miR-3059-3p Regulates Glioblastoma Multiforme Radiosensitivity Enhancement through the Homologous Recombination Pathway of DNA Repair

Background Glioblastoma multiforme (GBM) is one of the most deadly and recalcitrant illnesses of the neurocentral nervous system in humans. MicroRNAs (miRNAs) are a class of noncoding RNAs that play important roles in the regulation of gene expression and biological processes, including radiosensitivity. In this study, we demonstrated the relationship between miR-3059-3p and radiation in GBM. Materials and Methods Radioresistant (RR) cells were obtained by exposing GBM8401 cells to 80 Gy radiation in 20 weekly 4 Gy fractions. miR-3059-3p mRNA and DNA replication helicase/nuclease 2 (DNA2) protein expressions were detected using real-time polymerase chain reaction and immunoblotting. Using flow cytometry, colony formation and apoptosis were identified using miR-3059-3p mimic, miR-3059-3p inhibitor, DNA2 siRNA, and DNA2 plasmid. Immunoblotting was used to detect DNA repair proteins. Results Low levels of miR-3059-3p and high levels of DNA2 were observed in RR cells. Colony formation and apoptosis assays revealed that miR-3059-3p targeted DNA2 to regulate radioresistance. Immunoblotting revealed that miR-3059-3p regulated the homologous recombination (HR) pathway (Rad51 and Rad52) but not the nonhomologous end joining pathway (ku70 and ku80). Conclusion Downregulation of DNA2 via miR-3059-3p enhanced the radiosensitivity of GBM cells through the inhibition of the HR pathway.


Introduction
Glioblastoma multiforme (GBM) in humans is one of the most deadly and recalcitrant illnesses of the neurocentral nervous system. Approximately 12,120 patients in the United States alone were diagnosed with GBM in 2016 with a 5-year survival rate of 5%, and the peak age-adjusted inci-dence of GBM is estimated to be 3.2 per 100,000 [1]. The exact etiology of the disease is currently unknown, and only limited well-established research has indicated radiation as the cause [2]. Clinical results of GBM may present some obvious symptoms, including persistent weakness, numbness, loss of vision, or changes in language based on the neurological function. As the tumor size increases, symptoms such as headache, nausea, vomiting, and even loss of consciousness also appear. Magnetic resonance imaging is the standard radiographic imaging modality in the diagnosis and posttreatment management of patients with glioblastoma [3]. Current treatment approaches include surgical resection with radiotherapy (RT) as well as concomitant and maintenance chemotherapy, such as temozolomide [4]. The overall survival is still dismal, and the average survival time is <2 years [5,6]. Despite advancements in neurosurgery and RT, the development of potent chemotherapeutic drugs, and comprehensive genomic profiling and molecular diagnostics over the last several decades, there has been little improvement in increasing the overall survival rate [7].
Radioresistance (RR) is responsible for the poor therapeutic effect of RT on GBM tumors. GBM cells exhibit increased proliferation and insufficient vascularization, which induces local hypoxia in tumor sites [8,9]. Moreover, hypoxia is well known to play an important role in RR. Additionally, fractionated RT, epithelial-mesenchymal transition, and cancer stem cells can induce RR [10,11]. Therefore, inhibiting RR can improve the therapeutic effect of RT on GBM tumors.
MicroRNAs (miRNAs) are noncoding RNAs that play an important role in regulating mRNA expressions. The average length of a miRNA molecule is 22 nucleotides. They are transcribed from DNA sequences into primary miRNAs (pri-miRNAs) and processed into precursor miRNAs (pre-miRNAs), which then mature into miRNAs. miRNAs have been shown to regulate gene expressions following binding to the 3 ′ -untranslated region of target mRNAs to induce mRNA degradation or translational repression [12][13][14]. Recent studies have shown that miRNAs can regulate RR by targeting mRNAs to mediate many biological processes, including proliferation, cell cycle, aging, apoptosis, and DNA repair [15][16][17][18][19]. Several studies have shown that microRNAs can regulate the therapeutic effect of radiation. For example, miR-409-3p mediated radiosensitivity in non-small-cell lung cancer [20]. miR-31 induced RR by regulating reactive oxygen species in pancreatic ductal adenocarcinoma [21]. In addition, the tumor environment is associated with radioresistance. In colorectal cancer, exosomal miR-590-3p from cancerassociated fibroblasts regulated radioresistance [22]. At present, miR-3059-3p has been shown to regulate stress-induced depression and resilience [23]. However, there are no reports on the relationship between miR-3059-3p and radiation. This study is therefore aimed at investigating the relationship between miR-3059-3p and radiosensitivity and the underlying mechanisms.

Materials and Methods
2.1. Cell Culture. GBM8401cells were obtained from the Bioresource Collection and Research Center and cultured in RPMI medium supplemented with 10% fetal bovine serum under 5% CO 2 atmosphere at 37°C. The cells were exposed to 80 Gy radiation in 20 weekly 4 Gy fractions to yield RR cells.

Colony
Formation. GBM cells were seeded into 6-well plates at a density of 100, 200, 400, 1000, and 10,000 cells per well and exposed to radiation doses of 0, 1, 2, 4, and 8 Gy, respectively. A linear accelerator was used to irradiate cells, which was performed at room temperature. The cells were stained with 0.5% crystal violet after a 10-day incubation. The number of colonies formed was normalized to plating efficiency (PE) and represented as a surviving fraction (SF) relative to the control. The PE and SF were calculated as follows: PE = ðnumber of colonies formed/number of inoculated cellsÞ × 100%; SF = number of colonies formed/ðnumber of seeded cells × ½PE/100Þ.

MicroRNA Polymerase Chain Reaction (PCR).
Micro-RNAs were extracted and purified using the miRNeasy Mini kit (Qiagen, Hilden, Germany). miRNA expression levels were measured via quantitative reverse transcription-(qRT-) PCR using StepOne (Thermo, Waltham, USA). The cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s, and 60°C for 60 s. The relative miR-3059-3p expression level was calculated using the 2 −ΔΔCt method. U6 was used as an internal control.

2
Journal of Oncology 2.6. Flow Cytometry. A total of 1:5 × 10 5 GBM8401 cells were seeded into 6-well plates and incubated for 24 h followed by transfection with microRNA mimic, microRNA inhibitor, DNA2 siRNA, or DNA2 plasmid for 48 h and exposed to radiation. Both detached and attached cells were centrifuged at 1500 rpm for 5 min. Cells were washed once with phosphate-buffered saline and analyzed using the Muse® Annexin V and Dead Cell Assay Kit (Millipore, MCH100105, Burlington, USA).

Data
Analysis. The SPSS 24.0 (IBM, NY, USA) software was used for statistical analysis. A one-way analysis of variance followed by Tukey's post hoc test was used to analyze the results of colony formation, apoptosis percentage, and western blot. For all analyses, a P value of < 0.05 was considered statistically significant.

miR-3059-3p
Attenuated the HR Pathway to Reduce DNA Repair via Targeting DNA2. Both HR and nonhomologous end-joining (NHEJ) are the main pathways in doublestrand break (DSB) repair. RAD51 and RAD52 play key roles in HR pathway-mediated DNA repair. RAD51 was mediated on ssDNA in a form that is active for homologous pairing and strand invasion in humans. RAD51 also regulates dsDNA and prevents dissociation from ssDNA. RAD52 plays another crucial role in the repair of DNA DSBs at the active transcription sites during the G0/G1 phase of the cell cycle. Repair of these DSBs appears to use an RNA template-based recombination mechanism dependent on RAD52. In the NHEJ pathway, the KU70/80 heterodimer plays a vital role as it binds to DNA termini with high affinity, thereby protecting DNA ends from degradation, and recruits other NHEJ factors required for repair [25]. We used immunoblotting to determine which DNA repair pathway DNA2 could take. The results revealed that the protein expressions of Ku80 and Ku70 were similar in each group, but in the miR-3059-3p mimic and miR-3059-3p mimic + DNA2 siRNA groups, the protein expressions of both RAD52 and RAD51 were low. miR-3059-3p mimic + DNA2 plasmid, miR-3059-3p inhibitor + DNA2 siRNA, and miR-3059-3p inhibitor + DNA2 plasmid groups had high Rad52 and Rad51 protein expressions (Figure 7(a)). We found no significant difference in Ku80 and Ku70  Figure 4: The target of miR-3059-3p. (a) The binding relation between miR-3059-3p and DNA2 was predicted using miRDB, an online database for miRNA target prediction and functional annotations. (b) In immunoblotting, the RR group showed higher DNA2 expression. * * * P < 0:001 than the control group.  (Figures 7(b) and 7(c)). Moreover, in the miR-3059-3p mimic and miR-3059-3p mimic + DNA2 siRNA groups, the intensities of RAD52 and RAD51 were significantly lesser than those in other groups (Figures 7(d) and 7(e)) after radiation exposure. Our findings confirmed the association of miR-3059-3p with RAD52 and RAD51 and that miR-3059-3p increased radiosensitivity by targeting the DNA2 protein to affect the HR pathway in postradiation DNA repair.

Discussion
GBM in humans is still the most common primary malignant tumor of the central nervous system. Despite standard treatment including maximal surgical resection and RT with concomitant chemotherapy being well-established, the median progression-free and overall survival after the initial diagnosis is 6.2-7.5 and 14.6-20.5 months, respectively [26]. The main reason for these failures is the development of resistance to standard treatment regimens for GBM, including RR. Most studies over the years have elucidated the mechanisms of RR of GBM cells, and RR in these cells has been attributed to several mechanisms, including cell cycle, tumor microenvironment, hypoxia, apoptosis, cancer stem cells, microRNAs, and DNA damage and repair. In this study, RR cells exhibited downregulation of miR-3059-3p (Figure 1(a)) and upregulation of DNA2 (Figure 4(b)).
RT often results in DSB in cells. DNA damage response would induce RR in cancer cells. GBM cells develop RR via various DNA repair pathways, such as base excision repair, mismatch repair, nucleotide excision repair, homologous recombination repair, and NHEJ in glioma cells [27][28][29]. Inhibition of these pathways attenuated the RR cells and subsequent RT efficiency. Specific miRNAs can modulate proteins in the NHEJ pathway in gliomas. Blocking NHEJrelated proteins (KU70/KU80) was able to increase gene targeting efficiency [30].
The RAD51/RAD52 complex plays a key role in the HR pathway. Many studies have shown that inhibition of the HR pathway significantly enhances radiosensitivity in cancer cells. Chandler et al. showed that inhibition of Tatassociated T-cell-derived kinase-induced radiosensitivity       Journal of Oncology through the HR pathway, not via the NHEJ pathway, in breast cancer [31]. Tang et al. showed that both ATM and EGFR inhibitors promote radiosensitivity through the HR pathway, not via the NHEJ pathway, in lung adenocarcinoma, cervical cancer, GBM, and colorectal carcinoma [32]. Our results revealed that inhibition of the HR pathway, not the NHEJ pathway, via miR-3059-3p enhances the therapeutic effects of radiation.
In this study, we found a relationship between targeting DNA2 protein and RAD51/RAD52 complex and that the DNA2 protein was attenuated via miRNA-3059-3p. DNA2 protein, which was first identified in yeast, plays an important role in DNA replication because of helicase and nuclease activities in the nucleus and mitochondria [33,34]. DNA2 plays an important role in cell cycle, telomere maintenance, and DNA replication and repair [35]. Increased CHK1 expression has been shown to induce double-strand breaks (DSBs) through phosphorylation of DNA2 [36]. Gupta et al. showed that CHK1 inhibitor hypersensitizes osteosarcoma cells to radiation [37]. In our study, silencing DNA2 through miR-3059-3p targeting increased the percentage of apoptotic cells by inhibiting RAD51/RAD52 expression with radiation in GBM cells.

Conclusion
Currently, GBM remains a highly lethal cancer, despite several research efforts and clinical trials with agents designed to improve treatment outcomes. RR is among the reasons of treatment failure and tumor recurrence. Radiosensitizers have been considered and remain a viable option for improving the prognosis in patients with GBM. In our study, we focused on miR-3059-3p to target DNA2 and observed downregulated DNA2 expression. DNA2 plays an essential role in regulating the HR pathway and initiating DNA repair. Our data suggest that downregulation of DNA2 via miR-3059-3p could attenuate the HR pathway and decrease the possibility of DNA repair. Therefore, we believe that miR-3059-3p is an effective radiosensitizer candidate, which can inhibit GBM recurrence after RT.

Data Availability
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Conflicts of Interest
The authors declare that they have no conflicts of interests.  Journal of Oncology