Abstract

   Malignant glioma is the most common brain tumor in adults. Despite the
   great advances in anti-glioma treatments which have led to significant
   improvement in clinical outcomes, tumor recurrence remains the major
   cause of mortality. Increased cancer cell stemness and invasiveness are
   correlated with glioma progression. By searching the Cancer Genome
   Atlas, we showed that the expression of miR-7156-3p is significantly
   decreased in glioma tissues compared to the normal brain, and the
   decreased level of miR-7156-3p is closely correlated with glioma grade
   and patient survival. Clinical study consistently confirmed that
   miR-7156-3p is negatively correlated with glioma grade. Cell culture
   and animal experiments revealed that inhibition of miR-7156-3p
   effectively stimulates glioma cell stemness, invasion, and growth. In
   contrast, the augmentation of miR-7156-3p inhibits these phenotypes.
   Using Next-generation sequencing combined with target prediction
   approach, Homeobox D13 (HOXD13) is identified as the target gene of
   miR-7156-3p and further validated by luciferase reporter assay and cell
   transfection experiments. Additional in vitro and animal experiments
   demonstrated that miR-7156-3p regulates glioma cell stemness, invasion,
   and growth by mediating HOXD13. In conclusion, our findings provide new
   insight into the regulation of glioma stemness and invasiveness and may
   propose a potential strategy for anti-glioma treatment. Moreover,
   miR-7156-3p may serve as a candidate biomarker for predicting glioma
   progression in clinical practice.

   Keywords: miR-7156-3p, HOXD13, stemness, glioma, invasion, biomarker

Introduction

   Malignant glioma is the most common primary malignancy in the brain,
   accounting for 80% of all brain tumors [39]^1. Surgical resection
   adjuvant with chemoradiotherapy is the standard therapeutic regimen for
   glioma. However, patients with glioma often show a high rate of
   recurrence after treatment [40]^2. Even with the great advances in
   emerging surgical technology and therapeutic strategy, malignant glioma
   often recurs at the region within 1-2 cm of the original tumor site due
   to the tumor cells invading into the surrounding normal brain tissue
   where they can hide from surgical removal and chemoradiotherapy,
   suggesting that glioma cell invasiveness plays a critical role in tumor
   progression [41]^3. Previous studies have reported that cancer stem
   cells (CSCs) are the initialing factor responsible for glioma
   recurrence. CSCs are a small subpopulation of cancer cells that have
   high invasiveness and self-renewal ability to differentiate into tumor
   cells [42]^4^, [43]^5. Therefore, glioma invasion and stemness are a
   lethal combination, resulting in tumor progression and therapeutic
   resistance [44]^6. These findings indicate that inhibiting glioma cell
   invasion by specifically targeting CSCs is important with significant
   clinical implications for anti-glioma treatment. Unfortunately, the
   mechanisms of glioma cell invasion and regulation of CSCs are not fully
   understood and no effective treatment aiming at glioma cell invasion
   and CSCs exists thus far.

   MicroRNAs (miRNAs) are a class of small non-coding single-stranded RNAs
   that negatively regulate gene expression by stimulating the degradation
   of or inhibiting the translation of mRNAs by binding to 3'-untranslated
   region (3`-UTR) of the target genes. Dysregulated expression of miRNAs
   is identified in most cancer types including glioma [45]^7. It is
   well-documented that certain aberrantly expressed miRNAs are closely
   associated with glioma recurrence [46]^8, stimulation of glioma cell
   stemness [47]^9, and tumor invasiveness [48]^10. More importantly,
   inhibition or overexpression of miRNAs that are aberrantly upregulated
   [49]^11 or downregulated [50]^12^, [51]^13 in glioma significantly
   inhibits tumor growth and invasion, indicating that miRNAs have much
   potential as promising potential therapeutic targets. However, some
   miRNAs play opposite roles by targeting different target genes in
   different types of cancers, even in the same cancer type but at
   different grades or stages. For example, Korpal et al. [52]^14 found
   that miR-200s inhibit local invasion by targeting ZEB1 but stimulate
   metastatic colonization by targeting Sec23a in breast cancer. Also,
   studies show that even aberrant expression of a single miRNA is
   sufficient to cause cancer progression, suggesting the essential role
   of miRNA in tumorigenesis [53]^15. Therefore, studying individual miRNA
   that abnormally expressed in tumors is necessary to fully understand
   its functional roles in glioma and is the prerequisite for clinical
   translation of miRNA-targeted therapy.

   In this study, we reported for the first time that miR-7156-3p is
   significantly downregulated in glioma and closely associated with
   glioma grade, invasion, stemness, and survival in glioma patients.
   Moreover, we unveiled that miR-7156-3p overexpression dramatically
   suppressed glioma stemness, invasiveness, and tumor growth by
   downregulating HOXD13.

Materials and Methods

Cell culture and tumor specimens

   Human glioma cell lines (U373 and SW1783) were obtained from ATCC and
   cultured in MEM (Gibco) with 10% fetal bovine serum (FBS, Invitrogen).
   Formaldehyde-fixed, paraffin-embedded (FFPE) tumor specimens from 44
   newly-diagnosed glioma patients underwent tumor resection at Daping
   Hospital of Army Medical University were used in the study. The
   characteristics of the patients were summarized in Table [54]1. The
   study was approved by the Daping Hospital review boards.

Table 1.

   Characteristics of glioma patients
   Characteristics     Variable        Number
   Age (years)     Range (means±SD) 16-78 (60±12)
   Gender          Male             23 (52.3)
                   Female           21 (47.7)
   Family history  No               31 (70.5)
                   Yes              13 (29.5)
   Grade           II               14 (31.8)
                   III              15 (34.1)
                   IV               15 (34.1)
   IDH-1           Wild type        32 (72.7)
                   Mutant           12 (27.3)
   [55]Open in a new tab

mRNA analysis

   Total RNA from tumor cells and fresh tissues was isolated using TRIzol
   reagent (Invitrogen) following the manufacturer's instruction. Total
   RNA from FFPE tumor specimens was isolated using the RNA Prep Pure FFPE
   Kit (QIAGEN, Hilden, Germany). Reverse transcription was performed
   using a High-Capacity cDNA Reverse Transcription Kit (Applied
   Biosystems, Foster City, CA, USA). Mature miR-7156-3p and the U6 small
   nuclear RNA (RNU6) endogenous control were analyzed by PCR with a
   specific primer set and kit from RiboBio (Guangzhou, China). The
   primers used for HOXD13 amplification were 5'-CGCTGCCTCTGGCAAGTGGAGT-3'
   and 5'-TCGGTTATGGTACAAAGCGGAGAC-3'. The primers used for GAPDH
   amplification were 5'-GGAGCGAGATCCCTCCAAAAT-3' and
   5'-GGCTGTTGTCATACTTCTCATGG-3'.

Cell Counting Kit-8 analysis

   Glioma cells were transfected with the indicated oligonucleotides and
   plasmids. After 24 hours of transfection, cells were reseeded into
   96-well cell culture plates at a density of 1×10^4 cells per well, and
   cell viability was measured using a Cell Counting Kit-8 (CCK-8, Wuhan,
   China) assay according to the manufacturer's protocol at the indicated
   times.

Luciferase reporter assay

   A luciferase reporter assay was performed as described previously
   [56]^16. In brief, the 3`-UTR fragments of HOXD13 predicted to interact
   with miR-7156-3p were amplified by PCR from human genomic DNA and
   inserted into the Mlu І and Hind III sites of the pMIR-REPORTTM
   Luciferase vector (Thermo Fisher Scientific). Then, this firefly
   luciferase plasmid and Renilla luciferase plasmid were cotransfected
   into tumor cells. After 48 hours of transfection, the luciferase
   activity was examined using a Dual-Luciferase Assay System (Promega).

Apoptosis analysis

   Glioma cells were transfected with the indicated oligonucleotides and
   plasmids. After 48 hours of transfection, cells were harvested and
   stained with annexin V and 7-aminoactinomycin D (7-AAD), and then
   subjected to flow cytometric analysis.

Invasion assay

   The invasion assay was performed as described previously [57]^16. The
   indicated glioma cells were transfected with the indicated
   oligonucleotides and plasmids. After 48 hours, 1×10^4 cells in
   serum-free growth medium were seeded in the upper wells of transwell
   chambers. The lower wells contained the same medium supplemented with
   10% FBS. After 24 hours of culture, the cells on the upper side of the
   chamber were removed, and the cells on the lower side of the chamber
   were fixed with glutaraldehyde, stained with 0.1% crystal violet, and
   counted.

Western blotting and immunohistochemistry

   Western blotting and immunohistochemistry (IHC) were performed as
   described previously [58]^17. Antibodies against HOXD13, CD133, CD44,
   OCT4, SOX2, and β-Actin were purchased from Abcam.

Animal experiments

   Male BALB/c nude mice (6-8week, 20-25 g, obtained from the Experimental
   Animal Center of Daping Hospital, Chongqing, China) were used for
   xenograft modeling. Briefly, 1×10^6 U373 cells suspended in 10 μL DPBS
   were stereotactically implanted into the right cerebral cortex of mice
   using the Hamilton syringe. All the surgical procedures and handling of
   the animals were performed in accordance with the International
   Principles of Laboratory Animal Care and were approved by the Animal
   Use Subcommittee of Daping Hospital, Army Medical University.

   Assessments of tumor volumes were performed with a 7T animal magnetic
   resonance imaging (MRI) scanner (Biospin70/20, Bruker, Ettlingen,
   Germany) equipped with a four-channel mouse head transmit/receive coil.
   T[2]-weighted images (Turbo-RARE; TE/TR: 45ms/4000ms, field of view:
   25×25 mm, matrix size: 256×256, slice thickness: 0.5 mm) were conducted
   to evaluate tumor volumes according to the formula: [width]^2 ×
   [length] × ^1/[2].

Statistical analysis

   The differences between treatment groups were analyzed with one-way
   ANOVA using SAS software (SAS Institute Inc., Cary, NC, USA). All data
   were presented as the mean ± standard deviation (SD). The correlations
   between gene expression and glioma grade were determined using
   Pearson's chi-squared test. The overall survival (OS) of patients was
   calculated using the Kaplan-Meier survival curve. The results were
   considered significant only when P < 0.05.

Results

miR-7156-3p is downregulated in glioma and is associated with glioma
progression

   To identify glioma-associated miRNAs, we analyzed the Cancer Genome
   Atlas (TCGA) dataset and identified global miRNAs that were
   significantly dysregulated in glioma compared to normal brain tissues
   (Figure [59]1A). Among the top 10 miRNAs with the largest fold change
   (Figure [60]1B), the role of other miRNAs in glioma has been reported
   except miR-7156-3p [61]^18^-[62]^26. Therefore, we focused on
   investigating the role of miR-7156-3p in glioma. Clinical data analysis
   showed that low expression level of miR-7156-3p was closely correlated
   with poor prognosis in glioma patients (Figure [63]1C). More
   importantly, we observed a negative correlation between miR-7156-3p
   expression level and glioma grade in both TCGA dataset (Figure [64]1D)
   and our clinical cohort data (Figure [65]1E), indicating that
   miR-7156-3p has important clinical significance in glioma. However, no
   significant miR-7156-3p difference was seen between glioma with or
   without isocitrate dehydrogenase-1 mutation (IDH-1) (Figure [66]1F).
   Taken together, these data suggest that decreased expression of
   miR-7156-3p may be involved in the glioma progression and patient
   survival independent of IDH-1 mutation.

Figure 1.

   [67]Figure 1
   [68]Open in a new tab

   The decreased expression level of miR-7156-3p in glioma significantly
   correlates with glioma grade and survival rate of glioma patients. A.
   Heat map of differentially expressed miRNAs between grade II glioma
   (n=152) and normal brain tissues (n=5). Data were obtained from the
   TCGA database. B. List of the top 10 downregulated miRNAs in glioma
   tissues compared to normal brain tissues. Data were obtained from the
   TCGA database. C. Kaplan-Meier analysis of the overall survival rate of
   patients with glioma with low and high miR-7156-3p expression. Data
   were obtained from the TCGA database. D. miR-7156-3p expression levels
   are negatively correlated with glioma grade. Data were obtained from
   the TCGA database. Grade II, n=249; grade III, n=261; grade IV, n=174.
   E. Our clinical cohort analysis showed that miR-7156-3p expression
   levels are negatively correlated with glioma grade. The expression
   level of miR-7156-3p was measured using qRT-PCR. F. Our clinical cohort
   analysis showed that IDH-1 mutation did not affect miR-7156-3p
   expression in glioma. *, P<0.05. **, P<0.01; ***, P<0.001.

miR-7156-3p negatively regulates glioma progression

   To investigate whether the decreased expression of miR-7156-3p directly
   contributes to glioma progression, both U373 and SW1783 glioma cells
   were used and transfected with miR-7156-3p inhibitor and then subjected
   to in vitro functional analysis. Our data showed that inhibition of
   miR-7156-3p (Figure [69]2A) significantly increased cell growth (Figure
   [70]2B), invasion (Figure [71]2C), and suppressed apoptosis (Figure
   [72]2D) in both SW1783 and U373 cells. In contrast, overexpression of
   miR-7156-3p (Figure [73]2A) dramatically inhibited cell growth (Figure
   [74]2B), invasion (Figure [75]2C), and induced cell apoptosis (Figure
   [76]2D). These data indicate that inhibition of miR-7156-3p stimulates
   glioma progression and that upregulating miR-7156-3p may serve as a
   potential strategy for anti-glioma treatment.

Figure 2.

   [77]Figure 2
   [78]Open in a new tab

   miR-7156-3p negatively regulates glioma progression. A. miR-7156-3p
   expression levels were measured in glioma cell lines. The indicated
   cells were transfected with the indicated oligonucleotides and
   incubated for 72 hours. Then, the cells were subjected to qRT-PCR
   analysis. B. miR-7156-3p negatively regulated glioma cell growth. The
   indicated cells were transfected with the indicated oligonucleotides
   and incubated for 24 hours. Then, the cells were subjected to cell
   growth analysis. C. miR-7156-3p negatively regulated glioma cell
   invasion. The indicated cells were transfected with the indicated
   oligonucleotides and incubated for 48 hours. Then, the cells were
   subjected to an invasion assay. D. miR-7156-3p stimulated glioma cell
   apoptosis. The indicated cells were transfected with the indicated
   oligonucleotides and incubated for 48 hours. Then, the cells were
   subjected to apoptotic cell analysis. NC, negative control
   oligonucleotides; mimics, miR-7156-3p mimics; inhibitor, inhibitor of
   miR-7156-3p, ***, P<0.001.

miR-7156-3p negatively regulates glioma stemness

   To investigate the molecular mechanism of miR-7156-3p in the glioma
   progression, we performed mRNA sequencing using glioma specimens with
   high or low miR-7156-3p expression (Figure [79]3A) and conducted
   pathway enrichment and gene set enrichment analysis (GSEA). As shown in
   figures [80]3B and [81]3C, miR-7156-3p is closely involved in the
   regulation of stem cell-related signaling pathways. This result was
   further confirmed by the detection of stem cell marker proteins.
   Western blotting analysis showed that overexpression of miR-7156-3p
   significantly suppressed while silencing of miR-7156-3p increased the
   expression of stem cell marker proteins in SW1783 cells (Figure
   [82]3D). Overall these findings suggest that miR-7156-3p negatively
   regulates glioma cell stemness.

Figure 3.

   [83]Figure 3
   [84]Open in a new tab

   miR-7156-3p negatively regulates glioma stemness. A. Heat map of
   differentially expressed genes between miR-7156-3p high-expressing
   glioma specimens (n=5) and miR-7156-3p low-expressing glioma specimens
   (n=5). B-C. Pathway enrichment and GSEA analysis was performed using
   the mRNA sequencing data. D. miR-7156-3p negatively regulated glioma
   cell stemness. The indicated cells were transfected with the indicated
   plasmids. After 72 hours of transfection, cancer stem cell biomarker
   expression was detected by Western blotting.

HOXD13 is a target of miR-7156-3p in glioma

   miRNAs play an important role in the regulation of gene expression by
   inhibiting target gene translation. Thus, to identify the target genes
   of miR-7156-3p in glioma, we performed next-generation sequencing using
   miR-7156-3p-overexpressing U373 glioma cells and their control cells.
   As shown in figure [85]4A, we detected a total of 33 genes that were
   significantly downregulated in miR-7156-3p-overexpressing cells
   compared to the control. By applying these genes to miRNA target
   prediction database searches (TargetScan.org and miRDB.org), it
   revealed that the 3'-UTRs of HOXD13 and PERP sequences contained
   potential miR-7156-3p binding site (Figures [86]4A and [87]4B). Next,
   we used the TCGA dataset to investigate the correlations between
   miR-7156-3p expression levels and HOXD13 or PERP expression levels in
   glioma. TCGA dataset analysis showed that the miR-7156-3p expression
   level was negatively correlated with the HOXD13 expression level in
   glioma but not with the PERP expression level (Figure [88]4C),
   suggesting that HOXD13 may be a major target of miR-7156-3p in glioma.
   To determine whether miR-7156-3p is directly involved in the regulation
   of HOXD13 expression, miR-7156-3p mimics or inhibitors were transfected
   into SW1783 cells (Figure [89]2A) and the HOXD13 expression levels were
   measured. Our data showed that overexpression of miR-7156-3p
   significantly inhibited HOXD13 expression, while inhibition of
   miR-7156-3p increased HOXD13 expression in glioma cells both at the
   mRNA (Figure [90]4D) and protein levels (Figure [91]4E). Furthermore,
   we investigated the direct interaction between miR-7156-3p and the
   3'-UTR of HOXD13 using a luciferase reporter assay. The luciferase
   assay results showed that miR-7156-3p significantly inhibited
   luciferase activity in glioma cells transfected with the wild-type
   3'-UTR of HOXD13 but not in these transfected with the mutant 3'-UTR of
   HOXD13 (Figure [92]4F). Taken together, our findings indicate that
   HOXD13 is a target of miR-7156-3p in glioma and that miR-7156-3p
   suppresses HOXD13 expression by directly binding to its 3'-UTR.

Figure 4.

   [93]Figure 4
   [94]Open in a new tab

   HOXD13 is a target gene of miR-7156-3p in glioma. A. Candidate targets
   of miR-7156-3p in glioma cells. mRNA sequencing identified 33 genes
   (heat map) that were significantly downregulated in
   miR-7156-3p-overexpressing U373 cells relative to control cells. Then,
   through a search of the miRNA databases TargetScan and miRDB, two
   candidate target genes of miR-7156-3p were determined. B. Predicted
   binding sites of miR-7156-3p in the 3`-UTRs of PERP and HOXD13.
   Mutations in the 3`-UTR of HOXD13 are highlighted in red. C.
   Correlation between miR-7156-3p expression level and HOXD13 and PERP
   expression levels in glioma. Data from the TCGA database. D. HOXD13
   mRNA expression levels were measured in the indicated glioma cells.
   Cells were transfected with mimics or inhibitor of miR-7156-3p. After
   72 hours of transfection, cells were subjected to qRT-PCR analysis. E.
   miR-7156-3p inhibited HOXD13 protein expression in SW1783 cells. Cells
   were transfected with mimics or inhibitor of miR-7156-3p. After 72
   hours of transfection, the cells were subjected to Western blotting
   analysis. F. Luciferase activity of the reporter driven by the
   wild-type or mutant 3`-UTR of HOXD13 in U373 cells cotransfected with
   control oligonucleotides (NC) or miR-7156-3p mimics. A luciferase
   reporter containing the wild-type or mutant 3`-UTR of HOXD13 was
   constructed and transfected into U373 cells with or without miR-7156-3p
   mimics. After 48 hours of transfection, luciferase intensity was
   assessed. ***P < 0.001; ns, not significant.

HOXD13 stimulates glioma stemness and progression

   The role of HOXD13 in glioma is an uncharted territory currently.
   Therefore, we investigated the function of HOXD13 in glioma. The
   results of Kaplan-Meier analysis using TCGA dataset showed that a high
   expression level of HOXD13 was closely associated with a lower survival
   rate in glioma patients (Figure [95]5A). Moreover, the mRNA expression
   level of HOXD13 was closely correlated with glioma grade (Figure
   [96]5B). In accord with this result, our clinical cohort also showed
   that HOXD13 expression was positively correlated with glioma grade
   (Figure [97]5C). To further investigate the role of HOXD13 in glioma,
   we performed mRNA sequencing using glioma specimens with high or low
   HOXD13 expression (Figure [98]5D). Pathway enrichment analysis showed
   that HOXD13 was involved in the regulation of stem cell-related
   signaling pathways (Figure [99]5E). To further validate this finding,
   western blotting was performed showing that HOXD13 overexpression
   significantly increased, while silencing of HOXD13 decreased the
   expression of stem cell marker proteins in glioma cells (Figure
   [100]5F). Similarly, CCK-8 and invasion assays showed that HOXD13
   positively regulated glioma cell growth (Figure [101]5G) and invasion
   (Figure [102]5H). These findings suggest that HOXD13 plays an oncogenic
   role in glioma and it may be a potential therapeutic target.

Figure 5.

   [103]Figure 5
   [104]Open in a new tab

   HOXD13 positively regulates glioma stemness and progression. A.
   Kaplan-Meier analysis of the overall survival rate of patients with
   glioma with low and high HOXD13 expression. Data were obtained from the
   TCGA database. B. TCGA dataset analysis showed that the expression
   level of HOXD13 positively correlated with glioma grade. Grade II,
   n=249; grade III, n=261; grade IV, n=174. C. Our cohort samples
   analysis showed that the HOXD13 expression level is correlated with
   glioma grade. The expression level of HOXD13 was measured by IHC. D.
   Heat map of differentially expressed genes between HOXD13
   high-expressing glioma specimens (n=5) and HOXD13 low-expressing glioma
   specimens (n=5). E. Pathway enrichment analysis was performed using the
   mRNA sequencing results (data from Figure [105]5D). F. HOXD13
   positively regulated glioma cell stemness. The indicated cells were
   transfected with the indicated plasmids. After 72 hours of
   transfection, cancer stem cell biomarker expression was detected. G.
   HOXD13 positively regulates glioma cell growth. The indicated glioma
   cells were transfected with the indicated plasmid, and cell viability
   was measured at the indicated times. H. HOXD13 positively regulates
   glioma cell invasion. The indicated glioma cells were transfected with
   the indicated plasmids. After 48 hours of transfection, the cells were
   subjected to an invasion assay.

miR-7156-3p plays an anti-tumor role by mediating HOXD13 in glioma

   Next, we investigated whether HOXD13 is involved in the anticancer role
   of miR-7156-3p in glioma. Our data showed that overexpression of
   miR-7156-3p induced anti-tumor effects that were significantly
   attenuated by HOXD13 overexpression in glioma cells, including tumor
   cell stemness (Figure [106]6A), invasion (Figure [107]6B), growth
   (Figure [108]6C), and apoptosis (Figure [109]6D). These findings
   demonstrate that miR-7156-3p plays an anti-tumor role in glioma by
   mediating HOXD13 expression.

Figure 6.

   [110]Figure 6
   [111]Open in a new tab

   miR-7156-3p anticancer effects in glioma cells are attenuated by HOXD13
   overexpression. A. Stemness inhibition by miR-7156-3p was attenuated by
   HOXD13 overexpression in glioma cells. The indicated cells were
   transfected with the indicated oligonucleotides and/or plasmids. After
   72 hours, the cells were subjected to Western blotting. B. Invasion
   inhibition by miR-7156-3p was attenuated by HOXD13 overexpression in
   glioma cells. The indicated cells were transfected with the indicated
   oligonucleotides and/or plasmids. After 48 hours, the cells were
   subjected to an invasion assay. C. Growth inhibition by miR-7156-3p was
   attenuated by HOXD13 overexpression in glioma cells. The indicated
   cells were transfected with the indicated oligonucleotides and/or
   plasmids. Cell viability was measured using a CCK-8 kit. D. Apoptosis
   stimulation of miR-7156-3p was attenuated by HOXD13 overexpression in
   glioma cells. The indicated cells were transfected with the indicated
   oligonucleotides and/or plasmids. After 48 hours of transfection, cells
   were subjected to flow cytometry analysis.

Overexpression of miR-7156-3p significantly inhibits glioma progression in
vivo

   Finally, we established orthotopic xenograft models to confirm the
   effect of miR-7156-3p on glioma progression in vivo. Orthotopic glioma
   animal models were generated using U373 cells expressing miR-7156-3p or
   antisense miR-7156-3p. MRI scanning and HE staining showed that
   inhibition of miR-7156-3p significantly stimulated glioma growth, while
   overexpression of miR-7156-3p dramatically inhibited glioma growth
   (Figure [112]7A). IHC results of the cell proliferation marker protein
   Ki-67 showed that inhibition of miR-7156-3p enhanced glioma cell
   proliferation, but not in tumor tissues with overexpression of
   miR-7156-3p (Figure [113]7B). Additionally, we evaluated the tumor
   invasion depth from the border of the tumor mass to the invaded cells.
   The depth of tumor invasion was remarkably increased by miR-7156-3p
   inhibition but decreased when miR-7156-3p was overexpressed (Figure
   [114]7C). Moreover, histologic analysis showed that levels of HOXD13
   and CD133 reduced when miR-7156-3p levels were augmented but increased
   when miR-7156-3p was inhibited (Figure [115]7D). Notably, compared with
   the control group, inhibition of miR-7156-3p shortened the survival
   time of glioma xenograft model mouse, while overexpression of
   miR-7156-3p significantly prolonged the survival time (Figure [116]7E).
   Collectively, these findings indicate that the decreased levels of
   miR-7156-3p contribute to glioma progression in vivo and that
   overexpression of miR-7156-3p may be a promising strategy for
   anti-glioma treatment.

Figure 7.

   [117]Figure 7
   [118]Open in a new tab

   miR-7156-3p inhibits glioma progression in an animal model. A.
   miR-7156-3p negatively regulates glioma growth in an animal model.
   Tumor growth was detected using MRI. First, MR images at start time;
   Last, MR images after one month of tumor implantation. B.
   Ki-67-positive cells were detected by IHC in glioma tissues from animal
   models. C. Invasiveness was assessed using the distance between the
   tumor mass edge and the invasive lesion. D. HOXD13 expression level and
   CD133-positive glioma cells were detected using IHC in glioma tissues
   from animal models. E. miR-7156-3p positively correlated with survival
   time in xenograft models (n=6 per group).

Discussion

   Accumulating studies show that aberrant expression of miRNAs is
   associated with most cancer types, including glioma [119]^10^,
   [120]^27. Here, we first report that decreased expression levels of
   miR-7156-3p in glioma are closely associated with poor clinical outcome
   in glioma patients. Our in vitro and in vivo experiments demonstrate
   that inhibition of miR-7156-3p enhances glioma stemness, growth and
   invasion. Importantly, overexpression of miR-7156-3p significantly
   inhibits glioma stemness, growth, invasiveness and induce glioma cell
   apoptosis. Our findings also indicate that the level of miR-7156-3p is
   associated with glioma grade and patient survival. In this regard,
   miR-7156-3p may have much potential to serve as a prognostic candidate
   biomarker. Additionally, overexpression of miR-7156-3p may be a useful
   strategy for anti-glioma therapy.

   We next investigated the functional mechanism of miR-7156-3p in glioma.
   MiRNAs play a pivotal role by inhibiting target gene expression. In
   this work, we identify and validate that HOXD13 is the predominant
   target of miR-7156-3p in glioma by using a series of experiments. Our
   transcriptome analysis, qRT-PCR, and Western blotting analysis show
   that miR-7156-3p negatively regulates HOXD13 expression at both the
   mRNA and protein levels in glioma cells. Similarly, clinical data from
   the TCGA database shows that the expression levels of miR-7156-3p and
   HOXD13 are negatively correlated in glioma. Furthermore, the luciferase
   assay shows that miR-7156-3p inhibits HOXD13 expression by directly
   binding to the 3-UTR of HOXD13. Moreover, functional experiments
   demonstrate that the miR-7156-3p-induced anti-glioma effects are
   significantly attenuated by overexpression of HOXD13. These results
   indicate that miR-7156-3p plays its anti-tumor role through HOXD13
   inhibition.

   Upregulated expression of HOXD13 in glioma is previously identified,
   but the function of HOXD13 in glioma remains unclear. Clinical data
   analysis from the TCGA database showed that a high level of HOXD13 was
   closely associated with glioma grade and a low survival rate in glioma
   patients. Our experiments suggest that this association may be a
   functional result of HOXD13 in stimulating glioma cell stemness,
   invasion, and growth. These data demonstrate that HOXD13 plays an
   oncogenic role in glioma, but its specific mechanism in glioma needs
   further study.

   In conclusion, our study determines that miR-7156-3p acts as a tumor
   suppressor in glioma by targeting HOXD13. Moreover, a decreased
   expression level of miR-7156-3p is a prognostic marker candidate in
   glioma patients, and miR-7156-3p may be a promising therapeutic target
   by inhibiting tumor cell stemness for anti-glioma treatment.

Acknowledgments