DNMT1 facilitates proliferation and metastasis of breast cancer by inducing MEG3 promoter methylation in MEG3/miR-494-3p/OTUD4 regulatory axis

Background: To explore the mechanism by which DNMT1 potentiates proliferation and metastasis of breast cancer by inducing MEG3 promoter methylation in MEG3/miR-494-3p/OTUD4 regulatory axis. Methods: Human breast cancer cell lines (MCF-7, MDA-MB-231, SKBR3) and human breast epithelial cell line MCF10A were selected for the experiments. The expression levels of DNMT1, MEG3, miR-494-3p and OTUD4 were detected by qRT-PCR. Western blot was used to detect the protein expression levels of DNMT1 and OTUD4. ChIP assay was adopted to verify the binding relationship between DNMT1 and MEG3 promoter region. MethPrimer software was applied to identify MEG3 promoter methylation while methylation-specic PCR was conducted to examine its methylation level. The targeted binding sites of miR-494-3p on MEG3/OTUD4 were predicted by bioinformatics and further veried by RNA binding protein immunoprecipitation assay plus dual-luciferase reporter gene assay. Cell proliferative, migratory and invasive abilities were measured by CCK-8, wound healing and Transwell assays. Results: DNMT1 was highly expressed while MEG3 was poorly expressed in breast cancer cells. Silencing DNMT1 inhibited the proliferation, migration and invasion of breast cancer cells by increasing gene expression of MEG3 through demethylation. MEG3 was identied as a ceRNA that regulated miR-494-3p expression via RNA sponging in breast cancer. In addition, miR-494-3p could bind to the 3’-UTR of OTUD4 mRNA, thus negatively regulating OTUD4 expression. A regulator axis formed by MEG3/miR-494-3p/OTUD4 was thus established and identied to have an impact on proliferation, invasion and migration of breast cancer. Moreover, overexpression of MEG3 could suppress tumor growth of breast cancer in vivo. Conclusion: turn Western blot was used to detect the interference eciency of the shRNAs targeting DNMT1; (F) MEG3 methylation level was tested by MSP (U, unmethylated alleles; M, methylated alleles); (G) The expression of MEG3 was detected by qRT-PCR after DNMT1 was silenced; (H) Interference eciency of the shRNAs targeting MEG3 was detected by qRT-PCR; (I) The expression levels of DNMT1 and MEG3 were detected by qRT-PCR in three groups (sh-NC, sh-DNMT1+sh-NC, sh-DNMT1+sh-MEG3); (J) Cell proliferation, (K) migration and (L) invasion were tested by CCK-8, wound healing and Transwell assays, respectively. Each experiment was carried out in triplicate; * p<0.05. (B) (C) miR-494-3p expression levels in human breast cancer cell lines (MCF-7, MDA-MB-231, SKBR3) and human normal breast epithelial cell line MCF10A were detected by qRT-PCR; The binding relationship of MEG3 with miR-494-3p was veried by (D) RIP and (E) dual-luciferase assays; The expression levels of MEG3 and miR-494-3p in oe-NC group and oe-MEG3 group were detected The and MCF10A were detected (F) (G) dual luciferase assays were performed to verify the targeting binding relationship between miR-494-3p and OTUD4; (H) The expression of OTUD4 in NC inhibitor and miR-494-3p inhibitor groups was detected by western blot; (I) Interference eciency of sh-OTUD4 was tested by western blot; (J) The expression levels of miR-494-3p and OTUD4, (K) the cell proliferation, (L) migration and (M) invasion in NC inhibitor+sh-NC, miR-494-3p inhibitor+sh-NC, and miR-494-3p inhibitor+sh-OTUD4 groups were measured by qRT-PCR, CCK-8, wound healing and Transwell assays, respectively. Each experiment was carried out in triplicate; * p<0.05. and epithelial cell MCF10A were detected by (F) (G) dual luciferase assays were performed to verify the targeting binding relationship between miR-494-3p and OTUD4; (H) The expression of OTUD4 in NC inhibitor and miR-494-3p inhibitor groups was detected by western blot; (I) Interference eciency of sh-OTUD4 was tested by western blot; (J) The expression levels of miR-494-3p and OTUD4, (K) the cell proliferation, (L) migration and (M) invasion in NC inhibitor+sh-NC, miR-494-3p inhibitor+sh-NC, and miR-494-3p inhibitor+sh-OTUD4 groups were measured by qRT-PCR, CCK-8, wound healing and Transwell assays, respectively. Each experiment was carried out in triplicate; * p<0.05.

downregulation of DNMT1 has been reported to inhibit the proliferation and invasion of breast cancer cells [8], and DNMT1 also can downregulate maternally expressed gene 3 (MEG3) expression through increasing methylation level of MEG3 in breast cancer [9]. MEG3 is identi ed as an imprinted gene expressed according to the maternal originand encodes a long non-coding RNA (lncRNA) [10]. As reported, MEG3 is closely related to the progression of breast cancer, and studies have shown that high-expression of MEG3 can Istagnate cell growth and increase apoptosis of breast cancer cells [11]. Meanwhile, highexpression of MEG3 suppresses breast cancer cell proliferation, invasion and angiogenesis through AKT pathway [12].
In addition, many studies show that lncRNAs function as a competiting endogenous RNA (ceRNA) sponging miRNA and regulating the expression of miRNA targets [13,14]. It has been reported that lncRNA MEG3 inhibits cell epithelial-mesenchymal transition (EMT) by targeting miR-421 and regulating Ecadherin in breast cancer [15]. High-expression of miR-494-3p has an promoting effect on breast cancer by targeting and down-regulating TRIM21. Nevertheless, there is no report on MEG3 as a ceRNA regulating the expression of miR-494-3p in breast cancer. In addition, the targets of miR-494-3p were also studied in the present research.
In this study, we investigated the function of DNMT1 during MEG3 methylation process and the effect of MEG3/miR-494-3p/OTUD4 regulatory axis on the proliferation, migration and invasion of breast cancer, which could help develop treatment strategies for breast cancer.

Methods
Bioinformatics analysis RAID database and starBase database were used to obtain downstream regulatory miRNAs of MEG3 and the potential targeted binding sites of lncRNA-miRNA. GSE70905 dataset of breast cancer was obtained from GEO database, including 45 normal samples and 45 tumor samples. Normal samples were taken as control and differential analysis was conducted by using "limma" package in R. P-value was corrected by using FDR method. Differentilly expressed genes (DEGs) were screened with |logFC|>1 and p value<0.05. The downstram target genes of miR-494-3p were predicted by TargetScan, mirDIP and starBase databases. Targeted binding sites of miRNA-mRNA were consultated on the TargetScan database. SKBR3 cell line was cultured in McCoy's 5A Media medium (Modi ed with Tricine). The mediums were all purchsed from Hyclone and contained 10% fetal bovine serum (FBS).

Lentivirus vector construction
MEG3 cDNA was cloned into pcDNA4 vector, while the short hairpin RNAs (shRNAs) targeting DNMT1, MEG3 and OTUD4 were cloned into PLKO.1 vectors. pPAX2 and pVSVG along with target vectors were cotransfected into 293T cells to construct lantivirual vectors. Supernatant was harvested at 24 h and 48 h after transfection and ltered through a 0.45-μm membrane. The viral supernatant was added to medium in a ratio of 1:3 for viral infection. After 24 h, stably transfected cell lines were selected using 2 μg/ml purinomycin. All vectors, mimics and inhibitors were purchased from GenePharma (Shanghai, China). The scramble shRNA and empty pcDNA4 vector were used as negative controls, respectively. Sequences for sh-DNMT1, sh-MEG3 and sh-OTUD4 were detailed in Supplementary material 1. According to the preliminary experiments, the shRNA with a better interference e ciency was selected and the results were presented in the Results section.
Dual-luciferase reporter gene assay The 3'-UTR of MEG3 or OTUD4 was ligated to psiCHECK2 vector that was fused with luciferase gene and had been digested with XhoI and NotI restriction enzymes. The QuikChange multi-site-directed Mutagenesis kit (Stratagene, LaJolla, CA) was used to mutate the targeted sites of miR-494-3p on 3'-UTR. Luciferase activities were determined by dual-luciferase assay (Promega), and Renilla luciferase activity was used for normalization of Fire y luciferase activity.
RNA binding protein immunoprecipitation (RIP) assay RIP was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, Burlington, MA). MDA-MB-231 cells in each group were lysed, and then the cell extracts were cultured with protein magnetic beads and incubated with 2 μg of Ago2 antibody (ab186733, 1:30, Abcam, UK) or control IgG antibody (ab205718, 1:50, Abcam, UK) overnight at 4 ℃. The immunoprecipitated RNA was puri ed and the expression levels of MEG3, miR-494-3p and OTUD4 were detected by qRT-PCR.
Chromatin immunoprecipitation (ChIP) assay Enrichment of DNMT1 in MEG3 promoter region was analyzed using ChIP kit (Millipore, USA). When the MDA-MB-231 cells were grown to 70-80% in con uence, 1% formaldehyde was added to the cells and cells were xed at room temperature for 10 min to make the DNA and proteins in the cells immobilized and cross-linked. Then, the cross-linked products were randomly fragmented into fragments of appropriate size by 10 s of ultrasonication for 15 cycles with an interval of 10 s. After centrifugation at 13,000 rpm at 4 ℃, the collected supernatant was transferred into 3 tubes and cultured with positive control antibody RNA polymerase II, negative control antibody IgG of normal mice (ab6721, 1:30, Abcam, UK) and methylation transferase speci c antibody DNMT1 (ab13537, 1:50, Abcam, UK) overnight at 4 ℃, respectively. Protein Agarose/Sepharose was used to precipitate endogenous DNA-protein complexes, and the supernatant was adsorbed after a short centrifugation. The non-speci c complexes were washed and de-crosslinked overnight at 65 ℃. The DNA fragments were extracted and puri ed by phenol/chloroform. qRT-PCR was used to test the combination of DNMT1 and MEG3 promoter region. Primer sequences were detailed in Supplementary material 1.

Methylation-speci c PCR (MSP)
Genomic DNA was treated with sodium bisul te and DNA methylation was tested by MSP using EZ DNA Methylation-Direct kit (Zymo Research). Two primer groups were used to amplify the promoter region of MEG3 containing multiple CpG sites, and the primer sequences were shown in Table 1. PCR reaction conditions: pre-denaturation at 95 ℃ for 10 min, followed by 35 cycles of 95 ℃ for 45 s, 56 ℃ (methylation) /45 ℃ (non-methylation) for 45 s and 72 ℃ for 45 s, and nally extension at 72 ℃ for 10 min. The reaction products were subjected to agarose gel electrophoresis and images were captured for further analysis.

qRT-PCR
TRIzol reagent (Invitrogen) was used to extract total RNA from cells, and the OD260/280 value of each RNA sample was determined by an UV spectrometer. RNA concentration was calculated and samples were stored at -80 ℃ for subsequent experiments. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencis, CA, USA). cDNA of mRNA was obtained by reverse transcription kit (RR047A, Takara, Japan), while cDNA of miRNA was obtained by miRNA First Strand cDNA Synthesis kit (B532451-0020, Shanghai Sangon Biotech, China). The samples were loaded using the SYBR ® Premix Ex TaqTM II (Perfect Real Time) kit (DRR081, Takara, Japan) and subjected to qRT-PCR reaction on a real-time uorescence quantitative PCR instrument (ABI 7500, ABI, Foster City, CA, USA). The PCR ampli cation procedure was set as below: pre-denaturation at 95 ℃ for 30 s, followed by 40 cycles of 95 ℃ for 5 s and 60 ℃ for 34 s. Each sample treatment was repeated in triplicate. Primers were synthesized by Shanghai Sangon Biotech Company ( Wound healing assay Cell motility was assessed by a wound-healing assay, as described in a previous study [16]. In brief, 2×10 5 MDA-MB-231 cells were inoculated on 6-well plates and incubated in appropriate complete medium at 37 ℃ for 16 h. The monolayer was scraped and cells were cultured in a fresh medium without FBS for 24 h.
Finally, three different elds of each well were observed and photographed under an inverted microscope to measure the scratch width. Relative scratch width = 24 h scratch width / 0 h scratch width.

Transwell invasion assay
As mentioned previously, changes in cell invasion were analyzed through Transwell assay [17]. In this assay, 8.0-μm Millipore Transwell chambers containing Matrigel were used. Firstly, 1×10 5 MDA-MB-231 cells were resuspended in 200 μl medium without FBS and then inoculated into the upper chambers. Next, 500 μl medium containing 10% FBS was added to the lower chambers. After 48 h of culture, un-invading cells were removed from the upper surface of the membranes with a cotton swab, and the invading cells were xed and stained with crystal violet. Finally, 5 randomly selected elds were observed under an inverted microscope to calculate cell number.
Nude mice experiment [18] Ten 6-week-old BALB/c female nude mice were purchased from Beijing HFK bio-technology (Beijing, China). The mice were randomly divided into two groups with 5 in each group. Then, 5×10 6 MDA-MB-231 cells with sh-NC or sh-DNMT1 were resuspended in 100 μl PBS and sequentially injected into each mouse by tail vein injection. After the mice were fed for 5 days, tumor volume was measured by a caliper every 5 days and calculated as follow: V = D × d 2 × 0.5 (D, longer diameter; d, shorter diameter). After 35 days, the mice were euthanized by CO 2 inhalation. This experiment was approved by the Animal Care and Use Committee of Jinhua Municipal Central Hospital.

Statistical analysis
All data were processed by SPSS 21.0 statistical software (SPSS, Inc., Chicago, IL, USA), and the measurement data were expressed as mean ± standard deviation. The comparison between two groups was analyzed by Student's t-test, and the comparison among multiple groups was analyzed by one-way ANOVA. P<0.05 indicated the difference was statistically signi cant.

Results
Silencing DNMT1 inhibits the proliferation, migration and invasion of breast cancer cells by promoting the expression of MEG3 through demethylation Current studies nd that lncRNA MEG3 is regulated by DNMT1, and the promoter of MEG3 is methylated under the in uence of DNMT1, while MEG3 appears to be hypermethylated and lowly expressed in tumors [9,15,19]. In view of this nding, we further examined the effect of the interaction of MEG3 with DNMT1 on the breast cancer cells. DNMT1 and MEG3 expression levels in human breast cancer cell lines MCF-7, MDA-MB-231, SKBR3 and human normal breast epithelial cell line MCF10A were tested by western blot ( Figure 1A) and qRT-PCR ( Figure 1B), respectively. The results showed that protein expression of DNMT1 was signi cantly higher in breast cancer cells, while MEG3 was signi cantly lowly expressed (p<0.05). MDA-MB-231 cell line with the lowest MEG3 expression was chosen for subsequent experiments. ChIP assay was used to detect whether DNMT1 could bind to MEG3 promoter region ( Figure  1C). Compared with the IgG control group, DNMT1 enrichment in MEG3 promoter region was signi cantly increased (p<0.05). Besides, CpG islands were found in the MEG3 gene promoter region by analyzing 2100 bp nucleotide sequences near the MEG3 gene promoter region through the MethPrimer software, and the results indicated that MEG3 expression would be affected by promoter methylation ( Figure 1D).
Then, DNMT1 interference e ciency in MDA-MB-231 cells was examined by western blot (Figure 1E), and sh-DNMT1-1 was used in subsequent experiments for better interference e ciency. MEG3 methylation level detected by MSP ( Figure 1F) presented that MEG3 methylation level was signi cantly decreased upon DNMT1 knockdown. While qRT-PCR results revealed that MEG3 gene expression was remarkably up-regulated when silencing DNMT1 ( Figure 1G) (p<0.05). It indicated that silencing DNMT1 could promote MEG3 expression by inhibiting MEG3 promoter methylation.
It is reported that DNMT1 can promote the malignant progression of breast cancer [8], while MEG3 can elicit a suppressive effect [12]. In this study, we proved that DNMT1 could potentiate MEG3 promoter methylation in turn inhibiting MEG3 expression. Based on the above results, we put forward a hypothesis that DNMT1 regulates the proliferation, migration and invasion of breast cancer cells by inhibiting MEG3 expression. To validate the speculation, the level of MEG3 expression was rstly interfered, and sh-MEG3-1 with a better interference e ciency as directed by qRT-PCR was selected for subsequent experiments ( Figure 1H). Then, expression levels of DNMT1 and MEG3 in three groups (sh-NC, sh-DNMT1+sh-NC, sh-DNMT1+sh-MEG3) were detected by qRT-PCR ( Figure 1I). As demonstrated, the expression of DNMT1 was conspicuously down-regulated while the expression of MEG3 was remarkably up-regulated in the sh-DNMT1+sh-NC group relative to the sh-NC group. Besides, MEG3 expression was obviously downregulated when DNMT1 and MEG3 were both silenced, and there was no signi cant diversity in DNMT1 expression, with a comparison of those in the sh-DNMT1+sh-NC group. Then, the results of CCK-8 ( Figure  1J), wound healing ( Figure 1K) and Transwell ( Figure 1L) assays displayed that silencing DNMT1 decreased cell activity, migratory and invasive abilities while these abilities were increased when DNMT1 and MEG3 were silenced simultaneously. In conclusion, silencing DNMT1 inhibited the malignant progression of breast cancer via up-regulating MEG3.
miR-494-3p is a target of MEG3 in breast cancer cells Many studies have pointed out that MEG3 can play a regulatory role by acting as a ceRNA [20,21]. Further prediction of the downstream regulatory miRNAs of MEG3 (Figure 2A) discovered that miR-494-3p might be a target of MEG3 and there were potential targeted binding sites linking these two genes ( Figure 2B).
Moreover, a study indicated that the expression level of miR-494-3p in tumors is signi cantly increased [22]. Hence, expression level of miR-494-3p in breast cancer cell lines in vitro was further examined and found to be signi cantly higher than that in control cell line ( Figure 2C). Moreover, RIP was used to validate the binding relationship between MEG3 and miR-494-3p ( Figure 2D). Compared with the IgG group, the quantity of MEG3 and miR-494-3p bound by Ago2 was signi cantly increased. Dualluciferase reporter gene assay further veri ed the targeted binding sites of miR-494-3p on MEG3 3'-UTR ( Figure 2E). The results showed that overexpression of miR-494-3p markedly reduced the luciferase activity of the MEG3-wt group but had no effect on that of the MEG3-mut group. Expression analysis was conducted by qRT-PCR for the levels of MEG3 and miR-494-3p in the oe-NC group and oe-MEG3 group ( Figure 2F), demonstrating that the expression of miR-494-3p was remarkably down-regulated when MEG3 was overexpressed, indicating that MEG3 targeted and negatively regulated miR-494-3p.
Silencing miR-494-3p inhibits proliferation, migration and invasion of breast cancer cells by targeting and promoting OTUD4 expression Furthermore, DEGs in breast cancer dataset GSE70905 that was included in GEO database were analyzed ( Figure 3A), and downstream target genes of miR-494-3p were predicted on bioinformatics databases. It was found that OTUD4 was with speci c binding sites of miR-494-3p and it was lowly expressed in breast cancer ( Figure 3B-D). Similarly, in vitro cell experiments also showed that the expression of OTUD4 mRNA in breast cancer cell lines was signi cantly lower than that in normal cell line ( Figure 3E). To con rm the relationship of miR-494-3p with OTUD4, rstly, RIP assay was performed and the results shown in Figure 3F revealed that compared with the IgG group, the quantity of miR-494-3p and OTUD4 bound by Ago2 was signi cantly increased. Dual-luciferase assay was conducted for further veri cation ( Figure 3G). The result showed that overexpression of miR-494-3p signi cantly decreased the luciferase activity of the OTUD4-wt group (p<0.05) but had no effect on that of the OTUD4-mut group (p>0.05).

MEG3 negatively regulates miR-494-3p to promote OTUD4 expression and inhibits the proliferation, migration and invasion of breast cancer cells
For deeply understanding the in uence of MEG3/miR-494-3p/OTUD4 as the regulatory axis on breast cancer cells, the expression levels of MEG3, miR-494-3p and OTUD4 in 3 groups (oe-NC+sh-NC group, oe-MEG3+sh-NC group and oe-MEG3+sh-OTUD4 group) were tested by qRT-PCR ( Figure 4A), and the protein level of OTUD4 was detected by western blot ( Figure 4B). Overexpression of MEG3 signi cantly increased OTUD4 protein and mRNA expression levels, but reduced miR-494-3p expression. In comparison with the oe-MEG3+sh-NC group, the mRNA and protein expression levels of OTUD4 were greatly down-regulated in the oe-MEG3+sh-OTUD4 group, while the expression levels of MEG3 and miR-494-3p had no signi cant difference (p>0.05). The results of CCK-8 ( Figure 4C), wound healing ( Figure 4D) and Transwell ( Figure   4E) assays indicated that overexpression of MEG3 decreased cell viability, migratory and invasive abilities while silencing OTUD4 in MEG3-overexpressed cells could partially alleviate the inhibition.

Overexpression of MEG3 inhibits the tumorigenic ability of breast cancer in vivo
Finally, we overexpressed MEG3 in nude mice to observe the effect of MEG3 on the tumorigenicity of breast cancer. The tumor weight and volume of each group were detected as shown in Figure 5A-C.
Overexpression of MEG3 reduced tumor volume and weight. The expression levels of DNMT1, MEG3, miR-494-3p and OTUD4 upon MEG3 overexpression were detected by qRT-PCR ( Figure 5D), while the protein expression levels of DNMT1 and OTUD4 were detected by western blot ( Figure 5E). The results exhibited that miR-494-3p was signi cantly down-regulated when MEG3 was overexpressed, while MEG3 and OTUD4 were remarkably up-regulated (p<0.05). Besides, overexpression of MEG3 had no signi cant effect on DNMT1 expression (p>0.05).

Discussion
Aberrant DNA methylation plays an important role in gene expression [23]. This study explored the regulation of MEG3/miR-494-3p/OTUD4 axis in breast cancer from the perspective of DNMT1 promoting MEG3 hypermethylation, to clarify its potential effect on the biological behaviors of breast cancer cells. Previous studies demonstrated that DNMT1 (DNA methylase) can promote the methylation of lncRNA MEG3, and methylation of MEG3 promoter along with changes in gene region is the main reason for abnormal expression of MEG3 in tumors [24]. Another study showed that DNA methylation inhibitor (5'-Aza-2'-deoxycytidine) plays an important regulatory role in MEG3 expression in glioma cells [25]. Additionally, aberrant methylation promoted by DNMT1 can increase the resistance of breast cancer cells to anticancer drugs, and DNMT1 also can promote the development of breast cancer by reducing the expression of MEG3 [9]. Here, this study veri ed the role of DNMT1 in regulating MEG3, and the results were consistent with previous reports. The rescue experiments further veri ed that knockdown of DNMT1 could demethylate MEG3 and promote its expression, thus inhibiting the progression of breast cancer cells.
It is known that MEG3 can function as a ceRNA in tumors [15]. For example, MEG3 may regulate the progression of gastric cancer as a ceRNA binding to miR-181a [26], and regulate ischemic neuronal death by targeting miR-21/PDCD4 signaling pathway [27]. MEG3 modulates EMT of cells by targeting miR-421 in breast cancer [15]. This study further explored the downstream targets of MEG3, and found that MEG3 could target and bind to miR-494-3p. To verify this relationship, RIP and dual-luciferase assays were used and the results implied that MEG3 could regulate the expression of miR-494-3p as a ceRNA. Zhou et al. reported that miR-494-3p could promote cell proliferation and tumor growth in breast cancer through targeting and inhibiting TRIM21 expression [28], and miR-494-3p was also found to inhibit self-renewal of breast cancer stem/progenitor cells [29], which both indicate the important regulatory role of miR-494-3p in breast cancer. In the study, we overexpressed MEG3 expression in cancer cells and found suppressed cell activity, invasive and migratory abilities. In rescue experiments, overexpression of miR-494-3p reversed the inhibitory effect which functioned by MEG3 overexpression on the development of breast cancer cells. The results showed that high level of MEG3 inhibited the proliferation, invasion and migration of breast cancer cells by targeting and inhibiting miR-494-3p expression.
In order to further understand the underlying regulatory mechanism, target genes of miR-494-3p were predicted, and the DEmRNAs in breast cancer were screened using GEO database. The results suggested that OTUD4 was poorly expressed in breast cancer tissue samples, and the 3'-UTR of OTUD4 had potential binding sites of miR-494-3p. Furthermore, RIP and dual-luciferase assays were used to verify the binding relationship, and western blot assay results con rmed the down-regulation of OTUD4 expression in the case of miR-494-3p overexpression. This indicated that miR-494-3p targeted and down-regulated the expression of OTUD4 in breast cancer. OTUD4 has been found to be lowly expressed in non-small cell lung cancer and is able to inhibit the proliferation of cancer cells [30]. In addition, alkylation damage which is critical for cancer chemotherapy can also be regulated by OTUD4 [31]. However, the mechanism of OTUD4 in breast cancer has not been reported. Rescue experiments were performed in this study again to nd that silencing OTUD4 reversed the negative in uence of miR-494-3p inhibitor on breast cancer cells. Moreover, the downstream regulatory effects of DNMT1 and MEG3 were further investigated to determine their impact on breast cancer progression.

Conclusions
We explored the role of DNMT1 in reducing MEG3 methylation, and in turn regulating the expression of genes in the MEG3/miR-494-3p/OTUD4 axis to in uence progression of breast cancer cells ( Figure 6). This study reveals the mutual interactions among MEG3, miR-494-3p and OTUD4, providing a new approach for targeted therapy of breast cancer.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The data and materials in the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no potential con icts of interest.     invasion were measured by CCK-8, wound healing and Transwell assays, respectively. Each experiment was carried out in triplicate; * p<0.05.   the protein expression levels of DNMT1 and OTUD4 were tested by western blot. Each experiment was carried out in triplicate; * p<0.05.