miR-370 Sensitizes TMZ Response Dependent of MGMT Status in Primary Central Nervous System Lymphoma.

Primary central nervous system lymphoma (PCNSL) is an aggressive and rare subtype of non-Hodgkin lymphoma, arising exclusively in the CNS with a poor prognosis. Previous evidence has proved that MGMT was a promising target involving in TMZ resistance of PCNSL. Our study described a new miR-370-mediated mechanism of MGMT regulation in PCNSL. We first showed that miR-370 was downregulated in PCNSL tissues, while MGMT was inversely overexpressed. It was also observed that miR-370 suppressed the expression of MGMT. Additionally, upregulation of miR-370 significantly increased TMZ sensitivity dependent of MGMT, thus suppressed Raji cell proliferation and induced apoptosis in vitro. In conclusion, these results suggest that miR-370 is a potential target in PCNSL treatment.


Introduction
Primary central nervous system lymphomas (PCNSLs) are rare non-Hodgkin lymphomas only confined to the central nervous system, such as brain, eyes, leptomeninges, or spinal cord, which account for 2 to 3% of all primary intracranial tumours [1,2]. Diffuse large B cell lymphoma (DLBCL) is one of the most common types of the PCNSL. Age has been demonstrated as a significant prognostic factor in PCNSL patients [3]. PCNSL is much more sensitive to initial aggressive radiotherapy and chemotherapy, including high-dose based methotrexate, followed by whole-brain irradiation [4][5][6], compared to the other malignant CNS tumors. But the best therapeutic approach remains uncertain, due to the trouble of drug delivery across the blood-brain barrier [7]. In addition, the neurotoxicity, delayed diagnosis and treatment lead to final relapse of PCNSL. Thus the overall survival for PCNSL remains poor for most patients [8].
Temozolomide with good CNS penetration and low toxicity is an oral alkylating agent that affects O6methylguanine-DNA-methyltransferase (MGMT) status, which are also proved to be effective against non-Hodgkin's lymphoma [9,10]. Recently the usage of temozolomide as salvage therapy has been reported to treat the patients with refractory or relapsed PCNSL [11][12][13]. Temozolomide is also a good choice for the elderly patients with impaired renal function or other comorbidities, and can't receive HD-MTX. As MGMT gene promoter methylation and high expression are promising predictors in TMZ-treated glioblastoma patients, we further determined the MGMT status involving in the PCNSL patients.
MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally downregulate gene expression via binding to target mRNA [14,15], thus can function as tumour suppressors or oncogenes through downstream extensive network of signaling pathway [16]. Recent years, miRNAs have gained increasing attention from researchers due to their crucial functions in many cellular affairs, and also the potential to serve as clinical targets and markers. Though evidences have revealed the dysregulation of miRNAs in PCNSL, the understanding of functional roles of these abnormally expressed miRNAs are still limited.
In the present study, we sought to explore the association between miR-370 and MGMT, provide evidence that miR-370 regulates MGMT expression in PCNSL cells, further to increase the TMZ sensitivity.

Cell Culture, Plasmid Construction and Transfection
The Raji Burkitt's lymphoma cell line was purchased from Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China, and cultured in RPMI 1640 (Sigma) containing 10% fetal bovine serum. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. The pEGFP MGMT overexpression plasmid, MGMT siRNA, the scramble, miR-370 mimics and miR-370 inhibitors were constructed by GeneChem Co., Ltd. (Suzhou, China).
Raji cells were first seeded in 24-well plates. 24 h later, MGMT overexpression plasmid, MGMT siRNA, the scramble, miR-370 mimics or miR-370 inhibitors in 50 μL of medium was respectively mixed with 2 μL of Lipofectamine 2000 (Invitrogen, USA) dissolved in 50 μL of RPMI and then placed at room temperature for 20 min. The mixed 100-μL solutions were then added to each well containing 400 μL of medium. And cells were harvested for the next step.

Human PCNSL Samples
Twenty human PCNSL samples and twenty controls (cervical lymph nodes tissues) were obtained from the Department of Neurosurgery, Sir Run Run Shaw Hospital, Medical College, Zhejiang University, China. Informed consents were obtained from patients who had received a diagnosis of PCNSLs. Samples were gained by resection or biopsy, and immediately frozen in liquid nitrogen for subsequent total RNA extraction.

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated from, human PCNSL specimens, nodal tissues or raji cells with TRIzol reagent (Invitrogen).
The process quantitative of real-time polymerase chain reaction (qRT-PCR) was performed according to the manufacturer's instructions [17].

Immunohistochemistry and Western Blotting
Immunohistochemistry and Western blot were performed as previously described [15].

Flow Cytometry Analysis
The transfected Raji cells were incubated for 12, 24 and 48 h, then the cells were collected. After being washed with prechilled phosphate buffered saline (PBS) twice, the Raji cells were stained with FITC-labeled Annexin V and propidium iodide. FC-50 flow cytometry (Beckman Coulter) was used to assess the cell apoptosis.

Statistical Analysis
Statistics were performed using the SPSS version 11.0. Data were expressed as the mean ± standard deviation. Student's t test or analysis of variance was used to evaluate the comparisons between groups. All differences were considered to be statistically significant at the level of P < 0.05.

miR-370 and MGMT Expression in PCNSL Samples
To determine the expression of MGMT in PCNSL tissues, the immunostainings for MGMT were examined in eight PCNSL cases. It was shown in cell nuclei with an immunoreactivity of high intensity in all cases (Fig. 1a). Total RNAs were further extracted from twenty PCNSL and twenty nodal samples. The expression levels of miR-370 were analyzed by qRT-PCR. As shown in Fig. 1b, the levels of miR-370 expression in PCNSL tissues were significantly decreased, compared with nodal tissues. Taken together, our results revealed that miR-370 and MGMT were inversely expressed in human PCNSL samples.

miR-370 Regulates MGMT Expression
We next explored the correlation between miR-370 and MGMT in vitro. Using bioinformatic methods, we found that MGMT was a potential target of miR-370. As shown in Fig. 2, miR-370 inhibitors or mimics were transfected into raji cells, and 48 h later the transfected cells were harvested. Results revealed that miR-370 inhibitors suppressed both expression of MGMT mRNA and protein, while miR-370 mimics upregulated MGMT expression. Together, these results provide evidence that miR-370 regulates MGMT expression at both mRNA and post-transcriptional level.

miR-370 Affects PCNSL Cell Proliferation and Apoptosis Via MGMT
To confirm the modulation of miR-370 on TMZ sensitivity, we divided six groups in the experiment, including Control, TMZ, MGMT+TMZ, MGMT siRNA+TMZ, miR-370 mimics+TMZ and miR-370 inhibitor+TMZ groups. As shown in Figs. 3 and 4, TMZ obviously suppressed raji cell proliferation ability and induced cell death. Further we found that MGMT siRNA transfection increased the TMZ sensitivity to raji cells, while MGMT caused TMZ resistance. The similar results were also confirmed in miR-370 mimics+TMZ and miR-370 inhibitor+TMZ groups. Furthermore, the protein expression of apoptosis-related molecules (P53 and Bax) was consistent with the previous study (Fig. 5). In conclusion, TMZ sensitivity consistently correlated with the

Discussion
Though a number of evidences have supported that high-dose methotrexate (MTX)-based chemotherapy is the first-line therapy for patients with primary CNS lymphoma (PCNSL), the poor outcome still can't be improved for the majority of patients due to the reason of delayed neurotoxicity and renal dysfunction [18]. Because of well-toleration and good penetration of blood brain barrier, temozolomide has recently emerged as an important choice for PCNSL patients in elderly or salvage therapy after failure to high-dose MTX [19][20][21], or in combination with rituximab and methotrexate [10,22,23]. However current TMZ therapy is significantly impeded by TMZ resistance, in which MGMT was activated to repair DNA changes caused by these agents in PCNSL cells.
Dysregulation of miR-370 has been described in tumorigenesis and progression by functioning as a tumor   [24]. It was additionally demonstrated that expression of miR-370 was decreased in endometrioid ovarian cancer cells, and miR-370 suppressed proliferation and promotes endometrioid ovarian cancer chemosensitivity to cDDP by negatively regulating ENG [25]. miR-370 was also revealed to function as tumor suppressor gene in the gastric carcinoma and acute myeloid leukemia [26,27]. Researchers have shown that miRNAs were abnormally expressed in PCNSL, many of which were implicated in various molecular functions, involving in the network of signaling, such as Myc pathway, terminal B cell differentiation, and inflammatory cytokines, et al [28][29][30][31][32], but little was referred to drug resistance of PCNSL. In the glioblastoma multiforme, it was found that miRNA-370-3p affected the sensitivity of GBM cells to TMZ via MGMT expression, which was not identified in the PCNSL [33]. In the present work, our results have revealed that miR-370 was significantly down-regulated in PCNSL samples compared with the nodal tissues. Furthermore, MTT assay and apoptosis assay supported our hypothesis that ectopic overexpression of miR-370 enhanced the cytotoxic effect of TMZ on raji cells, caused cell proliferation suppression and induced apoptosis. Furthermore, miR-370-overexpressing cells exhibited an increased expression of apoptotic markers upon TMZ treatment, such as P53 and Bax. As MGMT participated in the repair of DNA, miR-370 may induce MGMT downregulation, and render the raji cells unable to repair DNA damage, finally lead to cell death. Collectively, our data suggested that miR-370 plays a vital role in TMZ resistance of raji cells via MGMT.
In conclusion, miR-370 was detected for the first time to be downregulated in PCNSL tissues, significantly correlated with the TMZ resistance and functioned as a tumor suppressor in raji cells depending on MGMT status. However, animal models, larger human PCNSL samples, and other studies, are required to validate the results from the present study.

Compliance with Ethical Standards
This study was approved by the ethics committee of Sir Run Run Shaw Hospital, Medical College, Zhejiang University (Hangzhou, China). All the participants have signed informed consent containing a detailed description of the purpose of this study. All specimens were handled and made anonymous to meet the ethical and legal standards.

Conflict of Interest
The authors declare no conflict of interest. Open Access This article is distributed under the terms of the Creative Comm ons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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