MiR-526b-3p Inhibits the Resistance of Glioma Cells to Adriamycin by Targeting MAPRE1

Background Cell resistance is the main reason for the high mortality in glioma. Adriamycin (ADR) is a treatment drug for glioma and often leads to chemoresistance. Previous studies have confirmed that the abnormal expression of microRNA (miRNA) affects the resistance of glioma cells. Methods RT-qPCR and western blot were conducted for detecting miR-526b-3p levels and related protein expressions. CCK8 assay, colony formation, flow cytometry, and Transwell were adopted to assess cell viability, proliferation, apoptosis, and metastasis. Moreover, downstream targets of miR-526b-3p were identified through a dual-luciferase reporter and RNA pull-down analysis. Results Nevertheless, miR-526b-3p functions on glioma cell resistance to ADR are not well characterized. This study demonstrated that miR-526b-3p levels were decreased within glioma cells and further decreased within ADR-resistant glioma cells. Then, miR-526b-3p overexpression repressed glioma cell proliferation and invasion while inducing cell apoptosis. Overexpression of miR-526b-3p within ADR-resistant glioma cells obtained similar results, which suggested miR-526b-3p suppressed glioma cell resistance to ADR. Mechanistically, miR-526b-3p targeted MAPKE1 and negatively regulated MAPKE1 expressions. Restoration of MAPKE1 levels reversed miR-526b-3p effects on the glioma process and resistance to ADR. Conclusion These results suggest that miR-526b-3p acts as a diagnostic marker in glioma development and therapeutic target of the glioma resistance to ADR.


Introduction
Glioma is an intracranial tumor that accounts for more than half of intracranial tumors and causes a high mortality rate [1]. At present, the treatment of glioma is mainly surgery, radiotherapy, chemotherapy, or combination therapy [2]. With the development of these treatment strategies, glioma treatment has been improved. However, the survival rate is still unsatisfactory [3]. Resistance of the glioma cells to chemotherapy is the primary reason for the poor prognosis [4]. Hence, exploring the target to overcome the resistance of the glioma is necessary. Adriamycin (ADR), also called doxorubicin (Dox), is the antibiotic used in tumor therapy, including glioma [5]. e treatment effect of ADR is weakened because of the resistance in glioma. However, the mechanism of ADR resistance in glioma is still unclear. erefore, we explore the molecular mechanism of the glioma resistance to ADR and expect to find new targets and pathways to decrease the ADR resistance of glioma.
MicroRNAs (miRNAs) exert vital impacts on various physiological and pathological processes through modulating mRNA degradation or posttranscriptional levels of targeted RNAs [6]. Nowadays, growing research has shown that miRNAs' abnormal expression is involved in the resistance of malignancies [7,8]. For example, miR-204 overexpression inhibits hepatocellular cancer's drug resistance and metastasis [9]. As a tumor suppressor miRNA, MiR-181b has less expression in human glioma and enhances chemosensitivity of TMZ [10]. e recovery or knockout of multiple miRNAs has also been shown to reduce or improve chemoresistance in glioma. MiR-195 levels are decreased within TMZ-resistant glioma cells, and miR-195 upregulation reverses TMZ resistance through regulating cyclin E1 expression [11]. Compared with parental cells, the expression of miR-125b is diminished in TRAILresistant glioma cells and increased miR-125b expression resensitizes glioma cells to TRAIL [12]. MiR-127 downregulation significantly increased glioma cell sensitivity to ADR [13]. Nevertheless, the potential role of miRNA in glioma ADR resistance was still largely unknown. erefore, finding key miRNAs for glioma ADR resistance was of great value to glioma treatment and prognosis.
MiR-526b-3p is considered a tumor suppressor gene involved in multiple biological processes of malignant tumors, including colorectal cancer and cervical cancer [14][15][16]. In glioma, miR-526b-3p has been reported as a prognosis factor and regulates the tumor process through WEE1 [17]. However, the functions of miR-526b-3p in resistance have not been elucidated.
is study investigated the roles and molecular mechanisms of miR-526b-3p within glioma resistance to ADR. Our findings showed that miR-526b-3p levels were decreased within glioma and ADR-resistant glioma samples. Furthermore, miR-526b-3p upregulation suppressed cell proliferation and cell metastasis, enhancing cell apoptosis. Meanwhile, miR-526b-3p overexpression reduced glioma cell resistance to ADR. Further study confirmed that miR-526b-3p increased the glioma cells' sensitivity to ADR by regulating the expression of MAPRE.

Tissues Sample.
All samples in this study were obtained from the Wujin People's Hospital. Glioma tissues and the adjacent normal tissues came from patients that clinically confirmed glioma and accepted the surgical treatment. e patients were treated with ADR and divided into the ADR resistance and nonresistance groups. e tissues were removed and frozen in −80°C refrigerator immediately. All patients who participated in the study were informed to sign the consent. e study was approved by the ethics committee of Wujin People's Hospital and conducted under its supervision.

Cell
Culture. Glioma cells T98G, U87, SHG44, and U251 and nonneoplastic glioma cell line H4 were purchased from ATCC (USA). T98G cells were cultured within MEM medium (Minimum Eagle's medium). U87 and U251 cells were cultured within MEM-EBSS medium. SHG44 and H4 cells were, respectively, cultured in the RPMI-1640 and DMEM-H medium. All media were purchased from the ermo-Scientific. ese media were added to 10% FBS (Gibico, NY, USA), and all cells were incubated within the humid incubator under 37°C and 5% CO 2 conditions. e ADRresistant cells (U87/ADR and U251/ADR) were obtained by adding an increasing concentration of ADR into the medium. e ADR concentration from 10 nM to 2 μM. e ADR-resistant cell line was successfully constructed when the cells tolerated 2 μM concentration of ADR, and the proliferation ability was similar to the wild-type cells. e culture medium of U87/ADR and U251/ADR cells was added to ADR (2 μM/L) to maintain the ADR-resistant property.

Cell Transfection.
Lentivirus vectors expressing MAPRE1, negative empty vector, and NC mimics/miR-526b-3p mimics were constructed from GeneChem (Shanghai, China). According to the instruction, each of the previously mentioned plasmids was transfected into cells for 48 h via Lipofectamine 6000 ( ermo Fisher Scientific, MA, USA). (RT-qPCR). Clinical tissues and cells were used to extract total RNA by utilizing Trizol reagent (Invitrogen, MA, USA), which was further reverse-transcribed to cDNA using Pri-meScript RT reagent Kit (Takara, Japan). e reaction procedure of RT-qPCR complied with the instruction. In addition, U6 or β-actin was normalized by the 2 −ΔΔCT approach. e RT-qPCR primers are displayed in Table 1.

Cell Viability.
CCK8 kit was used to analyze the cell viability. Briefly, transfected U87 and U251 cells were cultured in a 10% FBS medium until the logarithmic growth phase. Next, transfected U87/ADR and U251/ADR cells were cultured in a medium that was separately containing 0, 1, 2, 4, 8, and 16 μM ADR for 48 h. en, cells with a density of 10 3 cells/100 μL were inoculated in 96-well plates. CCK-8 kit (Sigma, USA) was utilized. Absorbance (OD) (450 nm) was detected using a spectrophotometer (Molecular Devices, San Jose, USA).

Colony Formation Assay.
e colony formation assay was performed to assess the proliferative potential of transfected U87 and U251 cells. Briefly, cells were cultured within 6-well plates (500 cells), and the medium (Procell Life Science & Technology Co., Ltd.) was replaced every two to three days for a total of two weeks. Subsequently, cells were stained with 0.5% crystal violet for 15 min for 1 h at room temperature. Colonies (>50 cells) were observed and counted under a light microscope (Nikon, Japan, ×40).

Flow Cytometry.
Cells from different groups were collected and washed twice with prechilled PBS and resuspended in a binding buffer. en, the cells were stained with 5 μL of Annexin V-FITC, and 10 μL of PI was utilized to treat cells for 15 min in the dark according to the protocol. A flow cytometer (BD Biosciences, USA) was utilized for determining apoptotic cells.

Transwell
Assay. Transwell migration and invasion assays (Corning, Inc.) were performed to assess transfected U87 and U251 cells' migratory and invasive abilities. Cells were cultured into the upper compartment with the basal medium in the migration assay. Lower chambers were supplied with 600 μL medium containing 10% FBS. After 48 h, and cells were immobilized with methanol and stained with 0.1% crystal violet and observed under a microscope (Leica, Germany) and counted. In invasion assay, the used membrane was precoated with the Matrigel (Franklin Lakes, NJ, USA). e other methods were like cell migration. e migratory and invasive cells were counted under an IX70 inverted optical microscope (magnification, x100; Olympus Corporation).
en, DNaseI was added to RNA solution, and incubation was for 5 min at 65°C. Afterwards, streptavidin-coated magnetic beads (New England BioLabs, USA) were incubated for another 4 h. Finally, the beads were washed with PBS and extracted RNA. MiR-526b-3p enrichment was detected via RT-qPCR analysis [19].

Statistical Analysis.
e average ± standard deviation (SD) represented data from three repetitions, further analyzing using GraphPad Prism 5.0. Differences between groups were compared by ANOVA and Tukey's post hoc analysis. P < 0.05 was considered statistically significant.
ese results suggested that miR-526b-3p participated in glioma development and ADR resistance.

MAPRE1 Is a Downstream Target Gene of miR-526b-3p.
MiRNAs mainly exert their regulatory abilities by regulating the expression of targeted RNA. To elucidate the molecular mechanisms of miR-526b-3p in glioma, the target genes of miR-526b-3p were predicted using the ENCORI, miRwalk, and TargetScan, and RT-qPCR detected these gene expressions. As shown in Figure 4(a), only MAPRE1 was dramatically elevated in glioma cells compared with other genes. e potential binding site of MAPRE1 and miR-526b-3p was displayed in Figure 4 NC mimic miR-526b-3p mimic NC mimic miR-526b-3p mimic   showed that miR-526b-3p upregulation reduced the luciferase activity in MAPRE1 WT-transfected cells. However, the luciferase activity in MAPRE1 Mut-transfected cells had no significant change (Figure 4(c)). Pull-down assay results also displayed that MAPRE1 bound with miR-526b-3p WT rather than miR-526b-3p Mut (Figure 4(d)). en, we detected MAPRE1 expression at the RNA and protein levels in miR-526b-3p overexpression cells.
e results showed that MAPRE1 levels were decreased within miR-526b-3p overexpressed cells compared with control cells (Figure 4(e)).
Further, MAPRE1 levels within glioma tissues and ADRresistant glioma tissues were analyzed. Based on Figures 4(f) and 4(g), MAPRE1 levels were increased in glioma tissues and ADR-resistant glioma tissues. e expression correlation analysis showed that MAPRE1 expression was negatively regulated by miR-526b-3p (Figure 4(h)). Consistently, increased expression of MAPRE1 in glioma cells was confirmed (Figure 4(i)). ese findings demonstrated that miR-526b-3p targeted binding to MAPRE1 and negatively regulated MAPRE1 expression in glioma.   Journal of Oncology

MiR-526b-3p Inhibits the Process and ADR Resistance of Glioma through MAPRE1.
To further verify the miR-526b-3p regulate glioma process through MAPRE1, we transfected the MAPRE1 vector in U87 and U251 cells. RT-qPCR detected the transfection efficiency, and the results are shown in Figure 5(a). MiR-526b-3p mimics and MAPRE1 vector were cotransfected in U87 and U251 cells. Cell viability assay and clone formation assay results suggested that the overexpression of MAPRE1 retarded the inhibition of cell proliferation induced by miR-526b-3p mimics (Figures 5(b) and 5(c)). Similarly, the increased cell apoptosis ratio induced by miR-526b-3p has attenuated in MAPRE1 overexpressed cells ( Figure 5(d)). Transwell assay results also suggested that MAPRE1 rescued the inhibition effects of miR-526b-3p on cell migration and invasion ( Figure 5(e)). ese results clarified that miR-526b-3p regulated the glioma process through MAPRE1.
To verify the miR-526b-3p regulating glioma resistance to ADR through MAPRE1, MAPRE1 expression in ADR-resistant cells was detected first. e results showed that MAPRE1 expression was increased in ADR-resistant glioma cells compared with the normal glioma cells ( Figure 5(f )). en, we transfected the MAPRE1 vector separately or transfected miR-526b-3p mimics and MAPRE1 vector simultaneously in U87/ADR and U251/ ADR cells. e transfection efficiency of the MAPRE1 vector is shown in Figure 5(g). e cotransfected cells were cultured with different concentrations of ADR. After 48 h, the cell proliferation inhibition ratio was assessed by CCK8. e results showed that MAPRE1 overexpression restored the cell inhibition ratio induced by miR-526b-3p upregulation ( Figure 5(h)). In line with the CCK8 assay results, flow cytometry results showed that MAPRE1 retarded the increase of apoptosis rate induced by miR-526b-3p within ADR-resistant glioma cells ( Figure 5(i)). Transwell assay results showed that MAPRE1 rescued the migration and invasion roles of miR-526b-3p in ADRresistant glioma cells (Figure 5(j)). ese findings demonstrated that miR-526b-3p inhibited the glioma resistance to ADR by downregulating the expression of MAPRE1.

Discussion
Resistance is the primary clinical obstacle to successfully treating cancer, including glioma [20]. e causes of tumor resistance are very complex. e abnormal expression of miRNAs has been reported to correlate with tumor resistance, such as exosomes secreted by tumor-associated M2 macrophages transferred miR-21 to the gastric cells. High levels of miR-21 promote the cisplatin resistance of gastric cancer [21]. In lung cancer, miR-146a has been proven not to affect the growth of the tumor but to alter chemotherapy sensitivity by regulating the CHOP expression [22]. Many reports confirmed that miRNAs expression dysregulation was involved in the process of glioma resistance. For instance, miR-130a-3p expression decreased in glioblastoma cell lines, and the target regulates the expression of Sp1. Increased levels of miR-130-3p inhibit the proliferation and TMZ resistance of glioblastoma cells [23]. Li et al. found that miR-186 inhibits the glioblastoma resistance to cisplatin by downregulating the expression of YY1. MiR-186 inhibits glioblastoma-initiating cell (GIC) phenotype formation in U87MG cells [24]. MiR-526b-3p has been reported to regulate multiple tumor progression [17,25]. For instance, upregulation of miR-526b-3p inhibits cell viability, migration, and tube formation of glioma-associated endothelial cells [26].
Nevertheless, miR-526b-3p roles in tumor resistance have not been reported. So, the functions of miR-526b-3p in glioma progress and ADR resistance were investigated. As expected, miR-526b-3p levels were decreased in glioma tissues and cells. Meanwhile, in ADR-resistant glioma tissues and cells, miR-526b-3p levels were also reduced. Upregulating miR-526b-3p levels repressed the proliferation, metastasis, and ADR resistance while promoting apoptosis in   glioma cells. Further, we explored the regulatory signal pathway of miR-526b-3p in glioma. We found that miR-526b-3p targeted binding to MAPRE1 and negatively regulated the expression of MAPRE1.
As an RP/EB family member, MAPRE1 encoded microtubule-associated protein. It has been reported that MAPRE1 is involved in mitochondrial membrane degradation [27]. Moreover, MAPRE1 works as a diagnosis and prognosis biomarker in cancer [28]. High levels of MAPRE1 are correlated with tumor malignancy, high histological grade, and poor clinical outcome [29]. Besides, Kim et al. found that MAPRE1 promotes cell apoptosis via reactive oxygen species, and Bax mediates mitochondrial dysfunction in NSCLC [30]. Wang et al. found that MAPKE1 overexpresses in ESCC and promotes cellular growth by affecting suppressor gene APC function and activating the beta-catenin/TGF pathway [31]. However, the relationship between MAPKE1 and tumor resistance remained unknown. In this study, MAPKE1 expression was increased in glioma tissues and cells. We also proved that MAPKE1 levels were increased in ADR-resistant glioma tissues and cells. Overexpression of MAPKE1 restored the miR-526b-3p-induced change in the glioma process and ADR resistance.
However, the present study had some limitations. For example, fluorescence in situ hybridization (FISH) could better detect the distribution and binding relationship between miR-526b-3p and MAPRE1. Furthermore, further in vivo experiments confirmed that miR-526b-3p increased the sensitivity of glioma cells to ADRs in vivo, which could increase the scientific relevance of this study.
In summary, this study proved that miR-526b-3p exerted a vital role in glioma development and ADR resistance. Furthermore, overexpression miR-526b-3p inhibited tumor procession and increased the sensitivity of glioma to ADR through downregulating the expression of MAPRE1.
ese results suggested that miR-526b-3p functioned as the diagnostic marker in glioma development and the potential treatment target of the glioma resistance to ADR. At the same time, these results can provide a new treatment strategy and experimental basis for glioma resistance to ADR.

Data Availability
All data generated or analyzed during this study are included in this published article. (g) e expression of MAPRE1 in U87/ADR and U251/ADR cells that transfected the MAPRE1 vector or control vector. U87/ADR and U251/ADR cells transfected miR-526b-3p mimics and NC vector or miR-526b-3p mimics and MAPRE1 vector simultaneously. Cell inhibition ratio (h), cell apoptosis ratio (i), cell migration, and cell invasion (j) were detected in transfected ADR-resistant cells. * * * <0.001, * * P < 0.01, * P < 0.05 vs the NC mimics and control vector cotransfected cells. ### P < 0.001, ## P < 0.01 P, # P < 0.05 vs. the miR-526b-3p mimics and control vector cotransfected cells. Error bars were represented the mean ± SD of triplicate experiments.

Consent
All patients who participated in the study were informed to sign the consent.