MIR2052HG regulates ERα levels and aromatase inhibitor resistance through LMTK3 by recruiting EGR1

Our previous GWAS using the MA.27 aromatase inhibitors (AIs) adjuvant trial identified SNPs in the lncRNA MIR2052HG associated with breast cancer free interval. Here we report that MIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing PKC activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor - dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibility of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer.


Introduction
Estrogens have long been recognized to be important for stimulating the growth of estrogen receptor α (ERα) positive breast cancer, a subtype that represents a large proportion of breast cancer patients. Estrogen action is mediated by ERα. Approximately 70% of breast cancers are ERα positive and rely on estrogen signaling to stimulate their growth and survival [1,2]. Its presence in breast tumors is routinely used to predict response to endocrine therapies that target 3 ERα, estrogen production or estrogen signaling. AIs suppress estrogen synthesis in postmenopausal women by targeting the aromatase enzyme, which converts precursor hormones to estrogens. The third-generation AIs (i.e., exemestane, anastrozole, and letrozole) have largely replaced tamoxifen as the preferred treatment for ERα positive breast cancer in postmenopausal women with early stage breast cancer because of their superior efficacy over tamoxifen [3,4].
However, both de novo and acquired resistance to AIs can occur, resulting in relapse and disease progression. It is estimated that approximately 30% of ER-positive breast cancer receiving adjuvant AI treatment eventually develop resistance [5][6][7], while nearly all patients develop resistance in the metastatic setting. The mechanisms for endocrine therapy resistance are complex and one mechanism includes dysregulation of ERα expression, encoded by ESR1 [8].
ERα is a member of the nuclear receptor superfamily of ligand-activated transcription factors [9], which regulates gene expression through direct binding to estrogen response elements (EREs) in promoters of estrogen-regulated genes and indirectly through recruitment to gene promoters by interaction with other transcription factors [10]. Previous studies have reported that ESR1 is upregulated during estrogen deprivation adaptation [11]. Overproduction of ERα leads to an enhanced response to low concentrations of estrogen, which is responsible for the acquisition of AI resistance or postmenopausal tumorigenesis [12,13]. In these AI-resistant tumors, ERα is either hypersensitive to low levels of estrogens [14] activated in a ligand-independent manner by phosphorylation via kinases in the growth factor receptor signaling pathways, or by acquired somatic ESR1 mutations [15,16]. ERα phosphorylation aids in regulating the transcriptional activity and turnover of ER by proteasomal degradation. Of particular importance are Ser118 and Ser167, which locate within the activation function 1 region of the N-terminal domain of ERα and are regulated by multiple signaling pathways [17][18][19][20]. The phosphorylation at Ser118 can be mediated by mitogen-activated protein kinase (MAPK) activation and induces ERα activity [15,21], whereas Ser167 can be phosphorylated by p90RSK [22,23] and plays a role in Lemur Tyrosine Kinase 3 (LMTK3)-mediated ERα stabilization [24,25]. LMTK3 has been implicated in both de novo and acquired endocrine resistance in breast cancer [26]. The phosphorylation of ERα at S167 is positively associated with pMAPK and pp90RSK in breast cancer patients and a predictor of better prognosis in primary breast cancer with reduced relapse and better overall survival [27].
Our previous genome-wide association study (GWAS) used samples from the Canadian Cancer Trials Group MA.27, the largest AI breast cancer adjuvant endocrine therapy trial (4,406 controls without recurrence of breast cancer and 252 cases with recurrence). In that study, we identified common SNPs in a long noncoding (lnc) RNA, MIR2052HG, that were associated with breast cancer free interval (HR= 0.37, P= 2.15E-07) [28]. The variant SNPs (minor allele frequency [MAF]= 0.32 to 0.42) were associated with lower MIR2052HG and ERα expression in the presence of AIs, and two of the top SNPs, rs4476990 and rs3802201, were located in or near an ERE [28]. MIR2052HG appeared to affect ERα expression both by promoting AKT/FOXO3mediated ESR1 transcription regulation and by limiting ubiquitin-mediated ERα degradation [28]. However, the underlying mechanisms by which MIR2052HG regulates ESR1 transcription and ERα degradation remain unknown.
LncRNAs are transcripts with no protein-coding functions. Accumulating evidence suggests that lncRNAs play critical roles in regulating a wide range of cellular processes through affecting various aspects of protein, DNA, and RNA expression and interactions [29][30][31]. Several lncRNAs have been implicated in breast cancer. For example, UCA1 is an oncogene in breast cancer that can induce tamoxifen resistance [32]. LncRNA HOTAIR is positively correlated with tamoxifen resistance [33]. In the current study, we sought to further investigate the mechanism of MIR2052HG action in the regulation of ERα and AI resistance. We found that MIR2052HG directly interacts with the early growth response protein 1 (EGR1) protein to enhance LMTK3 transcription and thus sustained ESR1 expression and stabilized ERα protein.

MIR2052HG regulates LMTK3 expression
We previously reported that MIR2052HG sustained ERα levels by promoting AKT/FOXO3 mediated upregulation of ESR1 transcript and by limiting proteasome-dependent degradation of ERα protein [28]. However, the mechanism involved in the regulation of MIR2052HG-mediated AKT activation and ERα ubiquitination remains unknown. Kinome screening previously identified LMTK3 as a potent ERα regulator, acting by decreasing the activity of protein kinase C (PKC) and the phosphorylation of AKT (Ser473), resulting in increased binding of FOXO3 to the ESR1 promoter [24]. LMTK3 also protected ERα from proteasome mediated degradation [24]. Given that the effects of LMTK3 on ERα were similar to our observations with MIR2052HG [28], we hypothesized that MIR2052HG might regulate LMTK3 to mediate ERα levels and in turn, response to AIs.
Previous studies have demonstrated that lncRNAs can function in trans to regulate expression of protein-coding genes; therefore, we examined the possibility that MIR2052HG may facilitate AI resistance by regulating LMTK3 expression. Consistent with this hypothesis, we found that knockdown of MIR2052HG using a pooled antisense oligonucleotides (ASO) in human ER positive CAMA-1 breast cancer cells resulted in a dramatic decrease of LMTK3 expression ( Fig   1A). A similar effect was also observed in an aromatase overexpressing cell line, MCF7/AC1 6 [34] (Fig 1B). We also observed that the changes in mRNA levels were confirmed at the protein level by the western blot analysis (Fig 1C), supporting the notion that MIR2052HG regulates LMTK3 expression. To determine whether LMTK3 is a major downstream target of MIR2052HG in regulating AI response, we first determined the transcriptome changes in MIR2052HG -knockdown cells and collected published RNA-seq data after LMTK3-knockdown [26]. Analysis of the RNA-seq data indicated that the changes induced by MIR2052HG knockdown and LMTK3 knockdown showed a large number genes overlapped, especially almost 1/3 genes regulated by LMTK3 were also regulated by MIR2052 ( Fig 1D, Table EV1).
The common dysregulated genes in both knockdowns included cell cycle genes, oocyte maturation and oocyte meiosis genes (Table EV1).
To further define the relationship between MIR2052HG and LMTK3, we transfected LMTK3 overexpressing constructs into ERα positive breast cancer cells with MIR2052HG -knockdown, followed by cell growth and colony forming assays. The cell proliferation and colony formation analysis demonstrated that the cell growth defect caused by downregulation of MIR2052HG could be successfully rescued by LMTK3 overexpression (Fig 2A-D), indicating that LMTK3 is a major target that mediates the MIR2052HG regulation on cell growth in ER positive breast cancer.

LMTK3 mediates MIR2052HG -regulation of ESR1 transcription and ERα protein stability
Previous studies demonstrated that MIR2052HG regulates ERα expression through transcription regulation of ESR1 and ER protein degradation [24,28]. However, the direct target of MIR2052HG in ERα regulation has not been fully elucidated. Therefore, we examined the role 7 of LMTK3 in ERα regulation. Our previous report indicated that the effect of MIR2052HG on ESR1 transcript is through AKT/FOXO3 [24]. In MCF7/AC1 and CAMA-1 cells, downregulation of MIR2052HG reduced ESR1 mRNA levels by promoting AKT-mediated downregulation of FOXO3 protein level, a transcription factor known to be involved in ESR1 transcription (Fig 3A, B). Whereas LMTK3 overexpression rescued the downregulation of ERα mRNA induced by MIR2052HG silencing (Fig 3A, B). LMTK3 overexpression resulted in a decrease in phosphorylated AKT (at Ser473), and an increase in FOXO3protein level but not mRNA level (Fig 3A, B, Fig EV1A). At the protein level, ERα protein was reduced by MIR2052HG knockdown, whereas LMTK3 overexpression stabilized ERα (Fig 3A, B, right panel). Our previous results showed that MIR2052HG also regulated ERα stability by regulating its proteasome dependent degradation process. Here, LMTK3 overexpression could increase protein level by decreasing ERα ubiquitination ( Fig 3C). Together, these data indicate that LMTK3, downstream of MIR2025HG, mediated MIR2052HG effect on the regulation of ESR1 transcription and ERα protein stability. Using the TCGA data set, LMTK3 showed higher expression level in ER positive breast cancer patients compared with other subtypes (Fig 3D), and RNA expression levels were also independently associated with disease-free survival and overall survival ( Fig 3E).

LMTK3/PKC/MEK/ERK/RSK1 pathway
Next, we investigated the mechanisms involved in MIR2052HG and LMTK3 regulation of ERα protein degradation. ERα phosphorylation, especially increased phosphorylation of ERα at Ser167, has been implicated in ERα proteasome-mediated degradation [24]. To explore the mechanism, we first determined the level of ERα pSer167 in MIR2052HG knockdown ERα 8 positive breast cancer cells. ERα pSer167 levels increased with MIR2052HG knockdown, despite decreased total ERα amounts ( Fig 4A). Furthermore, we observed that knocking down MIR2052HG increased wild type (WT) ERα ubiquitination, but not the mutant ERα with serine 167 to alanine (S167A) (Fig 4B), confirming the involvement of ERα Ser167 in ubiquitindependent and proteasome-mediated degradation. The phosphorylation of ERα at Ser167 is regulated by pp90 (RSK1) [22], which is activated by MAPK [35]. We thus hypothesized that MEK/ERK/p90RSK1 might be the signaling pathway that mediates ERα phosphorylation at Ser167 upon MIR2052HG knockdown. We first tested the effect of knockdown of MIR2052HG on MEK/ERK/p90RSK1 activity. As shown in Figure 4A, in MCF7/AC1 and CAMA-1 cells transfected with MIR2052HG ASO, coinciding with increased ERα pSer167, pMEK, pERK and pRSK1levels were also increased, indicating an increased MEK/ERK/p90RSK1 activity. We then examined the role of LMTK3 in MIR2052HG-mediated regulation of MEK/ERK/p90RSK1 activity, and found that LMTK3 overexpression abolished increased pMEK/pERK/p90RSK1 levels caused by MIR2052HG silencing (Fig 4C, D). These data further imply that MIR2052HG regulates LMTK3 expression, which then influences MEK/ERK/p90RSK1 activity, regulating ERα protein levels.
As protein kinase C (PKC) has been implicated to play a role in ERα protein degradation [36] and AKT -FOXO3 regulation [37], and LMTK3 inhibits PKC activity [24], we examined the effects of MIR2052HG and LMTK3 on PKC. In vitro kinase assays indicated that downregulation of MIR2052HG increased PKC activity (Fig 4E, F, Fig EV1B), whereas overexpression of LMTK3 decreased PKC activity (Fig 4E, F, Fig EV1B). In addition, LMTK3 overexpression dramatically reduced PKC activity that was induced by MIR2052HG silencing (Fig 4E, F, and Fig EV1B). Inhibition of PKC with the Go 6983 inhibitor reduced 9 MEK/ERK/p90RSK1 activity and ERα pSer167, which in turn, partially rescued ERα levels ( Fig   4G), suggesting that MIR2052HG regulated ERα protein level through the axis of LMTK3/PKC/MEK/ERK/RSK1. Our findings also confirmed that MIR2052HG effects on AKT/ FOXO3 activation and downstream ESR1 mRNA level were through the regulation of LMTK3/PKC pathway (Figs 3-4).

MIR2052HG contributes to LMTK3 transcription by facilitating EGR1 recruitment
Next, we wanted to address how MIR2052HG regulates LMTK3 transcription. First, we examined the localization of the MIR2052HG RNA transcript. RNA fluorescent in situ hybridization (FISH) demonstrated that MIR2052HG localized to a limited number of nuclear foci (one to two spots in most cases), suggesting that MIR2052HG had limited targets. We also checked the genomic location of the MIR2052HG transcript by RNA-DNA dual FISH, and the results showed that the MIR2052HG transcript was located at the LMTK3 gene locus (Fig 5A,   Fig EV2). Taken together, these data suggest that MIR2052HG is likely involved in LMTK3 transcription.
LMTK3 expression could be activated by several transcription factors based on the ENCODE database, including BHLHE40, CHD2, CTCF, EGR1, EP300, EZH2, HDAC6, POLR2A, REST, CREBBP, YY1, and STAT1. Therefore, we asked whether any of these transcription factors, together with MIR2052HG might be involved in the regulation of MIR2052HG expression.
Immunoprecipitation followed by qRT-PCR analysis demonstrated that MIR2052HG was significantly enriched in the EGR1 immunoprecipitates (Fig 5B, C). The enrichment of MIR2052HG by the EGR1 antibody was specific, as the antibody did not pull down another 10 lncRNA, LOC102724785 (Fig 5B, C). These data suggest that MIR2052HG regulation of LMTK3 transcription involves EGR1.
EGR1 was highly expressed in the Cancer Genome Atlas (TCGA) [38] ER positive breast cancer patients ( Fig EV3). We then confirmed EGR1 regulation of LMTK3 gene expression in the MCF7/AC1 and CAMA-1 cells. Knockdown of EGR1 reduced LMTK3 mRNA level (Fig 6A,   B). To examine whether binding of EGR1 to the LMTK3 promoter requires MIR2052HG, we first mapped the binding locations of EGR1 on the LMTK3 gene locus (

AIs modulate LMTK3 expression in a MIR2052HG SNP-dependent manner
Our previous GWAS showed that MIR2025HG SNPs regulate its own gene expression as well as ERα expression in an estrogen or AI dependent fashion [28]. Based on our finding showing MIR2025 regulation of LMTK3, we then determined whether the expression of LMTK3 might be also MIR2025HG SNP-and AI-dependent using the human lymphoblastoid cell lines (LCLs) system. This cell line model system, consisting of 300 individual LCLs for which we have extensive genomic and transcriptomic data, has shown repetitively to make it possible for us to study the relationship between common genetic variant and cellular phenotypes [28,39,40]. In the presence of androstenedione, LCLs with variant genotypes for both of the MIR2052HG SNPs, rs4476990 and rs3802201, showed dose-dependent increases in LMTK3 expression ( Fig   7A, B). However, addition of AI, either anastrozole ( Fig 7A) or exemestane ( Fig 7B) caused a "reversal" of the expression pattern with increased LMTK3 expression in LCLs with homozygous WT, but a marked decrease in LCLs homozygous for the variant genotypes. Of particular interest was the observation of a direct correlation between this pattern of expression for MIR2052HG and ERα [28] and that of LMTK3 (Fig 7A, B).
Since MIR2052HG regulated LMTK3 expression in a SNP-and AI-dependent fashion (Fig 7A, B), we determined if EGR1 binding to the LMTK3 promoter region was also SNP-and AIdependent. In the presence of androstenedione, cells homozygous for the variant SNP genotypes showed increased binding of EGR1 to the LMTK3 promoter (Fig 7C, D) relative to WT in ChIP assays using the EGR1 antibody. Anastrozole and exemestane could reverse this effect (Fig 7C,   D). These results suggest that MIR2052HG facilitates EGR1 recruitment to the LMTK3 promoter region in a SNP-dependent fashion to activate LMTK3 transcription.

Discussions
Resistance to endocrine therapy represents a major challenge for ERα positive breast cancer therapy. Therefore, the identification of biomarkers for endocrine response and understanding mechanisms of endocrine resistance should reveal possible strategies to overcome this problem.
We have previously demonstrated that germline genetic variations in MIR2052HG were associated with breast cancer free interval in the MA27 trial [28]. Downregulation of MIR2052HG reduced ERα positive breast cancer cell growth. The variant SNPs were associated with increased MIR2052HG expression due to increased ERα binding to EREs [28]. Therefore, MIR2052HG plays an important role in regulating ERα and endocrine resistance [28]. Recently, LMTK3, a serine-threonine-tyrosine kinase, has gained attention in breast cancer with respect to its roles in pathogenesis and therapy resistance of breast cancer [24,41,42]. The fact that overexpression of LMTK3 significantly rescued the cell growth defect caused by MIR2052HG depletion suggests that LMTK3 is one of the downstream targets of MIR2052HG (Fig 2). LncRNAs can play diverse roles in regulating gene expression as well as other cellular activities in breast cancer [43][44][45]. LncRNAs produce their cellular effects via several distinct mechanisms, including acting both in cis and trans [29,30]. Here, we demonstrated that MIR2052HG exerted its oncogenic role by regulating LMTK3 expression. LMTK3 is significantly elevated in high-grade breast tumors and is associated with poor survival rates in different breast cancer cohorts [24,26]. A prior study has shown that methylation is not a prevalent mechanism in the control of LMTK3 expression in breast cancer, and several somatic mutations in LMTK3 have been associated with overall survival [24]. However, we did not find any germline variations in LMTK3 associated with breast cancer recurrence in our MA.27 cohort, suggesting a LMTK3 upstream regulator such as MIR2052HG might be the driving factor influencing this clinical phenotype. We found that MIR2052HG was induced by hormone or AIs, and it was required for the LMTK3-mediated phenotypes, including cell growth in response to AIs (Fig 7). Current research into the potential role of LMTK3 as a therapeutic target is 13 underway [46,47]. At mechanistic level, we found that MIR2052HG positively regulated ERα at both mRNA and protein levels via LMTK3 to maintain the cancer cell growth. LMTK3 mediated the effect of MIR2052HG on AI response via ERα transcription through the LMTK3/PKC/AKT/FOXO3 signaling and protein levels via the LMTK3/PKC/MAPK pathway (Figs 3 and 4). We also found a positive correlation between the expressions of LMTK3 and ESR1 (Fig EV6) in the METABRIC and TCGA set data sample set [38], as well as in our LCLs model (p = 3.45E-04, rho=0.212). Due to the low expression levels of MIR2052HG in some of the patient samples (Fig EV3), we did not find strong correlation between the expressions of MIR2052HG and LMTK3.
EGR1 is an immediate-early gene induced by estrogen, growth factors, or stress signals [48]. The EGR1 protein binds to a specific GC-rich sequence in the promoter region of many genes to regulate the expression of these target genes including growth factors and cytokines. The mechanisms by which EGR1 activates downstream target genes appears to be cell-context dependent [49][50][51]. Although the DNA-binding domain of EGR1 is capable of binding to DNA through the GC-rich consensus sequence GCG (G/T) GGGCG, EGR1 can act as either an activator or a repressor of transcription through mechanisms that depend on interactions with distinct cofactors, and thus many partners, including DNA-binding proteins, have been reported to form complexes with EGR1 to activate EGR1 target gene expression [52,53]. In our study, we demonstrated that the association of MIR2052HG with EGR1 facilitated EGR1 binding to the LMTK3 promoter (Figs 5 and 6). Based on the current data, we propose a hypothetical model that may explain how MIR2052HG contributes to LMTK3 activation and AI resistance (Fig 6G). In the model, we showed that MIR2052HG facilitated the recruitment of EGR1 to the LMTK3 promoter through its interaction with EGR1and activated LMTK3 transcription. This process 14 might also involve other transcription co-factors. It is possible that other proteins are also required for the binding of MIR2052HG to EGR1, since some RNA-binding proteins have been shown to be able to regulate EGR1 [54]. Nevertheless, RNA-mediated EGR1 targeting represents one mechanism by which EGR1 is recruited to its targets.
In conclusion, our findings support a model in which the protective MIR2052HG variant

Quantitative real-time PCR assay (qRT-PCR)
QRT-PCR assays were performed for measuring gene expression using the TaqMan RNA-to-Ct 1-Step Kit. RNA was extracted using the miRNeasy mini Kit. RNA was measured by NanoDrops300. The TaqMan primers for MIR2052HG, ESR1 and GAPDH were purchased from Life Technologies. Primers for EGR1 targeted genes were purchased from IDT. QRT-PCR reactions were prepared following manufacturer's protocol. Samples were run using the StepOnePlus real-time PCR system.

Western blotting
Cells were washed with cold PBS and were lysed in cold NETN buffer (100mM NaCl, 20 mM All blots were visualized with the Supersignal WestPico or Supersignal WestDura chemiluminescent ECL kits and blue X-ray films or Gel Doc XR+ Gel documentation system.

RNAseq analysis and normalization
RNA was prepared from cells using the TRIzol extraction kit. Genomic DNA was removed using the Ambion DNA-free kit. NuGEN Encore reagents were used for library preparation of total RNA samples. One microgram of total RNA input was used for each sample. The libraries were sequenced on an Illumina HiSeq 2000 sequencing system using 100-bp single-ended reads.
After removing the poor-quality bases from FASTQ files for the whole transcriptome sequencing, paired-end reads were aligned by reads that were aligned to the human reference genome UCSC hg19 with Tophat 2.0.14 and the bowtie 2.2.6 aligner option. Transcript abundance was estimated using a count-based method with htseq-count.

Cell proliferation assays
Cells were seeded (3000 cells/100 µL/well) in a 96-well plate. The CyQUANT Direct Cell Proliferation Assay kit was used to determine the cell viability in six replicates. CyQUANT assays were performed to determine the cell viability every two days. In the assay, OD 490nm (optical density) represents the absorbance at the wavelength of 490 nm. Each absorbance was normalized to the media control without any cells.

Colony forming assays
Cells transfected with MIR2052HG ASOs or LMTK3 plasmids were plated (800~ 1000 cells/well) in 6-well culture clusters in triplicates. Subsequently, the cells were cultured for up to 14 days at 37°C, 5% CO2 to allow colony formation. Colonies were washed with cold PBS, fixed with 4% paraformaldehyde and stained by 0.05% crystal violet. Colonies (>50 cells) were accounted with the Image J software (version 1.42q) and colony-formation rates were calculated.

PKC kinase assay
MCF7/AC1 and CAMA-1 cells were transfected with indicated ASO or plasmids. 48 hours later, cells were lysed and the level of PKC-specific kinase activity was measured in 30 µg cell lysate using the PKC Kinase Activity Assay Kit (AbCam) as described by the manufacturer. Assays were performed in triplicates with the mean ± SD shown.

Chromatin immunoprecipitation (ChIP)
ChIP assays were performed using EpiTect ChIP OneDay kit (Qiagen). MCF7/AC1 and CAMA-1 cells were transfected with MIR2052HG ASO for 24 hours. Cells were then subjected to CHIP assay as described by the manufacturer. LCLs were cultured in 5% charcoal stripped FBS for 24 h, followed with RPMI1640 medium without FBS for additional 24 h. LCLs were then treated with 1 nM androstenedione, 1 nM androstenedione plus 100 nM anastrozole, 1 nM androstenedione plus 100 nM exemestane for additional 24 hours. Approximately 2×10 7 LCLs per every sample (different SNP genotypes with androstenedione or androstenedione plus anastrozole or exemestane treatment groups) were collected for the ChIP assay. Equal amount of chromatin from each sample (~ 2 million cells each IP) and 1µg control IgG or antibody against EGR1 were used. Quantitative PCR was carried out and the result was normalized to input. All primers are listed in Table EV2. 20 The sequential protein staining and RNA detection were performed as previously described [55,56]. Briefly, the cells were grown in chamber slides. LMTK3 staining was performed as usual until secondary antibody is labeled in the presence of RNase inhibitor. Slides were then dehydrated by serial treatment of ethanol with different concentrations. The Alexa Fluor 488labeled RNA probe was obtained using the FISH Tag RNA Kit (Invitrogen). In the first step, in vitro transcription is used to enzymatically incorporate an amine-modified nucleotide into the probe template. The modified nucleotide is UTP having an NH2 group attached through a linker to the C5 position of the base. In the second step, dye labeling of the purified amine-modified RNA is achieved by incubation with amine-reactive dyes. These active ester compounds react with the primary amines incorporated into the probe template, covalently conjugating the dye to the modified nucleotide base. The purified probe is then ready for hybridization to the specimen slides at 37°C overnight. Signal was then amplified using Tyramide Signal Amplification (TSA) kit (Life Technologies). LMTK3 DNA probe was produced using the FISH Tag DNA Kit (Invitrogen). In the first step, nick translation is used to enzymatically incorporate an aminemodified nucleotide into the probe template. The modified nucleotide is dUTP having an NH2 group attached through a linker to the C5 position of the base. In the second step, dye labeling of the purified amine-modified DNA is achieved by incubation with amine-reactive dyes. These active ester compounds react with the primary amines incorporated into the probe template, covalently conjugating the dye to the modified nucleotide base. The purified probe is then ready for hybridization to the specimen. For dual RNA-DNA-FISH, we used the protocol as previously described [56]. In brief, RNA-FISH was performed by using Nick translated Alexa Fluor 488labeled probe and followed by tyramide signal amplification kit as above. After RNA-FISH, the cells were treated by RNase A and denatured. Nick-translated BAC containing LMTK3 was 21 labeled with Alexa Fluor 594 and used as probe. Images were obtained with the LSM 780 inverted confocal microscope runs on Zeiss's Zen software package.

RNA-Binding Protein Immunoprecipitation
An RNA-binding protein immunoprecipitation (RIP) assay was performed using the Magna RIP kit according to the manufacturer's instruction. Cell lysates from 50 million cells and 2-5 μg of control IgG or antibody against BHLHE40, CHD2, CTCF, EGR1, EP300, EZH2, HDAC6, POLR2A, REST, CREBBP, YY1, and STAT1 were used. We validated the RIP assay using the SNRNP70 antibody, which can bind to U1 snRNA. Specifically, cells were washed on the plates twice with 10 mL of PBS, scraped off from plate and centrifuged at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Cell pellet was re-suspended in an equal pellet volume of complete RIP Lysis Buffer, and then incubated on ice for 5 min. Dispense ~200 μL each of the lysate into nuclease-free microcentrifuge tubes and store at -80°C. Immunoprecipitations were performed using antibodies of interest and IgG control. Anti-SNRNP70 served as controls. 50 μL of magnetic beads were washed and re-suspended in 100 μL of the RIP wash buffer. 2~5 μg of the antibody of interest were added to each reaction and incubated with rotation for 30 minutes at room temperature. The beads were then washed three times with RIP wash buffer. 900 μL of RIP immunoprecipitation buffer were then added to each tube. The RIP lysate were thawed and centrifuged at 14,000 rpm for 10 minutes at 4°C, and 100 μL of the supernatant were added to each beads-antibody complex in RIP immunoprecipitation buffer. 10 μL of the supernatant of RIP lysate were removed as "10% input", and stored at -80°C until starting RNA purification.
The immunoprecipitations were incubated with rotating overnight at 4°C, followed by six washes with 500 μL of cold RIP wash buffer. RNA purification was then performed. Each immunoprecipitate was re-suspended in 150 μL of proteinase K buffer. The input samples were The pellets were washed once with 80% ethanol, air dry, and re-suspended in 10 to 20 μL of RNase-free water. The RNAs were then analyzed by quantitative RT-PCR.

Luciferase activity assay
Transcription activity of EGR1 was measured using the dual luciferase assay with the Cignal      A Knockdown of MIR2052HG increased phosphorylation of MEK, ERK, RSK1, as well as ERα S167 and decreased LMTK3 total level in MCF7/AC1 and CAMA-1 cells.
B Knockdown of MIR2052HG promoted the ubiquitination of wild type ERα, but not ERα S167A mutant. 293T cells were transfected with HA-Ub plasmid and FLAG-ERα or FLAG-ERα S167A plasmid, and then transfected with either the MIR2052HG specific ASOs or the negative control ASO followed by MG132. Wild type or S167A mutant ERα proteins were immunoprecipitated and analyzed by western blot. Knock down efficiency in 293T cells was determined by qRT-PCR.