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SET and MYND domain containing 3 (SMYD3) is a histone methyltransferase (HMT) and transcription factor, which serves important roles in carcinogenesis. Numerous downstream target genes of SMYD3 have been identified in previous studies. However, the downstream microRNA (miRNA) s regulated by SMYD3 are yet to be elucidated. In the present study, the results of miRNA microarray demonstrated that 30 miRNA expression profiles were upregulated, whilst 24 miRNAs were downregulated by >2.0-fold in the SMYD3-overexpressed MCF-7 breast cancer cells. The HMT activity was demonstrated to be essential for SMYD3-mediated transactivation of miR-200c-3p and the overexpression of miR-200c-3p inhibited the transactivation effects of SMYD3 on myocardin-related transcription factor-A-dependent migration-associated genes. To our best knowledge, the current study is the first to report on the transcriptional regulation of SMYD3 on miRNAs, and miR-200c may be a downstream negative regulator of the SMYD3-mediated pathway in the migration of breast cancer cells. These results may provide a novel theoretical basis to understand the mechanisms underlying the initiation, progression, diagnosis, prevention and therapy of breast cancer.
SET and MYND domain containing 3 (SMYD3) is a novel histone methyltransferase gene identified in hepatoma and colon carcinoma cells by Hamamoto et al (1). SMYD3 is located on human chromosome 1 and encodes two protein isoforms that are composed of 428 and 369 amino acids. Previous studies have demonstrated that SMYD3 is frequently overexpressed in numerous types of cancer cells, including hepatic, colon, gastric and cervical carcinoma, and breast cancer (2–4), whilst the expression levels were lower in the corresponding normal tissue. A number of previous studies have demonstrated that SMYD3 has vital roles in the process of tumor development via its functions as a histone methylation enzyme and a transcription factor (5,6). SMYD3 modifies chromatin structure by catalyzing the methylation of histone H3 at lysine 4 (H3K4), H4K20 and H4K5 (5,6). Also, SMYD3 regulates the transcription of target genes via associating with RNA polymerase II or HELZ RNA helicase and binding at the motif CCCTCC or GGAGGG in the promoter (1).
MicroRNAs (miRNAs) are small, non-coding, endogenous RNA molecules of 18–22 nucleotides that were first identified in Caenorhabditis elegans. miRNAs suppress gene expression by binding the targeted mRNA transcripts, which causes translational repression or mRNA degradation. Previous studies demonstrated that miRNAs serve important roles in tumorigenesis through the regulation of genes involved in cancer development and maintenance (7,8).
A number of studies have identified that histone methylation and miRNAs are essential in the initiation and progression of cancer (7–9). However, the association between SMYD3 and miRNAs is yet to be elucidated. To investigate this further, the current study analyzed the global regulatory effects of SMYD3 on miRNAs in breast cancer cells using miRNA microarrays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
The MCF-7 human breast cancer cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The plasmid pcDNA5-TO/TAP-DEST-SMYD3 was a gift from Professor Philip Tucker from the Institute for Cellular and Molecular Biology, University of Texas (Austin, TX, USA). The CON049 (GeneChem, Co., Ltd., Shanghai, China) plasmid was used as a negative control for short hairpin (sh)SMYD3 (GeneChem, Co., Ltd.). The SMYD3-ΔNHSC plasmid was constructed by deleting the 205–208th amino acids (Asn-His-Ser-Cys) of pcDNA5-TO/TAP-DEST-SMYD3. The miR-200c-3p mimic and its negative control were obtained from Qiagen, Inc., (Valencia, CA, USA).
The MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM/F-12; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Tianjin Kangyuan Biotechnology Co., Ltd., Beijing, China) at 37°C in an atmosphere containing 5% CO2. For plasmid transfection experiments, MCF-7 cells were cultured in DMEM/F-12 medium without added hormones at 60% confluence for 12 h, and then transfected using TurboFect™ in vitro transfection reagent (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's protocol. Following incubation for 6 h, the medium was removed and replaced with normal culture medium (DMEM/F-12 medium without hormones) for 24 h. Then, the RT-qPCR was performed as described below. For miR-200c-3p mimic transfection experiments, Lipofectamine® 2000 reagents (Invitrogen; Thermo Fisher Scientific, Inc.) were used following the manufacturer's protocol.
Briefly, total cellular RNA was extracted from cultured cells using TRIzol reagent according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.) and 2 µg total RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocols. miRNA was isolated using the miRcute miRNA isolation kit (#DP501; Tiangen Biotech, Co., Ltd., Beijing, China), followed by cDNA synthesis using SuperScript First-Strand Synthesis system (Invitrogen; Thermo Fisher Scientific, Inc.), but with the specific stem loop primer primers. The miRNA RT-PCR primer sequences were as follows: JH6-miR200c-3p, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCCATC-3′; JH7-miR200c-3p, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCCATCA-3′; JH7-miR149-3p, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGGAGTG-3′. The total RNA and dNTPs were incubated at 65°C for 5 min, cooled on ice briefly. Subsequently, the miRNA specific primer, RNase inhibitor and reverse transcriptase were added. After being incubated at 25°C for 10 min, 37°C for 50 min and 70°C for 15 min, the cDNA was synthesized and used as a template of qPCR.
miRNA expression profiling microarray was completed using Agilent human miRNA (8×60K) V18.0 miRNA array (LC Sciences, LLC., Houston, TX, USA). The microarray probe sequence was derived from Sanger MiRBase version 15.0 (http://microrna.sanger.org). Each chip contained multiple quality control probes and employed dual-color chip to examine miRNA expression profiling in MCF-7 cells with overexpressed or normal endogenous expression of SMYD3. Probes were synthesized in situ with photosensitive photogenerated reagents. The sequence consisted of two fragments: A chemically modified oligonucleotide encoding fragment complementary to target miRNA; and an extension arm at the distance specific to the connected encoding sequence that reduced the hybridization spatial impairment. Slides were scanned on an Agilent microarray scanner (model G2565A; Agilent Technologies, Inc., Santa Clara, CA, USA) at 100 and 5% sensitivity settings. Agilent Feature Extraction software version 8.1 (Agilent Technologies, Inc.) was used for image analysis (10–12).
RT-qPCR was performed using a StepOne™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Bestar® SYBR-Green qPCR Mastermix was obtained from DBI Bioscience (http://www.xinghanbio.com/cpzs; Shanghai, China). The thermal profiles were 95°C for 10 sec and 60°C for 1 min. Melting curve analysis was performed for each PCR to confirm the specificity of amplification. At the end of each phase, fluorescence was measured and quantified. Data is presented as the relative expression level of mRNA following normalization to GAPDH or the relative expression levels of miRNA following normalization to U6 following calculations using the 2−ΔΔCq method (13). The PCR primer sequences were as follows: GAPDH forward, 5′-ATTCAACGGCACAGTCAAGG-3′ and reverse, 5′-GCAGAAGGGGCGGAGATGA-3′; zinc finger E-box binding homeobox (ZEB)1 forward, 5′-AAGGGCAAGAAATCCTGGGG-3′ and reverse, 5′-CTCTGGTCCTCTTCAGGTGC-3′; ZEB2 forward, 5′-AAATGCACAGAGTGTGGCAAGG-3′ and reverse, 5′-CTGCTGATGTGCGAACTGTAGGA-3′; SMYD3 forward, 5′-AAGTTCGAACCGCCAAGAG-3′ and reverse, 5′-AAGGCAGCGGTCGCAGACGA-3′; myosin light chain 9 (MYL9) forward, 5′-GAGCCCAAGCGCCTTCT-3′ and reverse, 5′-GTCAATGAAGCCATCACGGT-3′; cysteine rich angiogenic induced 61 (CYR61) forward, 5′-AAGGGGCTGGAATGCAACTT-3′ and reverse, 5′-TTGGGGACACAGAGGAATGC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′; and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; miR200c-3p forward, 5′-ACACTCCAGCTGGGTAATACTGCCGGGTAAT-3′; miR149-3p forward, 5′-ACACTCCAGCTGGGTCTGGCTCCGTGTCTTG-3′; and general reverse, 5′-TGGTGTCGTGGAGTCG-3′. All experiments were repeated three times.
In total 5 online prediction software programs, including miRDB (http://mirdb.org/), TargetScan (http://www.targetscan.org/), DIANA-microT (http://diana.cslab.ece.ntua.gr/microT/), microRNA.org (http://www.microrna.org/microrna/home.do) and RNA22 (https://cm.jefferson.edu/rna22), were used to predict the target genes of miRNAs. The intersection of 3/5 of these software programs were selected as the potential target genes.
Statistical evaluations were performed using GraphPad Prism (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA), using 3 independent experiments and were analyzed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference. The homologous alignment analysis was performed using DNAMAN (version 6.0; Lynnon LLC., San Ramon, CA, USA) and Primer Premier (version 5.0; Premier Biosoft International, Palo Alto, CA, USA).
To investigate the regulatory effects of SMYD3 on global miRNA in breast cancer cells, MCF-7 cells were transfected with SMYD3 and microarray analysis was performed using the Agilent human miRNA (8×60K) V18.0 microarrays chip that contains 1,871 miRNAs. As presented in Fig. 1A, the expression profile analysis identified 1,871 microRNAs with altered expression, including 191 miRNAs that were upregulated in SMYD3-transfected cells compared with mock transfected cells, whilst 1,680 miRNAs were downregulated. Amongst these, 30 miRNAs were upregulated >2.0-fold and 24 miRNAs were downregulated <2.0-fold (Fig. 1B and C). Furthermore, to investigate the function of these miRNAs in more depth, the potential target genes of these miRNAs and their functions were summarized using bioinformatic analysis (Table I) (14–57).
To confirm the results of miRNA microarray, miR-200c-3p, a predicted target that was upregulated by 37.9-fold, and miR149-3p, a predicted target that was downregulated by 20.9-fold by SMYD3 in the chip assay, were selected for further investigation. The pcDNA5-TO/TAP-DEST-SMYD3 plasmid and the shSMYD3 plasmid were transfected into MCF-7 cells for the overexpression and knockdown of SMYD3, respectively. In accordance with the results of miRNA microarray, the results of RT-qPCR identified that miR-200c-3p was significantly upregulated following SMYD3 overexpression (P<0.001) and downregulated following RNA interference (RNAi)-mediated suppression of SMYD3 (P<0.05; Fig. 2A and B), whereas the level of miR149-3p exhibited a significant negative association with the expression of SMYD3 (P<0.001; Fig. 2C and D).
The promoter sequence of miR-200c-3p from a number of species, including of Homo sapiens, Macaca mulatta, Pan troglodytes, Monodelphis domestica, Equus caballus and Rattus norvegicus, were analyzed using bioinformatic methods. The results identified that 8 conserved SMYD3 binding sites exist in the promoter region (−2500 to +1) of miR-200c-3p from numerous species, indicating that miR-200c-3p may be a target for the transcriptional regulation of SMYD3 (Fig. 3).
Furthermore, the results of RT-qPCR assay demonstrated that the mRNA level of ZEB1 and ZEB2, two predicted target genes of has-miR-200c-3p presented in Table I, was decreased by SMYD3-overexpression, whereas they were increased by RNAi-induced suppression of endogenous SMYD3 (Fig. 4A and B). Taken together, these results demonstrate that SMYD3 activates the transcription of miR-200c-3p and therefore indirectly decreases the mRNA levels of ZEB1/2.
Considering the results of the present study and the previous studies that focusing on the histone methylation activity of SMYD3, it was hypothesized that SMYD3 may function as a transactivator of miR-200c-3p via its histone methylation function. To investigate this, MCF-7 cells were treated with 100 µM methylthioadenosine, a histone methylation inhibitor, with or without physiological concentration of estradiol (10–11 M) and subsequently the transcription level of miR-200c-3p was detected using RT-qPCR. As presented in Fig. 5, the transcription level of miR-200c-3p was significantly downregulated by the histone methylation inhibitor (Fig. 5A). Consistently, SMYD3-ΔNHSC, a histone methyltransferase-activity-depleted mutant of SMYD3 (58,59), also decreases the transcription level of miR-200c-3p (Fig. 5B). These results suggested that the histone methylation activity may have vital roles in SMYD3-mediated transactivation of miR-200c-3p.
The authors' previous study demonstrated that SMYD3 promotes MRTF-A-mediated transactivation of MYL9 and migration of MCF-7 breast cancer cells (59). However, other previous studies have identified that miR-200c represses migration and invasion of breast cancer cells via suppression of the RhoA-MRTF-A signaling pathway (60,61). As the results of the present study demonstrated the overexpression of SMYD3 directly transactivates miR-200c-3p, whether SMYD3 and miR-200c-3p supports or opposes each others' effects on the transcription of MYL9 and CYR61, two MRTF-A-dependent migration-related genes, was investigated. The miR-200c-3p mimic and/or SMYD3-overexpression plasmid were transfected into MCF7 cells and the transcription level of MYL9 and CYR61 were detected using RT-qPCR. The results of RT-qPCR demonstrated that the transcriptional level of SMYD3 and the miR-200c-3p increased significantly following the transfection (Fig. 6A and B). As presented in Fig. 6C, the mRNA levels of MYL9 and CYR61 were suppressed by miR-200c-3p and increased by the overexpression of SMYD3. Furthermore, the upregulation of MYL9 and CYR61 remained after SMYD3 was co-transfected with miR-200c-3p mimics, but the effect in combination group was reduced compared with that of the SMYD3-transfected group. These results demonstrated that miR-200c-3p may be a downstream negative regulator of the SMYD3-mediated pathway in the migration of breast cancer cells.
The primary focus of epigenetics is to elucidate the heritable changes in gene expression and regulation, which occur without mutation to the DNA sequence, in the process of gene transcription in eukaryotic cells. The heritable alterations include DNA methylation, histone modification and regulation by noncoding RNAs. A number of studies have established that the majority of malignant tumor types exhibit cancer-associated epigenetics alterations (62). However, the interdisciplinary investigation of miRNA and histone modification has received increasing interest.
A previous study has analyzed downstream coding genes of SMYD3 using cDNA microarray and the results identified that there were 61 genes upregulated >3-fold and genes downregulated <3-fold by SMYD3 (1). However, to date, the downstream miRNAs regulated by SMYD3 are yet to be elucidated. Therefore, the present study focused on the effect of SMYD3 on the miRNA expression profile in MCF-7 breast cancer cells by analyzing microarray and RT-qPCR data. These results demonstrated that 30 miRNAs were upregulated >2.0-fold, whilst 24 miRNAs were downregulated <2.0-fold following the overexpression of SMYD3. The analysis of bioinformatic and previously published data identified that the majority of these potential downstream miRNAs of SMYD3 were associated with the proliferation, migration and therapy resistance of tumor.
To further validate the results of microarray, RT-qPCR was performed to detect the effects of SMYD3 on the expression of miR-200c-3p and miR-149, which are 2 representative target miRNAs of SMYD3 with opposing alterations, and the results were in accordance with those of the microarray. Previous studies have demonstrated that miR-149 and miR-200c are associated with the progression of cancer (63–65). miR-149 has been established as a tumor suppressor by inhibiting the spreading, migration and invasion of basal-like breast cancer cells (63). However, the roles of miR-200c in cancer remain to be fully understood. A number of previous studies have reported that miR-200c may inhibit the epithelial-mesenchymal transition and enhance the chemosensitivity and radiosensitivity of cancer cells (64,65), whereas another previous study reported a metastasis-promoting role of miR-200 in breast cancer (64). In addition, a separate study demonstrated that the expression level of miR-200c different between the subtypes of breast cancer (65). Compared with the normal breast epithelial cells (MCF-10A), the basal cancer cells (MDA-MB-231 and BT549) exhibited a reduced expression of miR-200c (65). However, the expression of miR-200c in luminal cancer cells (MCF-7 and BT474) was increased compared with that in the normal breast epithelial cells (65). Additionally, previous studies have suggested that miR-200c may repress migration and invasion of breast cancer cells via the suppression of RhoA-MRTF-A signaling pathways (60,61), whereas the current study suggests that SMYD3 promotes MRTF-A-mediated transactivation of MYL9 and migration of MCF-7 breast cancer cells (59). The results in the present study have demonstrated that the expression of miR-200c in MCF-7 breast cancer cells may be upregulated by SMYD3, and the overexpression of miR-200c partially inhibits the transactivation effects of SMYD3 on the MRTF-A-dependent migration-associated genes. Taken together, these results indicated that miR-200c may be a downstream negative regulator of the SMYD3-mediated pathway in the migration of breast cancer cells, and may promote a negative feedback loop to prevent excessive induction of migration of breast cancer cells. However, the underlying mechanism of miR-200c in breast cancer remains to be elucidated by future investigation.
Additionally, previous studies have identified that SMYD3 alters chromatin structure by catalyzing the di-/tri-methylation of histone H3 at lysine 4 (H3K4me2/3), H4K20me3 and H4K5me (5,6). As part of the established methylation marks at H3K4 or H3K36, mono-methylations of H3K9, H3K27, H3K79, H4K20 and H2BK5 are associated with transcriptional activation, whereas trimethylations of H3K9, H3K27, H3K79 and H4K20 are associated with transcriptional repression (5,6). The current study demonstrates that the histone methylation activity is essential for SMYD3-mediated transactivation of miR-200c-3p. Therefore, SMYD3 antagonistic regulation of the downstream miRNAs may also depend on the distinct modification on the histone adjacent to the promoter.
In conclusion, the effect of SMYD3 on the miRNA expression profile in MCF-7 breast cancer cells was analyzed using microarray and RT-qPCR in the present study. To the best of our knowledge, this is the first study focused on the transcriptional regulation of SMYD3 on miRNAs. These results may provide a novel theoretical basis to elucidate the mechanism underlying the initiation, progression, diagnose, prevention and therapy of breast cancer.
The current study was supported by grants from the National Natural Science Foundation of China (grant no. 31470816, 31300642 and 31000343), the College Students' Innovation and Entrepreneurship Training Program of Tianjin (grant no. 201510057057) and the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (grant no. IRT1166).