) and GAPDH (for mRNA) served as endogenous references. Data were analyzed using the 2
−ΔΔCT method. The primer sequence was listed in Additional file
1: Table S1.
Immunoblotting
Immunoblotting was used for measuring the protein levels of ADM, pro-caspase-3, pro-caspase-9, cleaved caspase-3/9, Bax, Bcl-2, cleaved PARP, Akt, p-Akt, Erk1/2, and p-Erk1/2. All cells were lysed in 1% PMSF-contained RIPA buffer. After extraction, protein samples were separated by loading onto SDS-PAGE gel, followed by transferring to PVDF membranes. After that, membranes were blocked with 2% bovine serum albumin in TBST, followed by an overnight incubation (4 °C) with primary antibodies: ADM (ab69117, Abcam, Cambridge, MA, USA), caspase-3 (19677-1-AP, Proteintech, Wuhan, China), caspase-9 (10380-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), Bcl-2 (12789-1-AP, Proteintech), cleaved PARP (13371-1-AP, Proteintech), Akt (Y409094, Applied Biological Materials Inc., Richmond, Canada), p-Akt (66444-1-1 g, Proteintech), Erk1/2 (67170-1-Ig, Proteintech), and p-Erk1/2 (sc-81492, Santa Cruz, Dallas, TX, USA). Next, membranes underwent an incubation with HRP-labeled secondary antibody, followed by visualization using ECL Substrates (Millipore, MA, USA). GAPDH served as an internal reference.
Flow cytometry
The apoptosis analysis was performed using the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN BioTech, Nanjing, China) following the aforementioned method [
28]. The excitation (Ex) and emission (Em) wavelengths are 488 nm and 530 nm, separately. The FITC positive/PI positive and FITC positive/PI negative cells were considered as apoptotic cells. The negative control was shown in Additional file
2: Fig. S1. The mitochondrial membrane potential (ΔΨm) change was also determined by flow cytometry. Briefly, cells were collected and incubated with 10 µg/mL JC-1 staining solution (Yeasen, Shanghai, China) for 15 min at a cell culture incubator. Then, cells were analyzed by flow cytometry immediately (Ex = 488 nm, Em = 530 nm). The rate of Q2 quadrant represented no loss of ΔΨm.
Dual-luciferase reporter assay
After ADM 3'-UTR amplification by PCR, wt-ADM 3'-UTR was constructed by cloning ADM 3'-UTR into the psiCHECK-2 vector (Promega, Madison, WI, USA) downstream of the Renilla gene. mut-ADM 3'-UTR was constructed by mutating ADM seed region for the removal of all complementarity to miR-1297. The above vectors were co-transfected with miR-1297 mimics/inhibitor into 293T cells. After transfection for 48 h, luciferase assays were carried out applying the Dual-Luciferase Reporter Assay System (Promega). The Renilla luciferase activities were normalized to firefly luciferase activities.
Nude mice tumorigenicity assay
All animal protocols were performed under the approval of the Animal Care and Used Committee of Sichuan Provincial People’s Hospital. LN-229 cells were randomly divided into 4 groups and transfected with ADM stable knockdown vector (lentivirus mediated shRNA-ADM) and shRNA control vector (lentivirus mediated shRNA-NC) were harvested at logarithmic phase and resuspended into serum-free medium at 1 × 10
7 per milliliter. Then, 0.2 mL cell suspension were subcutaneously injected into 4 weeks BALB/c nude mice (weighting at 18–20 g, purchased from Laboratory Animal Research Institute of Sichuan Provincial People’s Hospital), at the right side of armpit. When all the groups formed the tumor at approximately one week, mice were injected intraperitoneally with TMZ or DMSO (30 mg/kg/day) every 3 days for another 3 weeks [
29]. At end of the experiment, the length (mm), width (mm) and height (mm) of tumor tissues were measured by a vernier caliper, and tumor volume was calculated according to the following formula: Volume = long × width
2/2. All implanted mice were sacrificed and the tumor tissues were collected for the subsequent analysis.
Histological analyses
Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were performed as previously described [
30]. Mice tumor samples were fixed in 4% paraformaldehyde in PBS overnight, paraffin-embedded, and cut into 5-μm-thick sections. IHC analysis was performed for the protein level and distribution of Ki-67 using anti-Ki-67 (ab15580; Abcam).
Statistical analysis
Experimental data were represented as means ± standard deviation (SD). Differences among groups were identified using one-way analysis of variance (ANOVA), followed by the Tukey’s test. The GraphPad Prism software (ver. 7.0; GraphPad, La Jolla, CA, USA) was utilized for all statistical analyses. P < 0.05 was considered as statistically significant.
Discussion
In the present study, ADM expression has been found upregulated in glioma tissues and TMZ- resistant glioma cells based on bioinformatics and experimental analyses. Knocking down ADM in glioma cells enhanced the suppressive effects of TMZ on glioma cell viability, promotive effects on cell apoptosis, and inhibitory effects on mitochondrial membrane potential. Moreover, ADM knockdown also enhanced TMZ effects on Bax/Bcl-2, Akt phosphorylation, and Erk1/2 phosphorylation. miR-1297 directly targeted ADM and inhibited ADM expression. miR-1297 overexpression exerted similar effects to ADM knockdown on TMZ-treated glioma cells. More importantly, under TMZ treatment, inhibition of miR-1297 attenuated TMZ treatment on glioma cells; ADM knockdown partially attenuated the effects of miR-1297 inhibition on TMZ-treated glioma cells.
ADM, a member of the calcitonin-family of peptides, has approximately 24% homology with calcitonin gene-related peptide (CGRP) [
42]. Peptides of the CGRP family are extensively found in the body and exert crucial biological effects, such as mediating calcium regulation [
43], glucose metabolism [
44], and cardiovascular functions [
45]. ADM has been reported as one of 5 candidate genes associated with glioma resistance to TMZ, which showed to be consistently linked to higher expression of DDIT4 and lower prognosis in TMZ-treated cells [
46]. Adrenomedullin 2, another CGRP member, increased glioma cell invasion and proliferation via enhancing the formation of filopodia, which depends on the activation of Erk1/2 pathway [
47]. In this study, ADM expression has been found upregulated in TMZ -resistant glioma samples, suggesting the underlying effect of ADM on glioma cell TMZ resistance. As expected, ADM knockdown in glioma cells amplified the suppression of TMZ on glioma cell viability and promotive effects on cell apoptosis. Moreover, ADM knockdown also enhanced TMZ effects on Bax/Bcl-2, Akt phosphorylation, and Erk1/2 phosphorylation. These data suggest that ADM knockdown might sensitize glioma cells to TMZ treatment, and the Bax/Bcl-2, Akt, and Erk1/2 signaling might be involved. Notably, ADM knockdown also enhanced the inhibitory effects of TMZ on mitochondrial membrane potential. Reportedly, ADM upregulated the mRNA expression and peptide level of oncogene Bcl-2 within Ishikawa cells under the condition of normoxia and anoxia. It has shown that Bcl-2 has protective effects in response to many different apoptotic stimuli, such as hypoxia [
48]. Bcl-2 prevents apoptosis, which may be achieved by blocking the release of Cytochrome c from mitochondria, and then inhibiting caspase-3vactivation and the subsequent apoptotic effects [
49]. Herein, ADM knockdown or TMZ treatment induced caspase 3 and caspase 9 cleavage, as well as mitochondrial membrane potential impairment, suggesting that mitochondrial function might also be involved in ADM knockdown enhancing glioma cell sensitivity to TMZ treatment.
As promising agents that could inhibit the expression of downstream target mRNAs, miRNAs play a critical role in carcinogenesis through regulating different targets’ expressions. Various studies have indicated that miRNAs can be efficient biomarkers of glioma and used as therapeutic targets/agents [
50‐
52]. In this study, miR-1297 has been predicted as an upstream regulatory miRNA for ADM. Through direct targeting, miR-1297 inhibited ADM expression. It has been validated that miR-1297 exerts antitumor effects on glioma. Through regulating HMGA1, miR-1297 inhibited glioma cell growth in vivo and in vitro [
39]. Another group demonstrated that miR-1297 inhibited in vitro glioma cell invasion, migration and proliferation through targeting EZH2 [
38]. In this study, single miR-1297 overexpression enhanced TMZ effects on glioma cells, whereas miR-1297 inhibition attenuated TMZ effects; more importantly, ADM knockdown significantly attenuated miR-1297 inhibition effects on TMZ-treated glioma cells. These data indicate that miR-1297/ADM axis affects glioma cell sensitivity to TMZ treatment.
In conclusion, miR-1297 sensitizes glioma cells to TMZ treatment through targeting ADM. The Bax/Bcl-2, Akt, and Erk1/2 signaling pathways, as well as mitochondrial functions might be involved.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.