miR-639 Expression Is Silenced by DNMT3A-Mediated Hypermethylation and Functions as a Tumor Suppressor in Liver Cancer Cells

Please cite this article in press as: Xiao et al., miR-639 Expression Is Silenced by DNMT3A-Mediated Hypermethylation and Functions as a Tumor Suppressor in Liver Cancer Cells, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.021

Original Article

miR-639 Expression Is Silenced by DNMT3AMediated Hypermethylation and Functions as a Tumor Suppressor in Liver Cancer Cells Jing Xiao,1,5 Yankun Liu,1,2,5 Fuxia Wu,1,5 Ruiyan Liu,1,3,5 Yongli Xie,1 Qian Yang,1 Yufeng Li,2 Min Liu,1 Shengping Li,4 and Hua Tang1 1Tianjin

Life Science Research Center, Tianjin Key Laboratory of Inflammation Biology, Collaborative Innovation Center of Tianjin for Medical Epigenetics, Department of

Pathogen Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China; 2The Cancer Institute, Tangshan People’s Hospital, Tangshan 063001, China; 3Department of Laboratory Medicine, The First Teaching Hospital, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China; 4State Key Laboratory of Oncology in Southern China Department of Hepatobiliary Oncology, Cancer Center, Sun Yat-sen University, Guangzhou 510060, China

Emerging evidence has indicated that abnormal methylation of DNA contributes to hepatocarcinogenesis. However, the regulatory mechanisms are not well known. Here, we revealed that microRNA-639 (miR-639) expression is downregulated in liver cancer tissues and cells. The repression of miR-639 expression was attributed to hypermethylation in its promoter region, and DNA methyltransferase (DNMT3A) was found to mediate this hypermethylation. Repression of miR-639 expression promoted cell growth and migration/invasion in vitro and the growth of tumors in xenograft mouse models. Furthermore, miR-639 bound to the 30 UTR of both MYST2 and ZEB1 and suppressed their expression. MYST2 promoted the growth of liver cancer cells and ZEB1 facilitated the migration/invasion of liver cancer cells. Ectopic expression of MYST2 and ZEB1 counteracted the repression of malignancy induced by miR639, which coincided with the reciprocal correlation between miR-639 and MYST2 and ZEB1 expression in clinical hepatocellular carcinoma (HCC) tissues. Thus, DNMT3Amediated hypermethylation suppressed miR-639 expression, derepressing the expression of MSYT2 and ZEB1, which promoted tumorigenesis of liver cancer. These findings may shed light on the mechanism of abnormal expression of miRNAs involved in the malignancy of liver cancer and provide new biomarkers for liver cancer.

INTRODUCTION Liver cancer has a high incidence and mortality worldwide, and hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer.1 The development of liver cancer is a multistep and long-term process that involves various genetic and epigenetic alterations of many genes that are crucial to cellular processes, such as cell-cycle control, cell growth, apoptosis, cell migration, and metastasis.2 However, the detailed molecular mechanisms underlying liver cancer remain poorly understood. MicroRNAs (miRNAs) are endogenous small noncoding RNAs (20– 24 nt) that regulate gene expression at the posttranscriptional level.3

miRNAs complementarily bind to the 30 UTR of mRNA to mediate mRNA degradation or translation repression.4 miRNAs are involved in various biological processes,5 including tumorigenesis.6 For example, miR-21,7 miR-221,8 miR-222,9 and miR-490-3p10 are significantly upregulated in HCC and act as oncomiRs by stimulating tumor development and progression. Nevertheless, Let-7 family miRNAs11 and miR-101,12 miR-125b,13 and miR-615-5p14 act as tumor suppressors by negatively regulating oncogenes in liver cancer. However, the mechanism of miRNA dysregulation remains largely unknown. Epigenetic modifications involve DNA methylation and histone acetylation. DNA methylation is one of the most common epigenetic modifications and is processed by enzymes belonging to the DNA methyltransferase (DNMT) family, which are divided into three classes as follows: DNMT1, DNMT2, and DNMT3A\3B\3L. DNMT1 is predominantly responsible for maintaining the preexisting methylation pattern during DNA replication, and DNMT3A and DNMT3B act as de novo methyltransferases by establishing the methylation pattern during embryogenesis, but the functions of DNMT2 are not completely clear.15–17 DNA methylation participates in numerous biological events, such as embryonic development, parental gene imprinting, transposon silencing, X inactivation, and cancer.17 Aberrant DNA methylation of CpG islands at gene promoter regions plays important roles in cancer progression.18 Some miRNAs, such as the tumor suppressor miR-1, which is frequently silenced by DNA hypermethylation in both HCC cell lines and tissues, have been reported to be tightly regulated by DNA methylation.19 miR-122a, which is downregulated in HCC tissues and HCC-derived cell lines, plays a

Received 22 May 2019; accepted 23 November 2019; https://doi.org/10.1016/j.ymthe.2019.11.021. 5

These authors contributed equally to this work.

Correspondence: Hua Tang, Tianjin Life Science Research Center, Tianjin Key Laboratory of Inflammation Biology, Collaborative Innovation Center of Tianjin for Medical Epigenetics, Department of Pathogen Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China. E-mail: [email protected]

Molecular Therapy Vol. 28 No 2 February 2020 ª 2019 The American Society of Gene and Cell Therapy.

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Please cite this article in press as: Xiao et al., miR-639 Expression Is Silenced by DNMT3A-Mediated Hypermethylation and Functions as a Tumor Suppressor in Liver Cancer Cells, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.021

Molecular Therapy

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Figure 1. 5-Aza-dC Treatment Restores the Downregulation of miR-639 Expression by Enhancing Promoter Activities in Liver Cancer Cells (A) The relative expression level of miR-639 was measured by qRT-PCR in 30 pairs of HCC tissues. The expression level was normalized to the expression level in adjacent nontumor tissues. (B) The relative expression level of miR-639 was measured in the following liver cancer cell lines: QGY-7703, SMMC-7721, SK-Hep-1, Huh-7, LM-3, HepG2, and MHCC97-H, and in the L02 normal human liver cell line. (C) The relative expression level of miR-639 in different HCC cells and L02 cells (treated with 5-Aza-dC for 24 hr and untreated) was measured by qRT-PCR. (D) Detailed information on the promoter of miR-639 and primers for ChIP-PCR was shown. (E) Luciferase activity was measured to indicate the activity of the promoter in different cell lines with or without 5-Aza-dC treatment (10 mM) for 12 hr. (F) The promoter activity of miR-639 induced by 5-Aza-dC in DNMT3A knockdown background in liver cancer lines (ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001).

role in hepatocarcinogenesis by targeting Cyclin G1.20 Furthermore, miR-34a acts as a significant tumor suppressor in tumor cell proliferation and migration by negatively targeting Bcl-2 and SIRT1 in breast cancer cells.21 Our previous studies revealed that miR-10a promotes cancer cell migration and invasion in vitro but represses metastasis in HCC and colorectal cancer.22,23 We also found that methylation regulation of miR-941 regulates lysine (K)-specific demethylase 6B (KDM6B) in HCC.24 Recently, miR-639 is reported to exert oncogenic effects in human tongue cancer cells by targeting FOXC1.25 In contrast, miR-639 functions as a tumor suppressor in human thyroid cancer by suppressing CDKN1A expression.26 In the current study, we found that miR-639 expression was downregulated in HCC tissues and cells, and miR-639 suppressed the pro-

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liferation and migration/invasion of liver cancer cells, thereby functioning as a tumor suppressor in liver cancer. The promoter of miR639 was cloned and characterized by DNMT3A-mediated methylation. Upregulation of DNMT3A expression resulted in hypermethylation of CpG islands in the miR-639 promoter, thus repressing miR-639 expression. The miR-639 expression level was inversely correlated with the malignancy of liver cancer cells. Finally, MYST2 and ZEB1 were identified as target genes of miR-639 and were found to mediate the roles of miR-639 in HCC. Overall, these results, which might provide new insight into the mechanisms underlying tumorigenesis in HCC, indicated that DNMT3A-mediated downregulation of miR-639 expression contributes to the malignancy of HCC by increasing the expression levels of MYST2 and ZEB1.

Please cite this article in press as: Xiao et al., miR-639 Expression Is Silenced by DNMT3A-Mediated Hypermethylation and Functions as a Tumor Suppressor in Liver Cancer Cells, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.021

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RESULTS miR-639 Expression Is Silenced by Hypermethylation of Its Promoter in Liver Cancer Cells

To evaluate the potential role of miR-639 in HCC, we first examined the expression of miR-639 in 30 pairs of HCC and adjacent nontumor tissues by qRT-PCR. miR-639 expression levels were much lower in tumor tissues than in adjacent nontumor tissues, decreased by approximate 64% (Figure 1A). In addition, the expression levels of miR-639 were examined in seven different types of liver cancer cell lines (QGY7703, SMMC-7721, SK-Hep-1, Huh-7, LM-3, MHCC97-H, and HepG2) and in an immortalized normal hepatocyte cell line (L02) as a control. Consistent with the HCC tissue results, the miR-639 expression levels in all the liver cancer cells were less than that in L02 cells (Figure 1B). The downregulated expression of miR-639 in both HCC tissues and cells suggested that miR-639 might act as a tumor suppressor in HCC development. Because inactivation of tumor suppressor genes is closely related to epigenetic silencing, we speculated whether hypermethylation of the miR-639 promoter causes the downregulation of miR-639 expression in HCC tissues and liver cancer cells. To address this hypothesis, we used 5-Aza-20 -deoxycytidine (5-Aza-dC), which is an inhibitor of DNMT, to treat these liver cancer cells. The qRT-PCR assay showed that compared with the untreated cells, treating the HCC cells (QGY-7703, SMMC-7721, SK-Hep-1, Huh-7, LM-3, MHCC97-H, and HepG2) with 5-Aza-dC for 12 hr increased the expression levels of miR-639 by 4.73- to 16.4-fold (Figure 1C). Interestingly, HCC cells with lower expression levels of miR639 exhibited a greater increase in the expression level of miR-639 after treatment with 5-Aza-dC. These data indicate that the expression of miR-639 might be controlled by methylation of its promoter. Because miR-639 is imbedded in the exon of TECR gene, we subsequently addressed whether 5-Aza-dC affects the expression of TECR. Treated with 5-Aza-dC, cells increased TECR expression in SMMC-7721, SK-Hep-1, Huh-7, LM-3, and MHCC-97 cells, except L02 cells (Figure S1), suggesting that the activity of miR-639/TECR promoter could be induced by 5-Aza-dC in liver cancer cell lines. Next, bioinformatics analysis showed that miR-639 locates in the 50 UTR region of trans-2,3-enoyl-CoA reductase (TECR) on chromosome 19q13 and predicted that the core promoter of miR-639 might be within 750 bp upstream of the 50 UTR region of TECR (Figure 1D). The predicted fragment was amplified by PCR and cloned into the pGL3-basic vector to generate the pGL3-miR-639-P plasmid. Then, pGL3-miR-639-P was transfected into liver cancer cells and L02 cells before the luciferase activities were measured. As shown in Figure 1E, compared to the luciferase activities in the pGL3 group, the luciferase activities in the pGL3-miR-639-P group were increased by approximately 7- (SK-Hep-1 cells) to 20-fold (QGY-7703 cells). Except for L02 cells, when these cells treated with 5-Aza-dC, the luciferase activities were 1.33- (LM3 cells) to 7.57-fold (SMMC-7721 cells) greater than the luciferase activities in the nontreated pGL3-miR-639P group. Further, we confirmed the promoter activity of miR-639 induced by 5-Aza-dC in liver cancer lines with DNMT3A deletion. As shown in Figure 1F, both knockdown of DNMT3A and 5-AzadC treatment increased luciferase activity in HepG2, QGY-7703,

Huh-7, and L02 cells. Furthermore, the cells treated with 5-Aza-dC in the DNMT3A knocked down group exhibited higher luciferase activities in HepG2, QGY-7703, and Huh-7 cells, except L02 cells. These results demonstrated that the activities of the cloned miR639 promoter in all the liver cancer cells were activated by 5-AzadC, whereas the L02 cells did not demonstrate these activities. This result was substantially consistent with the elevated miR-639 expression levels in HCC cells treated with 5-Aza-dC (Figure 1C). Overall, these data indicate that the miR-639 promoter is regulated by methylation in liver cancer cells. Methylation of the miR-639 Promoter by DNMT3A Leads to Decreased miR-639 Expression Levels in Liver Cancer Cells and Tissues

To further explore the mechanism of miR-639 promoter methylation, we used the CpG island searcher prediction algorithm. Figure 1D shows that the promoter of miR-639 contains CpG islands with 14 CpG sites from 536 to 306. Subsequently, bisulfite genomic sequencing (BGS) was performed to detect the methylation status of these CpG sites in HCC tissues and cell lines. The methylation frequency in HCC tissues was 8.6%, whereas the methylation frequency was only 1.4% in the adjacent nontumor tissues (Figure 2A), suggesting that a lower expression level of miR-639 was inversely related to a higher methylation status in HCC tissues. As shown in Figure 2B, there were four continuous methylated CpG sites among the 14 CpG sites in the SMMC-7721, QGY-7703, Huh-7, and SKHep-1 cell lines and two continuous methylated CpG sites in the LM-3, HepG2, and MHCC97-H cell lines. However, no methylated CpG sites were found in the L02 cell lines with the highest level of miR-639 expression. When these cells were treated with 5-Aza-dC, methylation within the CpG sites disappeared (Figure 2B). These results indicated that the expression level of miR-639 is negatively correlated with its methylation level in liver cancer tissues and cell lines. Because the methylation frequency in cancer tissues is greater than that in nontumor tissues, we examined the relationship between the methylation status of the miR-639 promoter and tumor grades in liver cancer tissues. As shown in Figure 2C, there were four continuously methylated CpG sites in stage IV HCC tissues and two continuously methylated CpG sites in stage II HCC tissues, but none were found in nontumor tissues or in stage I HCC tissues. The data demonstrated that higher tumor grades were associated with higher methylation levels of the miR-639 promoter (Figure 2C). Consistently, the expression level of miR-639 was the highest in stage I HCC tissues and the lowest in stage IV HCC tissues (Figure 2D). These results indicated that the methylation level of the miR-639 promoter is positively correlated with tumor malignancy. To determine which molecule is responsible for the regulation of methylation of the miR-639 promoter, we examined miR-639 expression levels in HepG2 cells with overexpression or knockdown of DNMT1, DNMT2, and DNMT3A. The effectiveness of these plasmids in HepG2 cells is shown in Figure S2. In Figure 2E, the results

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Figure 2. Methylation of the miR-639 Promoter in Liver Cancer Cell Lines and Tissues and the Associated DNMTs (A) BGS was used to detect the DNA methylation status of the miR-639 promoter in 10 HCC tissues and paired non-tumor tissues. (B) The BGS results showed the DNA methylation status of the miR-639 promoter with (right) or without (left) 10 mM 5-Aza-dC treatment for 48 hr in the different HCC cells and in L02 cells. (C) The BGS results showed the DNA methylation status of the miR-639 promoter in HCC tissues and adjacent nontumor tissues in different pathology stages (TNM stages I, II, and IV). (D) The relative expression level of miR-639 in HCC tissues was measured by qRT-PCR. The expression level was normalized to the expression level in adjacent tissues. The black dots represent methylated CpG sites, and the white dots represent unmethylated CpG islands. (E) The expression level of miR-639 was measured by qRT-PCR in HepG2 cells with DNMT1, DNMT2, and DNMT3A overexpression or knockdown. (F) ChIP-PCR assay showed fragment of DNMT3A bound to miR-639 promoter. (G) The mRNA expression level of DNMT3A in 30 pairs of HCC tissues was measured by qRT-PCR, and the expression level was normalized to the expression level in adjacent tissues (left panel). The Pearson’s correlation analysis showed the inverse correlation of miR-639 and DNMT3A expression levels in liver cancer tissues (right panel) (*p < 0.05). (H) BGS analysis was used to evaluate the methylation of the miR-639 promoter in HepG2 cells with DNMT3A overexpression or knockdown. (I) The expression level of miR-639 was measured in HepG2 cells with DNMT3A overexpression or knockdown (ns, not significant; *p < 0.05, **p < 0.01).

showed that DNMT3A overexpression significantly reduced the miR639 expression level in HepG2 cells, whereas DNMT3A depletion dramatically increased the miR-639 expression level. We also detected the function of DNMT3B on methylation of miR-639 in liver cancer cells. Although DNMT3B induced activity of miR-639 promoter in HepG2 cells, it was weaker than DNMT3A and exhibited no significant difference between cancer tissue and adjacent non-tumor tissue of clinical HCC tissue samples (Figure S3). Thus, we focused on the study of DNMT3A’s function. Moreover, to determine a direct hypermethylation regulation on miR-639 promoter by DNMT3A, the

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chromatin immunoprecipitation (ChIP)-PCR assay was employed to confirm the role of DNMT3A on methylation of miR-639 promoter. We designed the primers for ChIP-PCR that located on the CpG island of miR-639 promoter (Figure 1D). As shown in Figure 2F, DNMT3A bond to the promoter region. To explore the correlation between DNMT3A and miR-639 expression in clinical HCC tissues, we used qRT-PCR to measure the expression level of DNMT3A in 30 pairs of HCC and adjacent tissue samples. The data showed that DNMT3A expression was upregulated by approximately 1.8-fold in the tumor tissues compared with that in the non-tumor tissues

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Figure 3. miR-639 Inhibits the Growth, Migration, and Invasion of Liver Cancer Cells In Vitro and Tumor Growth In Vivo (A) qRT-PCR was performed to evaluate the function of the pri-miR-639 plasmid in QGY-7703 and ASO-miR-639 in HepG2 cells. (B) MTT and (C) colony-formation assays were performed in both QGY-7703 and HepG2 cells transfected with pri-miR-639 or ASO-miR-639. Representative images are shown. (D) The cell-cycle distribution and (E) PI were analyzed by flow cytometry in HepG2 cells with miR-639 overexpression or knockdown. (F) In vivo tumor proliferation assay. The tumor growth curve (left), tumor size (middle), and statistical analysis of the tumor weight (right) are shown (n = 6). (G) Migration and (H) invasion assays were performed in QGY-7703 and HepG2 cells transfected with the indicated plasmids and oligonucleotides. Representative images are shown. (I) The protein expression levels of E-cadherin, cytokeratin, ICAM-1, vimentin, and Ncadherin were evaluated by western blotting in QGY-7703 cells with overexpression or knockdown of miR-639. The expression levels were normalized to GAPDH, which was used as a control. All the experiments are repeated at least three times (ns, not significant; *p < 0.05, **p < 0.01).

(Figure 2G, left panel), which was the opposite of the miR-639 expression pattern (Figure 1A). The relationship between miR-639 and DNMT3A in liver cancer tissues was inversely correlated (Figure 2G, right panel). Furthermore, to elucidate the influence of DNMT3A on miR-639 promoter methylation, the methylation status of the CpG sites in the miR-639 promoter was assessed by BGS in HepG2 cells with knockdown or overexpression of DNMT3A. As shown in Figure 2G, 5-Aza-dC treatment removed the methylation of the miR639 promoter. DNMT3A overexpression increased the methylation of miR-639, while DNMT3A knockdown reduced the methylation of miR-639. We also examined the miR-639 level under the same conditions. DNMT3A overexpression reduced the expression level of miR-639, while knockdown of DNMT3A increased the expression level of miR-639 (Figure 2I). Collectively, these data indicated that DNMT3A silences the expression of miR-639 through methylation of the CpG of the miR-639 promoter.

miR-639 Suppresses the Proliferation, Migration, and Invasion of HCC Cells

To determine whether miR-639 influences the malignancy of HCC cells, we constructed an miR-639 expression plasmid (pri-miR-639) and synthesized ASO-miR-639 and validated their efficiency in QGY-7703 cells and HepG2 cells (Figure 3A). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that compared to the control conditions in the QGY-7703 and HepG2 cell lines, pri-miR-639 impaired cell viability, whereas ASOmiR-639 increased cell viability (Figure 3B). Overexpression of miR-639 was significantly reduced, but ASO-miR-639 increased the colony formation rate in the QGY-7703 and HepG2 cell lines compared to that in cells treated with ASO-NC (Figure 3C). Flow cytometry analysis was performed to examine whether cell-cycle progression is influenced by miR-639. As shown in Figure 3D, overexpression of miR-639 led to an increase of HepG2 cells in the G1

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Molecular Therapy

phase and a decrease of HepG2 cells in the S phase, but ASO-miR-639 decreased the number of HepG2 cells in the G1 phase and increased the number of HepG2 cells in the S phase. The proliferation index (PI) was reduced when miR-639 was overexpressed, whereas ASO-miR639 treatment increased the PI in HepG2 cells compared to that in the control cells (Figure 3E). These results indicated that miR-639 suppresses the progression of the G1/S phase transition and cell growth in human liver cancer cells. To determine whether miR-639 acts as a tumor suppressor in vivo, a xenograft tumor model in nude mice was used. Pooled QGY-7703 cells overexpressing miR-639 were subcutaneously injected into the nude mice. After 1 week, the tumor volume was calculated every other day to generate a growth curve. The growth curve showed that miR639 suppressed tumors derived from QGY-7703 cells (Figure 3F, left panel). The mice were sacrificed 21 days after injection, and the tumors were collected and weighed separately. Overexpression of miR-639 significantly reduced both the tumor volume and weight (Figure 3F, middle and right panels). These results indicated that miR-639 inhibits the growth of tumors derived from liver cancer cells in vivo. To clarify the impact of miR-639 on the migration/invasion of HCC cells, Transwell migration and invasion assays were conducted. As shown in Figures 3G and 3H, both the migration and invasion were reduced for cells overexpressing miR-639 compared with those of the control cells. Conversely, ASO-miR-639 enhanced the migration/invasion abilities of QGY-7703 and HepG2 cells. Thus, miR639 suppresses the migration/invasion abilities of liver cancer cells. Epithelial-mesenchymal transition (EMT) is involved in the migration/invasion of cancer cells. Thus, we examined the influence of miR-639 on EMT markers (E-cadherin, cytokeratin, ICAM-1, vimentin, and N-cadherin) by western blotting analysis. Overexpression of miR-639 increased E-cadherin and cytokeratin expression levels but decreased the expression levels of vimentin, ICAM-1 and N-cadherin. Treatment with ASO-miR-639 had the opposite effect (Figure 3I). These results indicated that miR-639 suppresses the migration/invasion abilities and EMT of liver cancer cells. Overall, these data implied that miR-639 may function as a tumor suppressor by suppressing the proliferation and migration/invasion abilities of liver cancer cells. MYST2 and ZEB1 Are Direct Targets of miR-639

To identify the functional target genes of miR-639, Target-Scan, PicTar, and miRBase were employed to predict the potential targets of miR-639. MYST2 and ZEB1 were selected for further study based on their functions (Figure 4A). MYST2, which is a MYST domaincontaining HAT family protein, is involved in diverse cellular processes.27 Dysregulation of MYST family histone acetyltransferases induces malfunctioning cells, leading to cell death and uncontrolled growth and/or disease, including tumors.28,29 Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription repressor of E-cadherin that regulates the migration/invasion abilities of cancer cells.30 To identify the interaction of miR-639 and their candidate target genes, 30 UTR fragments or mutant 30 UTR fragments of MYST2 and ZEB1

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containing miR-639 binding sites were cloned into EGFP reporter vectors and transfected into QGY-7703 cells along with pri-miR639 or the control vector. Compared to the control cells, cells overexpressing miR-639 showed decreased EGFP intensities, whereas cells treated with ASO-miR-639 showed increased EGFP intensities (Figure 4B). However, EGFP intensities in cells transfected with pcDNA3/EGFP-MYST2/mut and pcDNA3/EGFP-ZEB1/mut were not influenced by alterations of miR-639 expression levels (Figure 4C). Overall, these results suggest that both MYST2 and ZEB1 are direct targets of miR-639. Next, we examined the influence of miR-639 on endogenous MYST2 and ZEB1 expression. qRT-PCR showed that compared to the control QGY-7703 cells, overexpressing miR-639 in QGY-7703 cells decreased the mRNA expression levels of MYST2 and ZEB1, whereas cells treated with ASO-miR-639 had increased mRNA expression levels of MYST2 and ZEB1 (Figure 4D). Considering that miRNAs typically negatively regulate their target genes, we examined the mRNA expression levels of MYST and ZEB1 in xenograft tumors. As expected, the expression levels of MYST and ZEB1 in the pri-miR-639 group were less than those in the vector control group (Figure 4E). Western blotting assays also showed that pri-miR-639 reduced MYST2 and ZEB1 protein expression levels, while ASO-miR-639 enhanced the expression levels of both proteins (Figure 4F). Moreover, immunohistochemistry (IHC) analysis of the xenograft tumors showed that the expression levels of MYST2 and ZEB1 were decreased in the pri-miR-639 group compared to those in the control group (Figure 4G). These data demonstrated that both MYST2 and ZEB1 are direct targets of miR-639 and are negatively regulated by miR-639.

MYST2 Mediates the miR-639-Induced Suppression of HCC Cell Growth

To determine the potential roles of MYST2 in liver cancer cells, we constructed a pcDNA3-MYST2 vector lacking the 30 UTR to avoid miRNA interference and a pSilencer-MYST2 vector to knockdown MYST2 expression, and the efficiency of the two plasmids transfected in HepG2 cells was evaluated by a western blotting assay and qRTPCR (Figure 5A). Then, the cell growth activities were detected by MTT and colony-formation assays. As shown in Figures 5B and 5C, MYST2 overexpressed for 72 hr in HepG2 and QGY7703 cells increased cell viability and growth but knockdown of MYST2 suppressed cell viability and growth. Consistently, pcDNA3-MYST2 promoted the G1/S phase transition and PI, but pshR-MYST2 repressed the G1/S phase transition and PI (Figures 5D and 5E). These data implied that MYST2 promotes the proliferation of HCC cells by increasing the rate of the cell cycle. Additionally, MYST2 was not observed to significantly affect the migration/invasion abilities (Figures 5F and 5G). In addition, we performed a rescue experiment and showed that MYST2 expression abrogated the miR-639induced suppression of cell viability and colony-formation rate of QGY-7703 cells (Figures 5H and 5I). Overall, these data indicated that miR-639 directly targets MYST2 to cause an antigrowth effect on liver cancer cells.

Please cite this article in press as: Xiao et al., miR-639 Expression Is Silenced by DNMT3A-Mediated Hypermethylation and Functions as a Tumor Suppressor in Liver Cancer Cells, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.021

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miR-639 Inhibits the Migration and Invasion of HCC Cells by Modulating the EMT Process through ZEB1

Because MYST2 did not affect the migration/invasion of HCC cells, we needed to identify another target gene that mediates the effect of miR-639 on the migration/invasion of liver cancer cells. Accordingly, another target gene, ZEB1, was involved in the motility of cancer cells. We examined the effect of ZEB1 on cancer cell migration/invasion. After we validated the overexpression plasmid and knockdown plasmid of ZEB1 in QGY-7703 cells (Figure 6A), we performed Transwell migration and invasion assays. Overexpression of ZEB1 significantly promoted the migration and invasion capabilities in QGY-7703 and HepG2 cells, but knockdown of ZEB1 repressed the migration and invasion capabilities in QGY-7703 and HepG2 cells (Figures 6B and 6C). Western blotting assays showed that overexpression of ZEB1 led to markedly increased expression levels of ICAM-1, vimentin, and N-cadherin, as well as a significantly reduced expression level of E-cadherin and cytokeratin. In contrast, knockdown of ZEB1 reduced the expression levels of ICAM-1, vimentin, and N-cadherin but increased the E-cadherin and cytokeratin expression level (Figure 6D). These data indicated that ZEB1 may promote the migration/invasion of HCC cells by enhancing the process of EMT. Furthermore, to confirm whether the suppression of migration/invasion caused by miR-639 directly depends on ZEB1, a rescue experiment was performed. As shown in Figures 6E and 6F, the migration/invasion abilities of QGY-7703 cells were promoted by inhibiting miR-639, and this effect was abolished when ZEB1 was knocked down. Overall, these results indicated that miR-639 suppresses the migration/invasion abilities of HCC cells at least partially by downregulating ZEB1 expression.

DISCUSSION Since Feinberg first reported the relationship between DNA methylation and human primary tumor tissues in the 1980s, the role of DNA methylation of specific genes rather than total levels of methylation in tumors has been extensively studied.31 Recent studies have shown that the expression of miRNAs (e.g., miR-1, miR-10a, miR9, miR-34, miR-124, miR-191, miR-203, and miR-125b) is deregulated by aberrant DNA methylation in liver cancer cells.19,32–35 Recently, miR-639 was found to be overexpressed in thyroid carcinoma and breast cancer tissues and cell lines.26,36 However, in our study, we found that miR-639 expression was downregulated in human HCC tissues and various liver cancer cells, suggesting that miR-639 may play a role as a tumor suppressor in liver cancer. In addition, liver cancer cells treated with 5-Aza-dC significantly increased miR-639 expression levels. These results indicated that demethylation could enhance the promoter activities of miR-639 and that cell malignancy is inversely correlated with the methylation status of the miR-639 promoter. Further studies demonstrated that the downregulation of miR-639 expression is due to hypermethylation of its promoter, and the promoter activity of miR-639 depends on the hypermethylation status in liver cancer cell lines (Figure 2). Aberrant methylation of CpG islands in the gene promoter region is one of the most common epigenetic alterations in cancer. Hyperme-

thylation of tumor suppressor genes leads to tumorigenesis, such as cervical cancer, sporadic parathyroid adenomas, and head-neck cancer.37–39 We found a substantial methylation status in the promoter of miR-639 in HCC tissues and liver cancer cell lines compared to adjacent nontumor tissues and L02 cells. Less methylation of CpG island sites was accompanied by increased activities of the miR-639 promoter. Clinical specimen analysis demonstrated that the methylation levels of CpG islands on the miR-639 promoter are inversely correlated with miR-639 expression but positively correlated with HCC tumor grades (Figure 2). It has been reported that DNMTs are significantly correlated with CpG island hypermethylation in tumor-related genes.40 In our study, DNMT3A was identified as a crucial methyltransferase that regulates the methylation of the miR-639 promoter in liver cancer cells by qRT-PCR and ChIP-PCR assay. Moreover, the influence of DNMT3A on the first 4 CpG island sites in this promoter was more notable than that on the other sites (Figure 2). The downregulation expression of miR-639 is inversely correlated with high DNMT3A expression in HCC clinical sample. Although DNMT3B has some effect on miR-639 expression and promoter activity, it is not as strong as DNMT3A. Thus, we concluded that DNMT3A mainly mediates the hypermethylation of the miR-639 promoter to downregulate the expression of miR-639 in liver cancer. Accumulated evidence has suggested that miRNAs have different functions in different tumor cells, which may account for the different roles of miRNAs in various cancers.41 In this study, we determined that miR-639 suppresses the growth and migration/invasion abilities of liver cancer cells (Figure 3). Because the target gene of miRNAs typically mediates its function, prediction algorithms were used to predict the putative mRNA targets of miR-639. Among the putative target genes, we selected MYST2 as a candidate gene based on the miR639 phenotype and functional characteristics of the candidate genes. MYST2 belongs to the family of histone acetyltransferases and plays crucial roles in various nuclear processes and in the control of cell proliferation. We confirmed MYST2 is a direct target of miR-639 by fluorescence reporter assay. Western blotting and qRT-PCR results also showed that miR-639 negatively regulates endogenous MYST2 expression at both the protein and mRNA levels. Consistently, the expression of MYST2 was repressed in tumors derived from QGY7703 cells overexpressing miR-639 in xenograft mice (Figure 4). MYST2 promotes cell-cycle progression and cell growth. Furthermore, the rescue experiment demonstrated that the suppression of cell growth induced by miR-639 could be counteracted by MYST2 in liver cancer cells (Figure 5). Thus, miR-639 suppressed the growth of liver cancer cells at least partially through targeting MYST2. However, MYST2 did not affect the migration/invasion of liver cancer cells (Figure 5), which suggested that other target genes may be involved in this process. Our previous studies showed that cervical cells undergo EMT to promote cell migration and invasion via ZEB1.42 Therefore, ZEB1 was selected as an additional target for further investigation. The reporter assay, qRT-PCR, and western blotting assays indicated that miR-639 directly targets and negatively regulates ZEB1 in liver cancer cells (Figure 4). Consistently, the migration/

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Figure 4. MYST2 and ZEB1 Are Direct Targets of miR-639 (A) A schematic showing the predicted complementary sequence of miR-639 in the 30 UTR of MYST2 and ZEB1 segments containing the wild-type or mutant binding sites. (B and C) The relative intensity of EGFP was analyzed after the wild-type (B) or mutant (C) 30 UTR ZEB1 or 30 UTR MYST2 plasmid was co-transfected with pri-miR-639 or ASOmiR-639 in QGY-7703 cells. (D) qRT-PCR analysis was used to evaluate the mRNA expression levels of MYST2 and ZEB1 in QGY-7703 cells with miR-639 overexpression or knockdown. (E) The expression levels of miR-639, MYST2, and ZEB1 in xenograft tumors were measured by qRT-PCR. (F) Western blotting was used to measure the protein expression levels of MYST2 and ZEB1 in QGY-7703 cells with miR-639 overexpression or knockdown. The expression levels were normalized to GAPDH, which was used as the control. (G) The expression levels of MYST2 and ZEB1 in the xenograft tumors were evaluated by IHC (200 magnification) (ns, not significant; *p < 0.05).

invasion assay demonstrated that overexpression of ZEB1 in QGY7703 and HepG2 cells suppressed the migration/invasion abilities. In addition, the rescue experiments indicated that the effect of suppression of miR-639 expression on migration/invasion and EMT was counteracted by ZEB1. Therefore, our results indicated that miR-639 suppresses migration/invasion by negatively regulating the expression of ZEB1, which modulates the EMT process in liver cancer cells.

miR-639 expression to augment the expression of its target genes, MYST2 and ZEB1, thereby contributing to tumorigenesis in liver cancer (Figure 6F). Our findings provide new insights into the relationship between methylation regulation and miR-639 in carcinogenesis and may provide potential new biomarkers for liver cancer.

MATERIALS AND METHODS Clinical Tissues and Cell Lines

In summary, we found that miR-639 expression is downregulated in liver cancer tissues and cells, which is attributed to hypermethylation of CpG islands on its promoter. Upregulated DNMT3A expression results in miR-639 promoter hypermethylation, which silences

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30 pairs of clinical specimens, including human liver cancer tissues and adjacent nontumor tissues, were obtained from patients undergoing hepatectomy for liver cancer without previous chemotherapy or radiotherapy. The collection of all of the specimens was

Please cite this article in press as: Xiao et al., miR-639 Expression Is Silenced by DNMT3A-Mediated Hypermethylation and Functions as a Tumor Suppressor in Liver Cancer Cells, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.021

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Figure 5. The miR-639-Induced Suppression of Liver Cancer Cell Growth Is Directly Mediated by MYST2 (A) The effects of the pcDNA3-MYST2 and pSilencer-shR-MYST2 plasmids in HepG2 cells were evaluated by western blotting and qRT-PCR. (B) MTT assay was performed in QGY-7703 and HepG2 cells with MYST2 overexpression or knockdown for 24, 48, and 72 hr. Representative images are shown. (C) Colony-formation assay was employed in QGY-7703 and HepG2 cells with MYST2 overexpression or knockdown. (D) The cell-cycle distribution and (E) PI were analyzed in HepG2 cells with MYST2 overexpression or knockdown. (F) Migration and (G) invasion assays were performed in QGY-7703 and HepG2 cells with MYST2 overexpression or knockdown. (H) MTT and (I) colony-formation rescue assays were performed in QGY-7703 cells in which the effects of miR-639 overexpression were rescued by MYST2. All the experiments are repeated at least three times (ns, not significant; *p < 0.05, **p < 0.01).

in accordance with the ethical standards of the institutional committee. Informed consent was obtained from each patient, and ethics approval was granted by the Ethics Committee at Sun Yat-sen University. Human liver cancer cell lines (QGY-7703, SMMC-7721, SK-Hep-1, Huh-7, LM-3, MHCC97-H, and HepG2) and the L02 immortalized human hepatocyte cell line were all routinely cultured in DMEM (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO BRL), 100 IU/mL penicillin and 100 mg/mL streptomycin at 37 C with 5% CO2. Plasmid Construction

MYST2, ZEB1, DNMT1, DNMT2, DNMT3A, DNMT3B, and primiR-639 cDNAs were amplified by RT-PCR and inserted into pcDNA3 or pcDNA3/Flag vectors to generate the following overexpression plasmids: pcDNA3-MYST2, pcDNA3-ZEB1, pcDNA3-

DNMT1, pcDNA3-DNMT2, pcDNA3-DNMT3A, and pcDNA3pri-miR-639 (pri-miR-639). The pcDNA3/Flag-ZEB1 vector contained a flag tag fused to the N terminus of ZEB1 (pcDNA3-ZEB1). Knockdown plasmids were constructed by inserting a DNA fragment encoding short hairpin RNA (shRNA) into the pSilencer vector to generate the following plasmids: pSilencer-shR-MYST2 (pshRMYST2), pSilencer-shR-ZEB1 (pshR-ZEB1), pSilencer-shR-DNMT1 (pshR-DNMT1), pSilencer-shR-DNMT2 (pshR-DNMT2), pSilencershR-DNMT3A (pshR-DNMT3A), pSilencer-shR-DNMT3B (pshRDNMT3B), and pSilencer-shR-MYST2 (pshR-MYST2). The antisense oligonucleotide of miR-639 was designed to block endogenous miR639 expression. The plasmids were validated by sequencing, and their effectiveness was confirmed by qRT-PCR and western blotting assays. The complementary DNA fragments of the 30 UTRs of ZEB1 and MYST2 were synthesized and inserted into the pcDNA3/EGFP vector to generate pcDNA3/EGFP-ZEB1-30 UTR (ZEB1-UTR) and

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Figure 6. miR-639 Inhibits the Migration and Invasion of Liver Cancer Cells by Targeting ZEB1 to Modulate the EMT Process (A) QGY-7703 cells transfected with pcDNA3-ZEB1 and pSilencer-ZEB1 plasmids were evaluated by western blotting. (B) Migration and (C) invasion assays were performed in QGY-7703 and HepG2 cells with miR-639 overexpression or knockdown. Representative images are shown. (D) The protein expression levels of E-cadherin, cytokeratin, ICAM-1, vimentin, and N-cadherin were measured by western blotting in QGY-7703 cells with ZEB1 overexpression or knockdown. (E) Migration and (F) invasion rescue assays were performed in QGY-7703 cells in which the effect of miR-639 knockdown was rescued by ZEB1. Representative images are shown. (G) A schematic showing the role of miR-639 in the regulatory network of HCC. All the experiments are repeated at least three times (ns, not significant; *p < 0.05).

pcDNA3/EGFP-MYST2-30 UTR (MYST2-UTR) plasmids. All the sequences are listed in Table S1.

turation 94 C 3 min, denature 94 C 20 s, anneal 60 C 30 s, extension 72 C 30 s, repeated for total of 32 times, final extension 72 C 2 min. The designed primers for ChIP-PCR are found in Table S1.

Fluorescent Reporter Assay

EGFP reporter plasmids for the target genes (MYST2-UTR and ZEB1UTR) were cotransfected with pri-miR-639 or ASO-miR-639 into QGY-7703 cells, and the pDsRed2-N1 RFP expression vector (Clontech, Mountain View, CA) was used as an internal control. The cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% NP-40, and 0.1% SDS; pH 8.0) after transfection for 48 hr, and the lysates were collected. The fluorescence intensities of EGFP and RFP were detected with an F4500 fluorescence spectrophotometer (HITACHI, Tokyo, Japan). ChIP-PCR Assay

The ChIP-PCR assay was conducted according to the Chromatin Immunoprecipitation Kit EZ-ChIP (Sigma, USA) instruction. Huh7 cells at 80%–90% confluency in a 150 mm culture dish were used to generate the chromatin for immunoprecipitations. The obtained lysate was sheared DNA by sonication method (3–5 s, for 16 times), next immunoglobulin G (IgG) (with 1 mL) and Anti-Myc-DNMT3A (10 mL, Tianjin, China) were added into the DNA lysate respectively, and then the crosslinked protein/DNA complexes was formed. Finally, to reverse the crosslinks of protein/DNA complexes to free DNA and conduct the PCR assay. The PCR program is as follows: initial dena-

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RNA Isolation and qRT-PCR

Trizol reagent was used to extract total RNA from liver cancer tissues and cell lines according to the manufacturer’s protocol. Small RNA (1 mg) or total RNA (3 mg) were reverse transcribed to cDNA by Moloney murine leukemia virus (M-MLV) (Promega, Madison, WI, USA). The levels of miR-639 and mRNAs were measured by qRT-PCR with SYBR Premix EX Taq (TaKaRa Otsu, Shiga, Japan) using an iQ5 real-time PCR system (Bio-Rad, USA). The relative fold changes in the transcripts were calculated with the 2-DDCt method. The results were normalized to endogenous U6 or b-actin levels. The sequences of the RT and qRT-PCR primers are shown in Table S1. Migration and Invasion Assays

QGY-7703 (3  104) or HepG2 (4  104) cells were seeded into the Transwell chambers (8 mm, Corning, Cambridge, MA) for migration assays. For the invasion assays, QGY-7703 (5  104) and HepG2 (8  104) cells were seeded into chambers with 40 mL of 2 mg/mL Matrigel (BD Biosciences, Bedford, MA). After incubating for indicated hr (12 hr for QGY7703 cells and 36 hr for HepG2 cells in the migration assays and 24 hr for QGY-7703 cells and 48 hr for HepG2 cells in the

Please cite this article in press as: Xiao et al., miR-639 Expression Is Silenced by DNMT3A-Mediated Hypermethylation and Functions as a Tumor Suppressor in Liver Cancer Cells, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.021

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invasion assays), the cells attached to the lower surface of the insert filter were counted after crystal violet staining. Dual-Luciferase Reporter Assay

To measure the activity of the promoter, we used a dual-luciferase reporter system. We inserted the promoter sequence of pri-miR-639 into the pGL3/luciferase-basic vector to construct pGL3/luciferasepri-639-p715 (pGL3-miR-639). The luciferase activity was measured according to the protocol of the Dual-Luciferase Reporter Assay System (Promega, MI, USA). MTT and Colony-Formation Assays

Cell viability and colony-formation assays were performed as previously described.22 FACS Assay

Flow cytometry was performed as previously described for cell-cycle analysis.43 Fluorescent Reporter Assay

EGFP reporter plasmids for the target genes (MYST2-UTR and ZEB1UTR) were co-transfected with pri-miR-639 or ASO-miR-639, respectively, into QGY-7703 cells, and the pDsRed2-N1 RFP expression vector (Clontech, Mountain View, CA) was used as an internal control. After 48 hr, the cells were lysed with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% NP-40, and 0.1% SDS; pH 8.0), and the lysates were collected. The intensity of EGFP and RFP was measured with a fluorescence spectrophotometer F-4500 (HITACHI, Tokyo, Japan). All experiments were performed more than three times. Immunohistochemistry Staining

Formalin-fixed paraffin-embedded (FFPE) tumor tissues harvested from mice were sectioned to a thickness of 4 mm. After routine deparaffinization, rehydration, blocking with hydrogen peroxide, and tissue antigen retrieval for 80 s, the samples were incubated with rabbit antiMYST2 and anti-ZEB1 antibodies (1:300 dilutions for both antibodies, Tianjin Saierbio, Tianjin, China) overnight at 4 C. The sections were stained with secondary antibodies and diaminobenzidine tetrahydrochloride (ZSGBBIO, Beijing, China), and then counterstained with hematoxylin.

fection for 48 hr. The primary antibodies were used as follows: GAPDH at a dilution of 1:2,000, E-cadherin, and N-cadherin at a dilution of 1:500, vimentin at a dilution of 1:3,000, intercellular cell adhesion molecule (ICAM), and cytokeratin at a dilution of 1:100, MYST2 at a dilution of 1:1,000, and ZEB1 at a dilution of 1:800. All antibodies were purchased from Tianjin Saierbio (Tianjin, China). The results were analyzed by LabWorks Image software. Tumor Growth in a Xenograft Mouse Model

To assess tumor cell proliferation in vivo, we suspended QGY-7703 cells (3  106 cells) stably expressing pri-miR-639 or pcDNA3 in 100 mL of serum-free RPMI-1640 medium and subcutaneously inoculated into the axillary fossae of female athymic nude mice (n = 6, 6– 8 weeks old). After 7 days, we measured the size of the tumor. 21 days after inoculation, the mice were sacrificed, and their tumors were harvested. All the studies were performed according to the American Association for the Accreditation of Laboratory Animal Care guidelines. Statistical Analysis

The data are presented as the mean ± SD of at least three independent experiments. A paired t test was used to perform statistical analyses of three independent experiments. Correlation between the expression of miR-639 and DNMT3A was assessed using Pearson correlation. *p < 0.05, **p < 0.01, and ***p < 0.001were considered statistically significant. All the tests were repeated more than three times.

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10. 1016/j.ymthe.2019.11.021.

AUTHOR CONTRIBUTION H.T. conceived the project and supervised the experiments. J.X., Y.L., F.W., R.L., Y.X., and Q.Y. performed the experiments. Y.L. and M.L. performed bioinformatics analysis. S.L. provided the clinical samples for the experiments. H.T., J.X., and Y.L. wrote the manuscript with help from all the authors. All authors reviewed the manuscript.

CONFLICTS OF INTEREST The authors declare no competing interests.

DNA Isolation and Methylation Detection

Genomic DNA was isolated from HCC cell lines and HCC tissues according to the instructions of the DNA extraction kit (BioTeke Corporation, http://www.bioteke.com). The DNA was treated with bisulfite (BS) according to the EZ DNA Methylation-Direct Kit protocol (Zymo Research, Orange, CA, USA). The miR-639 promoter was amplified by PCR with bisulfite sequence (BS) primers designed using MethPrimer and then cloned into the pMD18-T vector (TaKaRa, Japan) to assay the different sequences in the promoter. Western Blotting

Western blotting assays were performed according to a standard method. Cells were lysed in RIPA buffer to obtain proteins after trans-

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant numbers 81830094, 81572790, 81830094, 91629302, and 81573115), the Natural Science Foundation of Tianjin (grant number 19JCZDJC35900), and the Natural Science Foundation of Hebei Province (grant number H2018105049).

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