MicroRNA Expression Deregulated by Aberrant Methylation in B-ALL Mouse Model

The expression of microRNAs (miRNAs) in the serum of B-cell acute lymphoblastic leukemia (B-ALL) patients is abnormal. However, the mechanism is not clear. Recent studies have shown that the methylation state of circulating cell-free DNA (cfDNA) is different between tumor patients and normal people. Therefore, we speculate that abnormal expression of miRNA may be associated with methylation in cfDNA. The aim of this study was to establish a uorescently labeled B-ALL transplantation animal model, so as to explore the relationship between the serum miRNA expression and the related gene of cfDNA methylation. The expression levels of miRNAs were detected with quantitative real-time PCR (qRT-PCR). The cfDNA methylation levels of related gene were tested by bisulte sequencing polymerase chain reaction (BSP). The result showed that the expression levels of miR-196b, miR-203, miR-34a-5p, miR-335-3p, miR-34b-5p, miR-615, miR-375-3p and miR-193b-5p in the serum of the model mice were signicantly lower than those of the control (P < 0.05). The methylation level of miR-196b promoter in cfDNA of model group was signicantly lower than that of the control group (P < 0.05), and there was no signicant difference in miR-203 promoter. The methylation levels of miR-196b and miR-203 coding region in cfDNA of model group were signicantly higher than those of the control (P < 0.05). The results indicate that CpG island hypermethyation in the miRNA coding region of cfDNA is related to the low expression of miR-196b and miR-203.


Introduction
B-ALL is a hematological malignant disease result from the abnormal proliferation of primitive B lymphocytes in the bone marrow, which is the most commonly diagnosed childhood malignancy [1]. The patients with ALL are often accompanied by the occurrence of extramedullary in ltration, which is the main cause of the recurrence. MiRNA is a kind of non-coding RNA composed of 18-25 nucleotide fragments, which regulates gene expression patterns by targeting speci c mRNAs. They are epigenetic regulators of a variety of cell signaling pathways [2]. Data showed the expression of miRNAs in the serum of B-ALL patients are abnormal [3]. Most of miRNA genes are found in the intergenic region, called intergenic miRNA, and some of them are found in the exon and intron regions, called intragenic miRNA [4,5]. It is generally believed that miRNA genes are co-transcribed with their host genes. Some miRNA genes have independent transcriptional promoters [6,7]. DNA methylation is a kind of reversible epigenetic change, which is closely related to the progress of malignant tumor diseases. Hypermethylation of DNA causes gene expression to be down-regulated. DNA methylation usually occurs at the cytosine bases of CpG dinucleotides [8], short sequences of 5-10 CpG dinucleotides per 100 bp locate at gene promoters and regulatory regions [9]. It has been observed that most of CpG islands located near miRNA genes are involved in their transcriptional regulation [10]. These suggest that the miRNAs differences in B-ALL may be caused by different methylation status of the CpG island near their gene.
cfDNA is mainly derived from necrotic or apoptotic cells and the active release of intracellular DNA [11].
The content of cfDNA in serum of healthy people is about 1-10ng/mL [12], while was increased in patients with in ammatory diseases or tumor [13]. Recent study has shown that the methylation state of cfDNA is different between tumor patients and healthy people [14]. Therefore, we speculate that methylated cfDNA that affect miRNA expression may be found in the serum of B-ALL patients.
In this study, we established a uorescent labeled B-ALL transplantation animal model with the uorescent labeled cell lines. Meanwhile, we evaluated the expression levels of some miRNAs in serum, and identi ed the methyltion levels of the promoter and the coding region of two miRNA miR-203 and miR-196b in serum cfDNA.

Materials And Methods
Cell Culture and transfection According to the manufacturer's instruction, the miRNA was isolated from L1210 cell lines and serum of mice by miRcute miRNA Isolation Kit (Tiangen, China). The cDNA was synthesized by Prime Script RT Reagent Kit (TaKaRa, Japan). The qRT-PCR was conducted with the SYBR Premix Ex Taq Kit (TaKaRa, Japan). The primers used are given in Table 1. U6 was treated as internal control to quantify the expression levels of miRNAs by 2 −ΔΔct method. Table 1 The sequences of miRNA primers and BSP primers used in this study.

Primer
Sequence ( [15]. The modi ed DNA was subjected to PCR ampli cation. The primers are shown in Table 1 and Fig. 1. The puri ed PCR products were cloned into pTG19-T Vector (TaKaRa, Japan). The individual bacterial colonies were sequenced at least 20 colonies from each sample. The nal sequence results were processed by BiQ Analyzer.

Statistical analysis
Data are expressed as mean ± standard deviation (± SD). The expression levels of miRNA and DNA methylation among each group were tested by t-test using SPSS version 16.0 software. Differences were considered to be statistically signi cant at P < 0.05.

Characteristics of uorescently labeled B-ALL mouse model
After transfected with lentivirus and screening, uniform green light of L1210-GFP cells was observed with the uorescence microscope and ow cytometry (Fig. 2a, 2b). Thirty days after transplantation, ow cytometry results showed that the green uorescence intensity of peripheral blood mononuclear cells in the model group was higher than that of the control group (Fig. 2c). After dissection, splenomegaly could be seen in the model group (Fig. 2d). The weight of spleen in the model group (0.19 ± 0.03g) was signi cantly higher than that of the control group (0.14 ± 0.03g) (P < 0.05). There was no signi cant difference in the weight of heart, kidney and liver (Fig. 2e). The leukemic cell in ltration was observed under uorescence microscope in the model group (Fig. 2f).

Discussion
In this study, we modi ed L1210 cells with a lentivirus with uorescent protein gene, which express green uorescent protein stably (L1210-GFP). After 30 days of transplantation, the uorescence intensity of to the cell culture medium to combine with the glutathione in the cell to stain the cytoplasm red, which also has the characteristic of long-term non-decolorization. Jalalie et al. used this method to trace the distribution of therapeutic mesenchymal stem cells in ovarian tissue [19]. Compared with common immunohistochemistry, the model labeled with uorescence is more convenient to observe leukemia cells in ltration in various organs. Our results provide a good animal model for monitoring leukemia extramedullary in ltration.
We evaluated the expression of 9 miRNAs in the serum of this model. Among them, the expression of miR-196b, miR-203, miR-34a-5p, miR-335-3p, miR-34b-5p, miR-615, miR-375-3p and miR-193b-5p were signi cantly different between the model group and the control group (P < 0.05), while the expression of miR-320-5p was no different. This result suggests that most miRNAs were changed in the serum of B-ALL. The present studies revealed that aberrant miR-196b expression appears in multiple hematological malignancies [21,22]. Bhatia et al. showed a signi cant down-regulation of miR-196b accompanied by overexpression of the c-myc gene in peripheral blood mononuclear cells derived from B-ALL patients. It is proposed that miR-196b can down-regulate the expression of c-myc and play a role in inhibiting [23]. However, the factors affecting miR-196b expression in B-ALL are not clear. We observed the enrichment of CpG sites in the region near the miR-196b gene by UCSC (http://genome-asia.ucsc.edu) and MethylPrimer (http://www.urogene.org/methprimer2/). BSP results showed that in serum cfDNA of mouse model, the methylation level of miR-196b promoter region was signi cantly lower than that of the control (P < 0.05), while the methylation of miR-196b coding region was signi cantly higher than that of the control (P < 0.05), which is inconsistent with the result of Liu et al.. They showed that the methylation level of the miR-196b promoter region in the bone marrow of chronic lymphocytic leukemia (CLL) patients was higher than that of healthy control [24]. Tsai et al. also found that miR-196b expression was decreased in gastric cancer cells due to hypermethylation of its promoter [25]. The difference is the specimen, we used serum cfDNA as the analysis object, rather than cellular genomic DNA. The fragment of serum cfDNA is not complete, but it is easy to extract and more convenient for monitoring in B-ALL treatment. In addition, Popovic  reduced DNA methylation at CpG islands in the promoter regions of miR-196b in MLL gene-rearranged cases compared to the cases of non-MLL precursor B-cell acute lymphoblastic leukemia and normal bone marrow [27]. These observations suggest that the expression of miR-196b may be MLL-driven in B-ALL, which deserves further studies.
The recent study showed that a positive-feedback loop was found between miR-203 and its target DNA methyltransferases 3b (Dnmt3b) in breast cancer cells. Dnmt3b is a key enzyme in methylation, as restoring miR-203 suppressed Dnmt3b, while knocking down Dnmt3b up-regulated miR-203 [28]. Chim et al. observed that miR-203 was silent by its promoter hypermethylated in a variety of lymphoma cell lines [29]. Our results showed that the miR-203 coding region appears hypermethylated in the serum cfDNA of B-ALL model, whereas the methylation level of the miR-203 promoter region was not signi cantly different from the control. It may be for the Methyl CpG-binding protein 2 (MeCP2) is prone to bind to the miRNA coding region in mammalian cells, thereby affecting miRNA expression [30]. However, it is still controversial whether the effect of this response on miRNA is increased or decreased.
In conclusion, this study, for the rst time, clari ed the relationship between the serum cfDNA and miRNA and veri ed our previous hypothesis. What's more, we observed that the methylated region associated with the expression of miR-196b and miR-203 was coding region rather than promoter in serum cfDNA of B-ALL mouse model.

Conclusion
This study focused on the relationship between the abnormal miRNA expression and the serum cfDNA methylation in uorescent labeling B-ALL model. Most miRNAs were different between the model group and the control group. Out of these, miR-196b and miR-203 down-regulation are associated with the hypermethylation of related genes coding region in serum cfDNA. Simultaneous evaluation of serum cfDNA methylation level and miRNA will contribute to monitor leukemia better. Figure 1 The  Different methylation levels of miR-196b, miR-203 promoter and coding region a The methylation level of miR-196b promoter in cfDNA of model group was signi cantly lower then the control (P < 0.05). b There was no signi cant difference between the miR-203 promoter in cfDNA of model group and the control group. c The methylation level of miR-196b coding region in cfDNA of model group was signi cantly higher then that in the control (P < 0.05). d The methylation level of miR-203 coding region in cfDNA of model group was signi cantly higher then the control (P < 0.05). Bisul te genomic sequencing was depicted. Unmethylated (empty circle) and methylated ( lled circle) CpG dinucleotides were shown. *P < 0.05 vs control group.