1 Introduction
Glioma is one of the most major malignant tumors of the central nervous system, accounting for > 70% of all brain tumors [
1]. According to the classification criteria of the World Health Organization (WHO), there are four grades, including high-grade glioma (WHO grades III–IV) and low-grade glioma (LGG; WHO grades I–II) [
2]. Glioblastoma (GB; WHO grade IV) is the most lethal type, and the average survival time is 12–15 months [
3]. Despite significant progress in areas related to tumor diagnosis and treatment, including imaging, surgical skills, radiotherapy, chemotherapy and immunotherapy, glioma remains untreatable [
4]. Thus, novel treatment approaches are urgently needed.
Regulator of G-protein signaling (RGS) proteins were named for their ability to negatively regulate heterotrimeric G-protein signaling. This protein family acts as a GTPase-activating protein for certain Gα subunits, thereby accelerating the turn-off mechanism of Gα, terminating signaling via both the Gα and Gβγ subunits [
5]. To date, > 20 family members have been reported to be involved in the regulation of G-protein signaling pathways. As one family member, RGS16 has been implicated in negatively regulating the MAPK [
6], PI3K/AKT [
7], RhoA [
8], and SDF-1/CXCR4 [
9] oncogenic pathways, which are involved in various processes associated with cancer progression, such as migration, invasion, proliferation, chemoresistance, and metastasis. The dysregulation of RGS16 has been reported in breast cancer [
7], pancreatic cancer [
10], colorectal cancer [
11], and chondrosarcoma [
12]. Nevertheless, the role of RGS16 in human glioma has yet to be fully elucidated.
MicroRNAs (miRNAs/miRs) are a class of recently discovered endogenous small nonprotein-coding RNAs that are key posttranscriptional regulators of gene expression [
13]. By targeting or silencing target genes, miRNAs play crucial roles in controlling tumorigenesis [
14]. Let-7c-5p is hypothesized to function as a tumor suppressor by silencing targeted mRNAs and has been shown to be associated with clinical outcomes in cancer patients [
15,
16].
In the present study, the effect of RGS16 on glioma cells and its relationship with let-7c-5p were assessed. RGS16 function was investigated both in vitro and in vivo. The results showed that RGS16 may be a novel biomarker and that it is regulated by let-7c-5p. These results may improve our understanding of glioma progression and highlight a potential therapeutic target for gliomas.
2 Materials and methods
2.1 Cox proportional hazards model
A Cox proportional hazards model was used to select the genes which were relevant to survival and build a predictive model. The outcome time was defined as overall survival months and disease-free survival months. N genes were selected to construct a Cox proportional hazards model. For each gene Gj (j = 1, 2,…, N), the following model of vitality vs. death at time t was built:
where λ0, (t) is the baseline hazard function for gene Gj, and X1, X2,…, XP are covariates. The covariates that were adjusted included race, age, sex, Karnofsky performance score, and tumor status.
2.2 Tissue samples and cell lines
Human glioma cell lines, U87 MG cells (ATCC), which were authenticated by STR, and U-251 MG cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All glioma tissues were obtained from patients (n = 32) who underwent surgery in the Department of Neurosurgery, Qilu Hospital, Cheeloo College of Medicine, Shandong University. Patients who received an internal decompression surgery due to a past severe head traumatic injury were used as a control (n = 5). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.
2.3 Cell culture
Cell lines were cultivated in DMEM (Thermo Fisher Scientific, Inc.) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) at 37 °C in a humidified incubator with 5% CO2.
2.4 Cell transfection
RGS16-specific small interfering (si) RNA and scrambled control (NC) siRNA, miR let-7c-5p mimics and inhibitor and their respective negative control were designed and synthesized by Shanghai GenePharma, Co., Ltd., and cells were transiently transfected using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Stable knockdown of RGS16 in cells was generated via lentiviral transduction using short hairpin (sh) RGS16 (Shanghai GenePharma, Co., Ltd.), and the sequence of sh-RGS16 was 5′-GATCCGATCAGCTACCAAG-3′. When cell density reached ~80%, cell transfection was performed according to the manufacturer’s protocol. Knockdown efficiency was evaluated 24 h after transfection using reverse transcription quantitative (RT-q) PCR and 48 h after transfection by Western blotting. The sequences used for transient transfection were listed as follows: RGS16 siRNA sense, 5′-GAUCCGAUCAGCUACCAAGTT-3′ and antisense, 5′-CUUGGUAGCUGAUCGGAUCTT-3′; RGS16 NC siRNA sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′; let-7c-5p mimics sense, 5′-UGAGGUAGUAGGUUGUAUGGUU-3′ and antisense, 5′-CCAUACAACCUACUACCUCAUU-3′; mimics negative control sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′; let-7c-5p inhibitor, 5′-AACCAUACAACCUACUACCUCA-3′; inhibitor negative control, 5′-CAGUACUUUUGUGUAGUACAA-3′.
2.5 RNA extraction and RT-qPCR
TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA according to the manufacturer’s protocol. A PrimeScript 1st Strand cDNA Synthesis kit was used to reversely transcribe RGS16 and miR let-7c-5p. qPCR was performed using a SYBR Premix Ex Taq™ Kit (Takara Bio, Inc.). The sequences of the primers used were: RGS16 forward, 5′-TCCAGGGCACACCAGATCTT-3′ and reverse, 5′-TCGCAGTCTGCACGTTCATC-3′; miR let-7c-5p forward, 5′-CGTCATCCTGAGGTAGTAGGTTGT-3′ and reverse, 5′-TATGGTTTTGACGACTGTGTGAT-3′. Amplification was performed using a LightCycler 2.0 Instrument (Roche Applied Science). The expression levels of GAPDH were used as the loading internal controls. The expression levels of the fold changes were calculated using the 2-ΔΔCq method.
2.6 Western blot analysis
Total proteins were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). After resolving using SDS-PAGE, the proteins were transferred to a PVDF membrane (EMD Millipore). Blots were incubated with primary antibodies against RGS16 (Biorbyt); MMP2, MMP9, and GAPDH (all from Cell Signaling Technology, Inc.). Proteins were visualized using enhanced chemiluminescence (EMD Millipore). Densitometry analysis was performed using ImageJ (National Institutes of Health) and normalized to GAPDH. The relative integrated density values were measured using actin as the control.
2.7 CCK-8 assay
U87 or U251 cells were plated in 96-well plates at a density of 3×103 cells per well, transfected after 12 h, and 10 μL Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) solution was added to each well after 24, 48 or 72 h of culture according to the manufacturer’s protocol. After incubation for 1 h at 37 °C, the absorbance value was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).
2.8 EdU incorporation assay
Cell proliferation rate was analyzed using an EdU cell proliferation assay kit (Guangzhou RiboBio Co., Ltd.) according to the manufacturer’s protocol. Cells were incubated with 200 μL EdU solution for 2 h at 37 °C, fixed in 4% paraformaldehyde for 20 min, subsequently permeabilized using 0.4% Triton X-100 for 10 min and incubated with Apollo® reagent (100 μL) for 30 min. Nuclei were subsequently stained with DAPI, and visualized using a fluorescence microscope (Nikon Corporation).
2.9 Colony-formation assay
A total of 500 cells/well were plated in a 6-well plate and incubated for ~2 weeks. Subsequently, cells were stained using crystal violet (Beyotime Institute of Biotechnology) for 15 min and gently washed with PBS. Colonies visible with the naked eye were counted.
2.10 Transwell assay
A total of 48 h after transfection, cell migration assays were performed using Transwell chambers measuring 6.5 mm in diameter. Briefly, a total of 3×104 cells without FBS were added to the upper chamber. Medium in the lower chamber was eutrophic with 15% FBS. After 8 h (U87 cells) or 20 h (U251 cells), cotton swabs were used to gently remove the cells from the upper membrane, whereas cells which had successfully migrated were fixed and stained. A total of five fields of view were randomly selected and imaged, the results were quantified. Similarly, invasive ability was evaluated using chambers pre-coated with Matrigel®.
2.11 Wound-healing assay
Cells were seeded in 6-well plates. Following transfection, cell monolayers were scratched using a 200 μL sterile pipette tip. After removing the debris, each well was supplemented with DMEM without FBS, cells were then incubated at 37 °C. Images were taken after 0 and 24 h along the scratch under a microscope. Cell movement was expressed as the relative scratch width compared to the original scratch width.
2.12 Dual-luciferase reporter assay
TargetScan (targetscan.org) was used to predict the miRNAs that could bind to RGS16, and let-7c-5p was selected. pGL3 reporter constructs containing the target sequences of RGS16 and mutant RGS16 were purchased from BioAsia. Let-7c-5p mimics or NC and RGS16-wt or RGS16-mut 3′ untranslated region (UTR) sequences were co-transfected into cells. After 48 h of transfection, the relative luciferase activity was measured using a luciferase assay kit according to the manufacturer’s protocol (Promega Corporation).
2.13 Immunohistochemistry (IHC)
Paraffin-embedded samples from patient glioma tissues or tumors removed from nude mice were sectioned and mounted on microscopic slides. Sodium citrate buffer (pH 6.0) was used for antigen retrieval, and endogenous peroxidase activity was quenched by incubating the slides in methanol containing 3% hydrogen peroxide for 30 min, then washed in PBS for 6 min. Subsequently, the sections were incubated for 2 h at room temperature with normal goat serum and incubated at 4 °C overnight in a humidified chamber with an antibody against RGS16 (Biorbyt; 1:200). Subsequently, the sections were incubated with a horseradish peroxidase-conjugated secondary antibody and 3,3′-diaminobenzidine as the substrate in order to visualize. Slides were counterstained with hematoxylin, and representative images were obtained using an inverted microscope (Olympus Corporation). The total immunostaining score was evaluated according to the percentage of positively stained tumor cells.
2.14 Luciferase-transduced cell generation and in vivo assay
U87 luciferase cells were infected with sh-RGS16. After 24 h of addition of the viral suspension, cells were cultured in complete medium containing 1 µg/mL puromycin for a week to screen for stably knocked down clones, and stable clones were confirmed using a fluorescence microscope. A total of 3×104 sh-RGS16/ ov-miR let-7c-5p or control cells/10 μL were then implanted stereotactically into the brains of 5-week-old male nude mice (SLAC Laboratory Animal Center; Shanghai, China). In vivo bioluminescence imaging was performed every 7 days with the noninvasive IVIS™ bioimaging system with VivoGlo™ Luciferin (Promega Corporation) as a substrate according to the manufacturer’s protocol in each group. Living Image software was used to analyze the data. Next, we randomly chose 5 mice in each group and sacrificed them on the same day (21 days). The brains were fixed with paraformaldehyde for further study. The remaining mice (5/group) were kept until death for survival analysis. All procedures that involved mice were approved by the Animal Care and Use Committee of the Qilu Hospital of Shandong University.
2.15 AGO2 RNA immunoprecipitation (RIP) assays
A RIP-Assay Kit (Medical & Biological Laboratories Co., Ltd., Japan) was used to detect interactions between mRNA of RGS16 and miR let-7c-5p. Briefly, the prepared beads immunized with anti-Ig and AGO2 antibodies were mixed with the lysed U87 and U251 cells at 4 °C. After 3 h, the bead-antibody RIP complexes were collected for RT-qPCR.
2.16 Statistical analysis
All experiments were independently repeated at least three times. Data were analyzed using GraphPad Prism version 6 (GraphPad Software, Inc.), unless otherwise stated, Student’s t-test was used for two group comparison. For comparisons of more than two groups, one-way ANOVA tests were used. Unless otherwise stated, Sidak’s multiple comparisons test was used after ANOVA. Data are presented as the mean ± standard error of the mean. P < 0.05 was considered to indicate a statistically significant difference. Significance was represented using asterisks; ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
3 Results
3.1 RGS16 expression is associated with less favorable patient survival
Based on the Cox proportional hazards model using data acquired from The Cancer Genome Atlas (TCGA), significant correlations were observed between RGS16 and overall survival duration (P = 0.00054, regression coefficient = 0.17) as well as disease-free survival duration (P = 0.0047, regression coefficient = 0.16) (Table S1). Thus, RGS16 may serve as an oncogene.
3.2 RGS16 expression is upregulated in glioma tissues
After analyzing the data obtained from the TCGA, we focused on the gene expression levels of RGS16 in GBM, LGG, and normal brain tissues. The results showed that RGS16 mRNA expression levels were significantly higher in GBM tissue than in LGG and normal brain tissues. Furthermore, expression was higher in LGG tissue than in normal brain tissues (Fig.1). To investigate the clinical significance of RGS16 in gliomas, 37 paraffin-embedded archived human tissues (5 normal brain tissues and 32 glioma tissues) from Qilu Hospital (Jinan, China) were analyzed by IHC. Similar to the mRNA expression results from the TCGA database analysis (Fig.1), RGS16 levels were higher in GBM tissues than in LGG or normal brain tissues (Fig.1 and 1C). These results suggest that RGS16 expression is significantly associated with the histological grade of glioma.
3.3 Knockdown of RGS16 inhibits glioma cell proliferation
Subsequently, qPCR and Western blotting were used to evaluate the knockdown efficiency of RGS16-specific siRNA. The expression levels of RGS16 were lower in U87 and U251 cells after transfection with the siRNA compared with the control cells (Fig.2 and 2B). Cell proliferation was analyzed using a CCK-8 assay. The proliferation rates of U87 and U251 cells were significantly lower in the RGS16 knockdown group than in the control group (Fig.2 and 2D). To evaluate the long-term effects of RGS16 on cell proliferation, a colony formation assay was performed. As a result, fewer colonies were observed in the RGS16 knockdown group (Fig.2 and 2F). Additionally, the proportion of EdU-positive cells was significantly lower in cells transfected with RGS16 siRNA than in NC-transfected cells (Fig.2 and 2H). Protein electrophoresis and subsequent Western blotting revealed a significant decrease in proteins associated with proliferation progression, such as CDK2, cyclin B1, and cyclin D1. In contrast, the expression of the cyclin-dependent kinase inhibitor p21 was higher in the RGS16 knockdown group (Fig.2). These results suggest that RGS16 inhibits glioma cell growth in vitro.
3.4 Knockdown of RGS16 reduces the migration and invasion of glioma cells in vitro
To evaluate the migratory and invasive abilities of glioma cell lines with different levels of RGS16 expression, Transwell migration and invasion assays were performed using U87 and U251 cells. Compared with that in the NC group, the proportions of migrating cells in the RGS16 siRNA group were 66% and 64% lower in U87 and U251 cells, respectively (Fig.3 and 3B), and the proportions of invading cells were 51% and 27% lower, respectively (Fig.3 and 3D). MMP2 and MMP9 are key molecules involved in tumor progression related to migration and invasion; thus, MMP2 and MMP9 expression was examined following the knockdown of RGS16. As shown in Fig.3, knockdown of RGS16 induced downregulation of the MMP2 and MMP9 proteins in U87 and U251 cells. We next performed a Western blot assay and showed that the expression of AKT2 and p-AKT was downregulated after knockdown of RGS16, as shown in Fig.3. We hypothesized that RGS16 activates the PI3K-AKT pathway to regulate the progression of MMP2 and MMP9. Similar results were observed in the wound-healing assays. RGS16 knockdown attenuated migration in both U87 and U251 cells (Fig.3 and 3G). These results suggest that RGS16 inhibits glioma cell migration and invasion in vitro.
3.5 Overexpression of miR let-7c-5p inhibits glioma cell proliferation, migration, and invasion
To investigate the regulatory mechanism of RGS16, the TargetScan database was used to predict a potential binding miRNA, and miR let-7c-5p was selected for further analysis. pmirGLO vectors were constructed containing WT or mutant 3′-UTR sequences (Fig.4). To further verify the biological function of let-7c-5p, RT-qPCR was performed for 5 normal brain tissues and 12 glioma tissues, and the results showed that the expression of let-7c-5p was downregulated in glioma tissues (Fig.4). Before we conducted further experiments, the knockdown and overexpression efficiency of inhibitor and mimics of miR let-7c-5p was confirmed using RT-qPCR (Fig.4). Subsequently, CCK-8, EdU, and transwell assays were carried out, and the results showed that upregulating let-7c-5p obviously inhibited the proliferative, migratory, and invasive abilities of both U87 and U251 cells, and the trends were reversed by the inhibitor in vitro (Fig.4, 4E, and Fig. S1A). Moreover, in the in vivo study, overexpression of let-7c-5p markedly inhibited tumor growth and invasiveness and prolonged the survival of tumor-bearing mice (Fig.4, 4G, and Fig. S1B). In addition, immunohistochemistry (IHC) of the excised tumor sections revealed that the expression of Ki67 (a proliferation marker) and RGS16 was lower in the let-7c-5p overexpression group than in the vector group (Fig. S1C). Taken together, our data suggest that miR let-7c-5p plays an important role in inhibiting glioma cell proliferation, migration, and invasion both in vitro and in vivo.
3.6 RGS16 is regulated by miR let-7c-5p
To further confirm that RGS16 is the downstream target of miR let-7c-5p, we found that when let-7c-5p was overexpressed via mimics, RGS16 was downregulated significantly at the mRNA and protein levels according to the results obtained from RT-qPCR and Western blotting assays in both U87 and U251 cell lines, while the let-7c-5p inhibitor reversed this trend (Fig.4 and 4I). Moreover, a luciferase reporter assay was used to confirm the targeting relationship between let-7c-5p and RGS16 (Fig.4). Furthermore, we performed AGO2 rip assay to determine miR let-7c-5p and mRNA of RGS16 binding together with AGO2 (Fig.4). Functional rescue assays provided further proof that RGS16 is the downstream target of miR let-7c-5p. Let-7c-5p mimics impeded proliferation, migration, and invasion in U87 and U251 cells, but this effect could be rescued by RGS16 upregulation (Fig.5–5D).
3.7 Knockdown of RGS16 inhibits tumorigenesis in vivo
To confirm the growth-inhibiting effect of RGS16 on glioma cells in vivo, a nude mouse orthotopic xenograft model was established by implanting sh-RGS16 or control cells into the brain. Before the model was generated, the silencing efficiency was validated by RT-PCR assay (Fig.6). Animals implanted with sh-RGS16 cells exhibited significantly improved outcomes, for example, smaller tumor sizes (Fig.6 and 6C) and longer survival times than the controls (27 vs. 22 days; P < 0.05; Fig.6). When the mice died, the brains were removed, and RT-qPCR proved that the expression of RGS16 was stably knocked down by sh-RGS16 (Fig.6). Because the tumor size was no longer a sensitive indicator when the mice died, hematoxylin and eosin staining of the xenograft tissues was applied and showed that the borders of tumors in the control group were blurred, and tumor cells were more aggressive and infiltrated into the peripheral normal tissue. In the sh-RGS16 group, the borders between the tumor and peripheral tissues were distinct, and tumor infiltration was rare (Fig.6). RGS16 was downregulated in sh-RGS16 xenografts compared with the controls (Fig.6 and 6H), and there were fewer positive Ki-67 cells in the sh-RGS16 group than in the control group (Fig.6 and 6J). According to the results, knockdown of RGS16 impeded the growth and invasion of glioma cells in vivo.
4 Discussion
Malignant gliomas are characterized by uncontrollable cell proliferation and growth [
17]. Numerous studies have focused on gliomas due to their high morbidity and mortality rates. Unfortunately, the molecular mechanisms underlying glioma development and progression are incompletely understood, and consequently, new therapeutic treatments are urgently needed. The roles of RGS16 are beginning to be elucidated in several malignancies, whereas its function in brain tumorigenesis has not been investigated to the best of our knowledge. In the present study, by investigating the function of RGS16 expression, we found that it was more highly expressed in GBM than in LGG. High RGS16 expression was associated with a poor prognosis in patients with glioma. Therefore, RGS16 appears to serve as an oncogene in gliomas. Further analysis confirmed that knockdown of RGS16 attenuated migration, invasion, and proliferation both
in vitro and
in vivo. Using gene ontology (GO) analysis, Huang
et al. revealed that RGS16 was significantly correlated with oncogenic processes, including immune and inflammatory responses, angiogenesis, cell proliferation and migration, T cell activation, cell-matrix adhesion, and epithelial to mesenchymal transition (EMT) [
18]. Therefore, to explore the underlying mechanisms of RGS16 in glioma cells, the expression levels of cell proliferation-related proteins (cyclin B1, cyclin D1, CDK2, and P21) [
19], apoptosis-related proteins (p-AKT and AKT) [
20], and migration/invasion-related proteins (MMP2 and MMP9) [
21] were assessed by Western blotting. According to the results of the present study, knockdown of RGS16 significantly downregulated the expression of downstream oncogenic factors, including cyclin B1, cyclin D1, CDK2, MMP2, and MMP9. The expression of p21, a tumor suppressor in the cell cycle pathway, was upregulated when RGS16 expression was knocked down. As in the phenotype assays, all results indicated that silencing RGS16 attenuated the migration and proliferation abilities. These results may provide a potential molecular target/pathway for therapy. The same oncogenic effects were also confirmed in colorectal cancer, and the RGS16 high expression group had a lower overall survival rate than the low expression group [
12].
In contrast, RGS16 has been reported to function as a tumor suppressor in several tumors due to its clear negative inhibitory effect on the MAPK, RhoA, and SDF-1/CXCR4 pathways [
7–
10,
12]. It is worth mentioning that the PI3K/Akt pathway is important in gliomas [
20,
22]. Numerous studies have demonstrated that the expression of p-Akt is elevated in gliomas; as a consequence, the upregulated expression of p-Akt is thought to be associated with poor prognosis [
23,
24]. Liang
et al. [
7] demonstrated that RGS16 inhibits breast cancer cell growth by modulating the PI3K/Akt pathway. From this point of view, RGS16 should be a tumor suppressor gene in gliomas, so what is the reason for this discrepancy?
Humans have three AKT isoforms: AKT1, AKT2, and AKT3. A previous study performed a comprehensive analysis of the three isoforms in gliomas. The results showed no difference in the mRNA and protein levels of AKT1 in glioma tissues compared with normal tissues, and the expression of Akt2 was significantly higher in high-grade glioma (WHO grades III–IV) than in LGG; however, AKT3 was hardly expressed in gliomas. Knockdown of AKT2 in glioma cell lines suppressed colony formation and induced apoptosis; however, AKT1 knockdown had no such effect [
25]. Therefore, we hypothesized that RGS16 might be related to AKT2. p-Akt expression levels were investigated following RGS16 knockdown in U87 and U251 cell lines. RGS16 silencing downregulated the expression of AKT2 and p-AKT, and the effect on p-AKT was more pronounced (Fig.3). This finding suggests that tumor growth is an extremely complicated process and that the tumor microenvironments vary depending on the tumor type [
26]. One molecule may play different roles in diverse tumors.
miRNAs are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. They have been shown to play roles in various physiologic and pathological events [
14,
27]. A growing body of evidence has suggested that various aberrantly expressed miRNAs in glioma tissues and cells function as either tumor suppressors or tumor promotors by targeting various genes [
28,
29]. Therefore, understanding their functions and mechanisms may highlight novel targets. According to the TargetScan database, let-7c-5p was predicted to be an upstream miRNA for RGS16. In the present study, RGS16 expression was significantly downregulated following transfection with exogenous let-7c-5p mimic, indicating that let-7c-5p acts as a tumor suppressor in gliomas. The present study showed that overexpression of let-7c-5p suppressed the proliferation and metastasis of glioma cells. Conversely, deletion of let-7c-5p significantly promoted these behaviors. To the best of our knowledge, this study is the first to reveal the negative regulatory effects of let-7c-5p on RGS16 expression in glioma cells, highlighting the antitumor mechanisms of let-7c-5p in gliomas.
In conclusion, RGS16 expression is upregulated in glioma cell lines and clinical samples and can be regulated by miR let-7c-5p. Thus, as an oncogenic molecule in glioma, RGS16 plays an important role in tumor progression, suggesting that RGS16 may be a novel potential therapeutic target for the treatment of gliomas. Undoubtedly, there are limitations in the present study. For example, RGS16 is not the only target gene of let-7c-5p, and the precise molecular mechanisms of the crosstalk between RGS16 and let-7c-5p require further investigation to adequately understand the functions of RGS16 in human gliomas.
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