miR-186 Regulates the Initiation and Development of Breast Cancer via SHP-1 Methylation

Background Change in the methylation status of genomic DNA, especially in CpG islands in the promoter region, is considered to be an early event in tumor initiation, leading to silencing of gene expression, subsequent abnormalities in gene structure and function, and malignant transformation of the cell. Due to the abnormal expression of miR-186 and SHP-1 in breast cancer tissues and cells, we propose that miR-186 is closely related to the methylation of SHP-1 Method Using 5-azacytidine as a de-methylation agent and Validating with Setylation-specic polymerase chain reaction (MSP) after treatment. Measurement of the viability of breast cancer cells using the CCK-8 method Measurement of the apoptotic rate of breast cancer cells using annexin V-FITC/PI double labeling. Cell metastasis were measured by wound healing assay. Luciferase reporter assays was used to conrm the target of MiR-186. SHP-1 and miR-186 expression was measured by RT-PCR and western blot. Results In the present study, we found that SHP-1 expression was reduced to various degrees in all 5 cell lines (UACC-812, MDA-MB-213, MDA-MB-468, SK-RB-3 and MCF-7). 5-azacytidine can remove the methylation from the SHP-1 promoter region. Apoptosis was observed in MCF-7 cells after demethylation of the SHP-1 gene promoter region by 5-azacytidine, and the effect was time- and concentration-dependent. Luciferase reporter assays showed that miR-186 promotes methylation through binding with the 3’ UTR of the SHP-1 promoter region.Western blot showed miR-186 regulates the initiation and development of tumor cells through the SHP-1-JAK-STAT axis. In animal models, low expression of miR-186 can cause signicantly limited tumor growth. Conclusion important and that miR-186 excellent effects of SHP-1 on tumors have been conrmed, the effects of SHP-1 on breast cancer cells remain unclear, and the regulatory mechanisms of its expression have not been completely elucidated. This study used the breast cancer cell line MCF-7 to assess the methylation status of the SHP1 gene in breast cancer; performed demethylating treatment on MCF-7 cells and observed the effects of the SHP-1 gene in tumor cells on the gene and protein expression of SHP-1 in MCF-7 cells, as well as on cell proliferation and apoptosis; discussed the causal factors of SHP-1 gene methylation and the association of SHP-1 with miR-186 and the roles of these 2 factors in the initiation and development of breast cancer; and provided epigenetic information for breast cancer diagnosis.

We detected elevated miR-186 expression in breast cancer tissues, and elevated miR-186 expression was also reported in esophageal squamous cell carcinoma 4 and pancreatic cancer 5 . This type of abnormal miRNA expression is frequently concurrent with the progression and malignant transformation of tumors.
There is abundant evidence indicating that cancer-related miRNAs can serve as reliable diagnostic markers as well as potential therapeutic targets. It has been noted in the literature that miRNAs can inhibit the degradation or translation of target mRNA by recognizing and binding to complimentary sites on the 3'-untranslated region (3'-UTR). 6 Therefore, we speculate that miR-186 may regulate the initiation and development of breast cancer cells through its effects on SHP-1 methylation.
5-Azacytidine is a cell cycle-speci c agent with anti-metabolic and anti-tumor effects. Due to its ability to associate with DNA and RNA, it interferes with ribonucleic acid metabolism and subsequently causes a decrease in protein synthesis. 5-Azacytidine is also a DNA methyltransferase inhibitor and an epigenetic modi er that activates multiple tumor suppressor genes by inhibiting DNA methylation of the promoters of these genes and induces tumor cell apoptosis. We utilized this function of 5-azacytidine to explore the relationship between miR-186 and SHP-1.
Although the inhibitory effects of SHP-1 on tumors have been con rmed, the effects of SHP-1 on breast cancer cells remain unclear, and the regulatory mechanisms of its expression have not been completely elucidated. This study used the breast cancer cell line MCF-7 to assess the methylation status of the SHP-   denaturation (40 cycles), 94 C for 30 s, 55 C for 40 s, and 72 C for 1 min; and annealing, 72 C for 5 min.
The products were analyzed via 20 g/L agarose gel electrophoresis, and the results were visualized with an ultraviolet light gel imaging system.

Preparation and storage of 5-azacytidine
The instructions for preparation were as follows: 1 mg of 5-azacytidine was dissolved in 1 ml of 50% acetic acid solution (acetic acid:water, 1:1) for a stock solution of 4100 µmol/L; the stock solution was diluted to the desired concentration for immediate use or stored at -20 C for future use.
6. Demethylation treatment MCF-7 cells were cultured in high-glucose DMEM containing 100 mL/L fetal bovine serum. The cells were cultured at an initial density of 5 10 4 /mL in tissue culture asks, and 4-6 h later, the demethylating agent 5-azacytidine was added to the medium, with a nal concentration of 5 µmol/L. Fresh medium and demethylating agent were replaced every 24 h. Cells cultured in the absence of the demethylating agent were used as a control.

Methylation-speci c polymerase chain reaction (MSP) after treatment
Cells were cultured in medium containing 5 µmol/L 5-azacytidine (demethylating agent) as described above, and fresh medium and demethylating agent was provided every 24 h. After 3 d of culture, methylation modi cation and DNA isolation were performed as described above, and PCR was carried out as previously described.

Analysis of the mRNA expression of SHP-1 in MCF-7 breast cancer cells via RT-PCR
Total RNA from MCF-7 cells was isolated using Trizol, and cDNA synthesis was performed using random primers and MMLV reverse transcriptase according to the product manual. The primers for SHP-1 were as follows: forward, GACTGTGACATTGACATCCAG; reverse, CTTCCTCTTGAGGGAACCCTT; the product length was 350 bp. The primers for β-actin were as follows: forward, 5'-CACTGTGTTGGCGTACAGGT-3'; reverse, 5'-TCATCACCATTGGCAATGAG-3'; the product length was 154 bp. The ampli cation conditions were as follows: initial denaturation, 95 C for 5 min; 35 cycles of 94 C for 45 s, 60 C for 45 s, and 72 C for 1 min; and extension, 72 C for 10 min. β-actin was used as the endogenous reference gene. The products were analyzed via 20 g/L agarose gel electrophoresis, and the results were visualized using an ultraviolet light gel imaging system.

Measurement of the viability of breast cancer cells using the CCK-8 method
The stock solution of 5-azacytide was diluted to 0.1, 1, 5, 10 and 20 μmol/L. Plates (96-well) were labeled as blank (equal volume of medium without cells), control and experimental, and each group contained 6 replicates. MCF-7 breast cancer cells in the log growth phase were collected, digested and seeded in 96well plates at 1 10 4 cells per well in 200 μl of medium, with 5 plates total. MCF-7 cell suspension (50 μl) was seeded into each well of the plates for the control and experimental groups; the plates were shaken gently, placed in a 37 C incubator with 5% CO 2 and incubated for 4h. Then, the medium was removed, and 200 μl of medium containing 0.1, 1, 5, 10, or 20 μmol/L 5-azacytidine was added to each experimental well; 200 μl of medium was added to each blank and control well. After shaking the plates gently, they were placed in a 37 C incubator with 5% CO 2 , and fresh medium with the same concentration of 5-azacytidine was provided every 24 h. One plate was removed every 24 h, and 10 μl of CCK-8 solution was added into each well. After an additional incubation of 2.5 h in the CO 2 incubator, the plate was placed in an enzyme-linked immunosorbent assay (ELISA) analyzer, and the A values (OD) at 450 nm were measured. Cell viability was calculated as follows: viability (%) = (experimental A value -blank A value)/(negative control A value -blank A value) 100%. Each experiment was repeated 3 times.

Measurement of the apoptotic rate of breast cancer cells using annexin V-FITC/PI double labeling
Annexin V-FITC is a calcium-dependent phospholipid-binding protein conjugated with uorescein. It has a very strong a nity for phosphatidylserine, to which it speci cally binds while the cell maintains cell membrane integrity. Therefore, it was used as a more sensitive tool to detect early apoptotic cells and calculate the percentage of apoptotic cells. MCF-7 breast cancer cells in the log growth phase were digested with 2.5 g/L trypsin for passage, and the medium was replaced with high-glucose DMEM containing 5-azacytidine at 1, 5, or 10 μmol/L after 24 h. Cells were provided with fresh medium containing 5-azacytidine (at the same concentrations) every 24 h, and after 3 d of treatment, the cells were cultured in fresh medium without 5-azacytidine for 24 h before measurement. Cells incubated in the same volume of complete media without 5-azacytidine were used as controls. MCF-7 cells treated with 5 μmol/L 5-azacytidine for 1, 3 or 5 d were analyzed in the same manner, with cells incubated in the same volume of complete medium without 5-azacytidine as controls. Each experiment was repeated 3 times.

Statistical analysis
The data were analyzed using SPSS 11.0 software, and all measurement data are presented as the mean standard deviation (±s). One-way ANOVA was performed, and the least signi cant difference (LSD) test was used for pairwise comparisons. P<0.05 was considered to be statistically signi cant.  Table 2).

miR-186 promotes methylation through binding with the 3' UTR of the SHP-1 promoter region
The hypermethylation of the SHP-1 promoter region in breast cancer cells appears to be a key causal factor of decreased gene expression and the initiation and development of tumors. What is the cause of the decreased expression? There are indications in the literature that miR-186 is closely associated with SHP-1. miR-186 is a microRNA that can target SHP-1 and destabilize the target mRNA or inhibit its translation. First, we used RT-PCR to assess the difference in miR-186 expression in breast cancer tissues and adjacent normal tissues (Figure 3 A, p < 0.05), and the results suggested that the elevated expression of miR-186 is closely associated with tumor initiation. Subsequently, to con rm the interaction between miR-186 and the SHP-1 gene, we designed small interfering RNA (simiR-186) to knock down miR-186 and 2 breast cancer cell lines harboring SHP-1 mutations (Figure 3 C). First, 2 types of control miR-ctrl and simiR-186 were transfected into MCF-7 breast cancer cells after serum starvation, and Western blot analysis was conducted to con rm the function of simiR-186 in the 3 groups of cells (Figure 3 B). Then, the interaction between miRNA-186 and SHP-1 was explored through a luciferase reporter assay (Figure 3 D). The 3 cell lines, wildtype, SHP-1 mut-1 and SHP-1 mut-2, were each divided into 2 groups that were transfected with miR-ctrl and miR-186, respectively, and the changes in SHP-1 gene activity was assessed by measuring the correlating luciferase activity. We found that the binding of miR-186 to the SHP-1 promoter region is key to SHP-1 gene methylation (p < 0.05).

miR-186 regulates the initiation and development of tumor cells through the SHP-1-JAK-STAT axis
As a tumor suppressor gene, the anti-tumor effects of SHP-1 are weakened by the methylation of its promoter, and this process is closely related with miR-186. Therefore, to further investigate the effects of miR-186 on breast cancer cells, we knocked down miR-186 using simiR-186 and assessed the effects on the viability of tumor cells through a phenotypic analysis. Figure 4 A-1, 2 shows the results of a wound healing assay. After 24 h, the wound areas in the simiR-186 group were signi cantly larger (p < 0.05). To further investigate whether a similar reduction in migration ability would also occur across a membrane, we employed a transwell cell assay (Figure 4 B-1, 2). The number of cells in the same-sized measurement area was lower in the simiR-186 group (p < 0.05), indicating that the migration ability of breast cancer cells decreased after miR-186 was knocked down.
Which cellular pathways or molecular targets are involved in this phenomenon? The JAK/STAT signal transduction pathway participates in the regulation of biological processes such as cell proliferation, differentiation, embryonic development, and immunity. Preliminary results (Figure 4 C) from this study showed that that SHP-1 gene is closely related to the JAK/STAT cellular signaling pathway and that the elevated expression of this gene inhibited the expression of downstream proteins of this pathway, including CREB-binding protein (CBP), CIS, interferon alpha (IFN-α), proviral integration site for Moloney murine leukemia virus-1 (Pim-1), phosphorylated Janus kinase (p-JAK) and phospho-signal transducer and activator of transcription 3 (p-STAT3), leading to a reduction in the ability of tumor cells to grow, migrate and invade. In addition to breast cancer, the methylation of the SHP-1 gene promoter occurs at a high frequency in B-cell lymphoma, and further study is necessary to elucidate whether this phenomenon is related to the deregulation of the JAK/STAT signal transduction pathway due to the downregulation of SHP-1 gene expression.

Explorative studies in animal models
To verify these results in animal models, we used normal MCF-7 breast cancer cells (control group) and MCF-7 breast cancer cells in which miR-186 was knocked down (treatment group) as tumor sources and injected the cells subcutaneously into the inguinal regions of nude mice (4-6-week-old nude mice, 1*10^7 cells/200 µl/mouse). The results in Figure 5 B reveal that the treatment group exhibited smaller tumor sizes, slower tumor growth, and longer latency before tumor formation. Subsequent Western blot and immunohistochemistry analyses of the expression of SHP-1 in tumor tissues ( Figure A, C) con rmed that the change in tumor size stemmed from the relatively elevated expression of SHP-1.

Discussion
The most basic genetic model of tumor formation is that the overampli cation of proto-oncogenes and the silencing of tumor suppressor genes result in the disruption of the balance of cell proliferation, inducing cell malignancy. Studies have found that inactivation or deletion of tumor suppressor genes will place tumor suppressor genes at a disadvantage in the battle against oncogenes, usually resulting in the overproliferation of cells and subsequent occurrence of malignant tumors. Epigenetic mechanisms are closely related to the initiation and development of tumors. Changes in the methylation status of genomic DNA, especially CpG islands in the promoter region, can lead to gene expression silencing, which results in abnormalities in gene structure and function and subsequent carcinogenic transformation of cells. CpG islands are found in the 5' promoter regions of 60% of human genes. The methylation of these CpG islands regulate the con guration, structure and stability of DNA and alter normal gene expression 7 .
At the end of the 20th century, several research groups, including the group at Washington University, isolated and puri ed the human SHP-1 gene in succession. The SHP-1 protein can speci cally bind to phospho-tyrosine (Tyr-P) and catalyze its dephosphorylation 8  Since we had previously uncovered SHP-1 gene hypermethylation in breast cancer through extensive experimental studies, we have further explored the endogenous cause of this phenomenon -the upstream factor miR-186 -and further con rmed the association between miRNA-186 and SHP-1 through a luciferase reporter assay. miR-186 binds to the 3'UTR of the SHP-1 promoter region, leading to the methylation of SHP-1 and the inhibition of the function of the tumor suppressor gene SHP-1, as well as subsequent tumor initiation and development. Although the elevated expression of miR-186 in breast cancer suggested its association with SHP-1, the expression of miR-186 was suppressed in other tumor types 17 . Recent studies on miR-186 found that it regulates the growth, invasion, cell cycle, and apoptosis of tumor cells; it can also serve as a noninvasive marker for malignant tumors, and it is closely related to patient prognosis. miR-186 provides a new direction to explore tumor treatment.
In summary, miR-186 induces the abnormal methylation status of the SHP-1 gene, which is closely associated with tumor initiation and development, and provides new diagnosis and treatment methods for breast cancer. Currently, investigations into various new targets through increasingly more detailed studies on the mechanisms of action of gene methylation in tumor cells is of great importance.

Conclusion
In summary, in this study, we found that SHP-1 gene has reduced expression in breast cancer cells and breast cancer tissue, and that methylation of the gene is an important cause of low expression. In addition, we have found that miR-186 is highly expressed in breast cancer tissue and is the key to the methylation of SHP-1 gene. Once the methylation of the SHP-1 gene is removed, or the expression of miR-186 is inhibited, the growth and invasion of breast cancer cells is signi cantly inhibited, and animal models are validated. This process is associated with JAK/STAT pathway.

Declarations
Authors' contributions Xia Zhang and Weiguo Zhang conceived of the study. Yongjian Zheng and Lifei Tong contributed the materials used in this study. Kun Wang, Chunxia Zhou,Di Wang and Bin Zhang performed research and analyzed data. Di Wang wrote the paper. All authors read and approved the nal manuscript.

Funding
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Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Ethics approval and consent to participate Not applicable.

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Competing interests
The authors declare that they have no competing interests.