LncRNA MACC1-AS1 associates with DDX5 to modulate MACC1 transcription in breast cancer cells

Summary MACC1 is a master oncogene involved in multiple aspects of cancer metastasis in a broad variety of tumors. However, the molecular mechanism by which MACC1 transcription is regulated remains unclear. Here, we show that in breast cancer cells, lncRNA MACC1-AS1 serves as a cis-factor to up-regulate MACC1 transcription and this regulation increases the cell proliferation potential. Mechanistically, MACC1-AS1 forms a complex with DEAD-Box helicase 5 (DDX5) and simultaneously interacts with the distal region of the MACC1 promoter. The interaction allows its associated DDX5 to spatially contact the MACC1 core promoter and shift from MACC1-AS1 to the core promoter. Moreover, binding of DDX5 to the core promoter results in local recruitment of the transcription factor SP-1, thus enhancing MACC1 transcription. Our findings reveal a molecular mechanism by which MACC1-AS1 cis-regulates MACC1 transcription by interacting with the distal promoter region and delivering DDX5 to the core-promoter of the gene.


INTRODUCTION
Breast cancer is the most frequently diagnosed cancer and is the second cause for cancer-related death in women. 1 Since metastasis is responsible for the high mortality among breast cancer patients, the metastatic process is directly related to patient survival and requires the identification of targets for treatment.][4][5][6] A major function of MACC1 is to regulate the transcription of the MET gene via binding to the EMT gene promoter, resulting in epithelialmesenchymal transition (EMT) and metastasis. 7,8MACC1 is located on human chromosome 7 (7p21.1)and contains seven exons and six introns. 9Studies indicate that in human colorectal cancer cells, the proximal flanking region of MACC1 (À426 nt to À18 nt) is the core promoter that harbors functional elements for binding of transcriptional factors and is responsible for basal transcription of MACC1 mRNA. 10However, the potential mechanism by which MACC1 gene transcription is regulated during tumorigenesis remains unclear.
Long noncoding RNAs (lncRNAs) are defined as a group of abundant transcripts longer than 200 nucleotides and have no protein-coding ability. 11,12To date, numerous lncRNAs have been reported to perform roles in cancer-related gene expression and shown to be responsible for cancer progression. 13,14Depending on their subcellular localization, lncRNAs impact gene regulation through a variety of pathways at both transcriptional and posttranscriptional levels. 15,16LncRNAs that exhibit distinct nuclear localization patterns often function as important modulators to regulate local gene expression either by chromatin reorganization or recruiting transcription factors to the gene promoter, 17,18 whereas lncRNAs that are exported to the cytoplasm play regulatory roles to post-transcriptionally mediate mRNA translation or to act as ceRNAs for particular miRNAs. 19,20ACC1-AS1 is an antisense RNA of the sixth intron of the MACC1 gene.Recent studies have reported that in gastric cancer cells, MACC1-AS1 promotes metabolic plasticity via AMPK/Lin28-mediated stability of MACC1 mRNA and induces fatty acid oxidation-dependent chemoresistance through regulating miR-145-5p activity.21,22 In breast cancer cells, we have previously shown that MACC1-AS1 is distributed in both the cytoplasm and nucleus.The cytoplasmically localized MACC1-AS1 coordinately interacts with PTBP1 and multiple miRNAs to modulate the expression of target mRNAs.23 In this study, we mainly used MDA-MB-231 and BT-549 breast cancer cell lines to reveal the function of nuclear localized MACC1-AS1 in upregulation of MACC1 transcription.We identified that in the nucleus, MACC1-AS1 forms a complex with DEAD-Box helicase 5 (DDX5) to interact with the distal region (À2000 nt to À1500 nt) of the MACC1 promoter.Interaction with the distal promoter allows MACC1-AS1-associated DDX5 to contact and invade the core promoter of MACC1, thus increasing recruitment of ll OPEN ACCESS transcription factor SP-1 and activating MACC1 transcription.Our study reveals a molecular mechanism by which association of MACC1-AS1 with DDX5 cis-regulates MACC1 transcription.

MACC1-AS1 promotes MACC1 expression in breast cancer cells
MACC1-AS1, a 639 bp lncRNA, is a cognate antisense RNA of the sixth intron of the MACC1 gene (Figure 1A).MACC1-AS1 is expressed in various breast cancer cell lines and has been shown to promote cell proliferation and invasion. 23Investigation of the relationship between MACC1-AS1 and MACC1 mRNA indicated that MACC1-AS1 can effectively induce MACC1 expression not only in breast cancer cell lines, but also in mouse xenograft tumor models (Figures S1A and S1B).Knocking down MACC1-AS1 by siRNA or by ASO (antisense oligonucleotide) either in BT-549 cells, which express relatively higher endogenous MACC1-AS1 or in MACC1-AS1-expressing MDA-MB-231 cells, reduced MACC1 mRNA expression (Figures 1B, 1C, and S1C).Moreover, MACC1-AS1 also increased levels of primary MACC1 RNA (Figures S1D and S1E), eliminating the possibility that splicing effect could involve in MACC1-AS1-mediated MACC1 expression.Correlated expression of MACC1-AS1 and MACC1 mRNA was as well shown in human breast tumors by analyzing the GEPIA RNA-seq database (Figure S1F, r = 0.28).Notably, the role of MACC1-AS1 in inducing breast cancer cell proliferation could be rescued by knocking down MACC1 mRNA (Figures 1D and 1E).To analyze whether MACC1-AS1-mediated MACC1 upregulation resulted from mRNA stability, we treated breast cancer cell lines with actinomycin D (5 mg/mL) for 24 h to block de novo transcription and measured the endogenous levels of MACC1 mRNA by RT-qPCR.Results showed that cellular levels of MACC1 mRNA were barely affected post-transcriptionally in responding to MACC1-AS1 expression (Figures 1F and 1G).These data suggest the potential of MACC1-AS1 to transcriptionally upregulate MACC1 expression and this regulation facilitates breast cancer progression.

MACC1-AS1 interacts with the distal region of the MACC1 promoter
In colorectal cancer, a core promoter region (À426 to À18) of the MACC1 gene is essential for transcriptional activity. 10We determined whether MACC1-AS1-induced MACC1 expression is through the core promoter.Transfecting the psiCHECK-2 luciferase reporter driven by the core promoter (À450 to À1) into BT-549 cells displayed no obvious change in luciferase activity when the endogenous MACC1-AS1 was silenced by siRNA (Figure 2A).In comparison, luciferase activity was significantly lower in MACC1-AS1 silenced BT-549 cells when using a reporter driven by a region spans the nucleotides À2,000 to À1 upstream of the MACC1 gene (MACC1p 2000 ) (Figure 2A).Moreover, transfecting the MACC1p 2000 reporter into MACC1-AS1-expressing MDA-MB-231 cells showed increased luciferase activity (Figure S2A).These results allowed us to hypothesize that MACC1-AS1 modulates MACC1 transcription through the region in the À2,000 bp outside of the core promoter.We made a series of luciferase constructs driven by different truncated MACC1p 2000 promoters (Figure 2B, left), ranging from À1,500 nt to +1 (MACC1p 1500 ), À1,000 nt to +1 (MACC1p 1000 ), À450 nt to +1 (MACC1p 450 ), and À1,000 to À500 nt (MACC1p nC ).Luciferase activity was much lower when the core proximal promoter region was deleted.Interestingly, luciferase activity was also decreased by about 40% in MACC1-AS1-expressing cells following transfection of MACC1p 1500 reporter lacking the À2,000 to À1,500 nt region (distal region) of the promoter (Figure 2B, right), suggesting that the distal region of the promoter could be the regulatory region mediated by MACC1-AS1.We next performed ChIRP (chromatin isolation by RNA precipitation) assays to investigate the potential interaction between MACC1-AS1 and the MACC1 promoter.We used MDA-MB-231 and MCF-7 stable cells, in which exogenously expressed MACC1-AS1 was tagged with repeated hairpin motifs (MS2) that allow us to use a recombinant fusion protein (MBP-MCP) to pulldown its associated partners. 24We have also used a biotin-labeled antisense oligonucleotide (ASO) that hybridizes to the 5 0 -end of endogenous MACC1-AS1 in BT-549 cells. 25We designed two sets of PCR primers (Figure 2C, upper), one set amplified the distal promoter region (raging from À1,840 to À1,500 nt), and the other set amplified the core promoter region (raging from À450 to À1 nt).ChIRP followed by PCR experiments revealed that either exogenously expressed MACC1-AS1 or endogenous MACC1-AS1 favorably associated with the distal region of the MACC1 promoter (Figures 2C, lower panel; S2B, and S2C).Recently, CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing has revolutionized biomedical research, allowing to precisely edit the gene of eukaryotic cells by removal or insertion of genetic information at a desired locus. 26To confirm that MACC1-AS1 is the genetic interactant of the distal promoter of the MACC1 gene, we established a BT-549 cell line in which the MACC1 distal promoter (À1,840 to À1,527) was knocked out by using CRISPR-Cas9 system (upper panels of Figure 2D).Genetic deletion of the distal promoter region, which was further identified by Sanger sequencing (Figure S2D), reduced MACC1 expression and this reduction was not able to be rescued by overexpression of MACC1-AS1 (lower panel of Figure 2D).As a results, reduced MACC1 expression in BT-549 cells caused by knocking out of the distal promoter region decreased cell proliferation (Figure 2E).These results indicate that the distal promoter region is responsible for activating MACC1 transcription by MACC1-AS1.

Full-length MACC1-AS1 is required for mediating MACC1 transcription
To further investigate the molecular mechanism by which MACC1-AS1 mediates MACC1 transcription, we divided MACC1-AS1 into five segments, each of them was tagged with six MS2 hairpin loop repeats (Figure 3A), and separately cloned them into pCIP2 lentiviral vector.MDA-MB-231 cell lines stably expressing the full-length and truncated MACC1-AS1 lncRNAs were verified by qRT-PCR (Figure S3A).Unlike the fulllength MACCl-AS1, all truncated MACC1-AS1 had little effect on increasing MACC1 expression (Figure 3B).We then transfected the MACC1p 2000 luciferase reporter into the cells expressing full-length or truncated MACC1-AS1.Luciferase activity was significantly higher in cells expressing full-length MACC1-AS1 than the cells expressing truncated MACC1-AS1 (Figure 3C).Co-transfection of the MACC1p 2000 luciferase reporter and individual vectors expressing full-length or truncated MACC1-AS1 into breast BT-549 cells gave similar results (Figure S3B), indicating that the full-length MACC1-AS1 was required for facilitating MACC1 mRNA transcription.To determine which region of MACC1-AS1 interacts with the distal promoter, ChIRP experiments were performed in stable cell lines expressing full-length (1-639 nt) or truncated MACC1-AS1 (150-639 nt) (Figure 3D, upper).In contrast to the full-length MACC1-AS1, little amount of the distal promoter was co-precipitated with the truncated MACC1-AS1 (Figure 3D, lower).Moreover, in comparison to cells expressing full-length MACC1-AS1, the cells expressing 5 0 -truncated MACC1-AS1 has less ability to activate MACC1 transcription (Figure 3E), suggesting that the 5 0 -part of MACC1-AS1 interacts with the MACC1 distal promoter and this interaction promoted MACC1 transcription.

Association of MACC1-AS1 with DDX5 enhances MACC1 transcription
RNA binding-proteins often play roles in mediating the activity of their binding partners. 27Using in vitro RNA pulldown assays combined with protein sequencing, we identified DDX5, PTBP1, as well as some hnRNPs to be complexed with MACC1-AS1 in breast cancer cells (Figure S4A). 23We hypothesized that these proteins, particularly DDX5, could associate with MACC1-AS1 to mediate MACC1 transcription.To validate the potential interactions between MACC1-AS1 and the precipitated proteins, we prepared cell nuclear extract (Figure S4B) and performed RNA immunoprecipitation (RIP) assays using DDX5 antibody.MACC1-AS1 was directly bound to DDX5 in both BT-549 and MDA-MB-231 breast cancer cells (Figures 4A and S4C).MACC1-AS1 pulldown assays also showed that DDX5 was specifically precipitated with MACC1-AS1, whereas hnRNPD was barely detected in the MACC1-AS1 precipitates (Figure 4B).Further RIP assays using nuclear lysates from cells expressing truncated MACC1-AS1 indicated that DDX5 bound to the 3 0 -part of MACC1-AS1 (310-639 nt) (Figure 4C).Interestingly, either knocking down MACC1-AS1 or DDX5 mRNA decreased MACC1 expression (Figures 4D and 4E), as well as cell growth ability (Figure 4F).In addition, luciferase activity was decreased in DDX5 silenced BT-549 cells when using a reporter driven by MACC1p2000 promoter (Figure S4D).These results suggest the significance of MACC1-AS1/DDX5 complex in mediating MACC1 transcription.

MACC1-AS1 mediates its associated DDX5 to bind to the core promoter of MACC1
Next, we investigated the underlying mechanism of MACC1-AS1/DDX5 complex in mediating MACC1 transcription.DDX5 is a potent RNA-or DNA-binding protein involved in many aspects of gene regulations. 28,29A recent study has demonstrated that DDX5 modulates transcription of the Myc gene by refolding its promoter. 30We rationalized that interaction of MACC1-AS1 with the MACC1 distal promoter would eventually allow its associated DDX5 to approach and invade the MACC1 core promoter.To test this hypothesis, we performed ChIP assays to determine whether MACC1-AS1 could mediate DDX5 binding to the MACC1 core promoter.By measuring the enrichment of the MACC1 core promoter or the MMP2 promoter (internal control) in the DDX5 ChIP precipitates, we observed that the binding potential of DDX5 to the MACC1 core promoter was largely increased in MDA-MB-231 cells expressing MACC1-AS1 compared to the cells without MACC1-AS1 expression (Figure 5A).No enrichment was seen for a control MMP2 promoter.Moreover, binding of DDX5 to the core promoter was markedly reduced in BT-549 cells either the endogenous MACC1-AS1 was silenced by siRNA (Figure 5B), or the distal region of the MACC1 promoter was genetically knocked out (Figure 5C), indicating both MACC1-AS1 and the MACC1 distal promoter are required for facilitating DDX5 to binds to the core promoter.We next using stable cells expressing truncated MACC1-AS1 to determine which part of MACC1-AS1 would mediate the binding of DDX5 to the core promoter.PCR and agarose gel electrophoresis indicated that in contrast to the full-length MACC1-AS1, truncated MACC1-AS1 either lacking the 5 0 -region or the 3 0 -region lost the ability to assist DDX5 binding to the MACC1 core promoter (Figure 5D).qPCR also detected that the enrichment of the core promoter in DDX5 precipitates was about 3-folds more in the cells expressing full-length MACC1-AS1 than the cells expressing truncated MACC1-AS1 (Figure 5E).These results suggested that interaction of the 5 0 -region of MACC1-AS1 with the MACC1 distal promoter would shift its 3 0 -associated DDX5 to the core promoter of the MACC1 gene.

Binding of DDX5 to the MACC1 core promoter increases the recruitment of SP-1 and activates MACC1 transcription
Using RNAInter, an online database (http://www.rna.socirty.org/raid/),we identified that the MACC1 core promoter harbors potential binding sites for transcriptional factors SP-1 and c-Jun (Figure S5A).Luciferase reporter assays indicated that SP-1 performed a major role for the core promoter activity and mutation on the SP-1 binding site apparently decreased basal promoter activity (Figures S5B and S5C).ChIP experiments further demonstrated that SP-1 bound to the core promoter containing the SP-1 binding site (Figure 6A).We hypothesized that the shift of DDX5 from MACC1-AS1 to the core promoter would remodel its structure thus to increase the recruitment of SP-1.By comparing relative enrichments of the core promoter in SP-1 precipitates in cells with or without MACC1-AS1 expression, we observed that SP-1 was more efficiently associated with the core promoter in cells expressing full-length MACC1-AS1 (1-639) than the cells expressing truncated MACC1-AS1 (310-639) that lost the ability to interacts with the distal promoter (Figure 6B).Moreover, binding of SP-1 to the core promoter could be reduced when the MACC1 distal promoter region was knocked out or MACC1-AS1 was silenced by siRNA (Figure 6C).We then constructed a mutant reporter (MACC1p 2000m ), in which the potential SP-1 binding site on the core promoter was mutated (Figure 6D, upper).In comparison to the wild-type promoter, luciferase activity was notably decreased when the mutant promoter was used (Figure 6D, lower), indicating the significance of SP-1 binding for core promoter activity.IP (immunoprecipitation) assays showed that DDX5 and SP-1 were not coprecipitated, eliminating the possibility that the recruitment of SP-1 to the core promoter was resulted from DDX5/SP-1 interaction (Figure S5C).Taking together, these results suggest that MACC1-AS1-mediated binding of DDX5 to the core promoter enhanced recruitment of SP-1 to activate MACC1 transcription.

A model of MACC1-AS1/DDX5-mediated MACC1 gene transcription
Based on our findings, we predict a model by which MACC1-AS1 associates with DDX5 to regulate MACC1 transcription in cis (Figure 7).First, the 3 0 -region of nuclear MACC1-AS1 forms a complex with DDX5, and afterward its 5 0 -region interacts with the distal region of the MACC1 promoter.Second, this interaction enables MACC1-AS1-associated DDX5 to come in contact with the MACC1 core promoter and shift from MACC1-AS1 to the core promoter.Third, binding of DDX5 to the core promoter unwinds the local DNA architecture and recruits transcriptional factor SP-1 to activate MACC1 transcription.MACC1-AS1 truncates with impaired ability to bind to either DDX5 or to the distal promoter is unable to regulate the transcription of the MACC1 gene.

DISCUSSION
MACC1 was originally shown to be a key regulator of the HGF-MET pathway in colon cancer 8 and has since been identified as a biomarker in a variety of tumors. 31MACC1 is involved in many aspects of cancer progression including promotion of epithelial-to-mesenchymal transition (EMT), acceleration of cancer metastasis and induction of cell proliferation.In this study, we revealed a novel molecular mechanism by which MACC1-AS1 serves as a cis-acting lncRNA to activate transcription of the MACC1 gene in breast cancer cells.Mechanistically, nuclear localized MACC1-AS1 interacts with a distal region of the MACC1 promoter.This interaction allows MACC1-AS1-associated DDX5 to contact and invade the core promoter of MACC1, thus increasing the recruitment of SP-1 to enhance MACC1 transcription.
Studies accumulated to date show that lncRNAs are widely expressed in mammalian cells and have key roles in regulating gene expression.Depending on their subcellular localization and their specific interactions with proteins, DNAs or RNAs, lncRNAs can modulate chromatin structure, recruit transcription factors, and mediate the stability and translation of cytoplasmic mRNAs. 16,32LncRNAs, such as MACC1-AS1 that localizes in both cytoplasm and nucleus would perform multiple biological functions.We have previously reported that, in breast cancer cells, MACC1-AS1 functions as a ceRNA (competitive endogenous RNA) for multiple miRNAs to post-transcriptionally regulate the expression of genes targeted by these miRNAs in the cytoplasm. 23In this study, we identified a nuclear function of MACC1-AS1 to modulate MACC1 transcription.While increasing evidence demonstrated the contribution of lncRNAs in oncogenicity, MACC1-AS1 cis-regulates MACC1 transcription provides a key insight toward understanding the molecular mechanism by which lncRNAs execute their oncogenic function.
The functions and mechanisms of lncRNAs to regulate gene transcription are diverse. 32One of the major functions of lncRNA is to regulate gene transcription, and this regulation often occurs through association with RNA-binding proteins.LncRNAs could either serve as molecular scaffolds to form functional regulatory complexes via interacting with specific proteins, 33,34 or recruit chromatin modifiers to promote gene activation. 35,36Our study indicates that activation of MACC1 transcription relies on MACC1-AS1 to form an RNA/protein complex with DDX5 via its 3 0 -region, and then to interact with the MACC1 distal promoter through its 5 0 -region.Either knocking out the distal region of the endogenous MACC1 promoter, or silencing MACC1-AS1/DDX5, decreases the MACC1-AS1-mediated MACC1 activation.The interaction of MACC1-AS1 with the distal promoter is most likely to allow MACC1-AS1-associated DDX5 to spatially close and invade the core promoter, therefore remodeling the local DNA architecture and making the core promoter more accessible for transcriptional factor SP-1.In this issue, MACC1-AS1 functions as a carrier lncRNA to transport a protein-activator to a gene core promoter through interacting with the gene distal promoter.
DDX5, a member of the DEAD-box RNA helicase family, regulates gene expression in diverse pathways. 298][39] Recently, DDX5 regulates MYC gene transcription through interaction with the MYC promoter and unfolding the local DNA G4 structure has been reported. 30Based on our results, DDX5 functions not only as a lncRNA-binding protein but also a DNA-binding partner to mediate MACC1 transcription.We propose two roles of DDX5 in the process of MACC1 activation.First, association of DDX5 with MACC1-AS1 would retain and stabilize MACC1-AS1 in the nucleus and assist MACC1-AS1 to interact with the MACC1 distal promoter.Second, binding of MACC1-AS1 to the MACC1 distal promoter allows DDX5 to spatially close and shift to the core promoter, resulting in the unwinding of the local DNA architecture and recruiting transcriptional factor SP-1 to the core promoter.
In conclusion, our study provides a key insight into the lncRNAs in regulation of gene transcription and reveals a molecular mechanism that MACC1-AS1/DDX5 can serve as a cis-acting complex to activates MACC1 transcription.

Cell lines
Primary MDA-MB-231, BT-549, T47D and MCF-7 cell lines were purchased from ATCC (the American type culture collection).Cells were cultured and passaged according to standard instructions of the ATCC.Cell lines were tested by determination of short-tandem repeats profiling through PCR following the instructions of the ATCC.The latest test was performed at October 2020.

Cell culture
Breast cancer cell lines and stable cell lines expressing MS2-tagged MACC1-AS1 and MACC1-AS1 truncates were grown in DMEM medium supplemented with 10% FBS, 100 units of penicillin/ml and 100 mg of streptomycin/ml, and were incubated at 37 C and 5% CO2 in a humidified chamber.

METHOD DETAILS PCR primers
PCR primers and chimeric antisense oligonucleotide (ASO) were purchased from IGE Biotech (Guangzhou, China).MACC1-AS1 siRNA and DDX5 siRNA were purchased from Gene Pharma (Suzhou, China) and are listed in Table S1.

Plasmid constructs
Human MACC1-AS1 cDNA was amplified by RT-PCR from MDA-MB-231 cells as previously described. 23Individual MACC1-AS1 truncates were PCR amplified and cloned into the pCIP2 lentivirus plasmid at the Not I and Bam HI sites.MS2-tagged MACC1-AS1 and its truncated variants were constructed by introducing a six-repeat MS2 hairpin structure into the BamHI site of a pCIP2 lentivirus plasmid.PsiCHECK 2 was purchased from Promega (USA) and used for luciferase reporter construction and luciferase assays.To construct the luciferase reporters, difference lengths of the MACC1 promoter were PCR amplified and cloned into the 5' end of the Renilla luciferase gene of the PsiCHECK 2 plasmid.Primer pairs used in the cloning experiments are described in Tables S1 and S2.All constructs were verified by sequencing analysis.

SgRNAs designing and cloning
CRISPR guide RNA (sgRNA) sequences were selected based on the criteria of having high on-target score 41 and low off-target effects. 42Sequences of sgRNA#1, TTCATTTCGGGTACTGTCAAGGG, is located at -1840 bp to -1818 bp of the MACC1 promoter and sgRNA#2: ATGAGGCCTTGCCTTCAAATAGG, located at -1549 bp to -1527 bp of the MACC1 promoter.The sgRNAs were subcloned into a lentiviral plasmid pLC5-NC (IGE Biotech (GuangZhou, China), which contains a GFP marker.All the procedures for cloning of sgRNAs and preparing lentiviral particles for cell infection were commissioned manufacturing at IGE Biotech.

CRISPR genome editing procedure
The two vector approach was used to create CRISPR-induced knockout of the MACC1 distal promoter in BT-549 cells. 43In this approach, a Cas9-expressing plasmid pCDH-CAG-CAS9-T2A-HygR was first transduced into the cells according to the manufacturer's instruction.Following selection by HygR (400mg/ml) for 5 days, Cas9-expressing stable cells were collected and verified by RT-PCR.Cas9-expressing cells were then infected with sgRNA-expressing pLC5-NC lentiviral particles to generate knockout cells.24-hours post transfection, cells were cultured in regular DEME medium.According to the green fluorescence intensities, stable cells with green color were sorted by FACS (fluorescence-activated cell sorting), then cultured and expanded by plating to progressively larger plates until there were enough cells to freeze and to use.Knockout efficiency of the MACC1 distal promoter was verified by PCR and by Sanger seducing of the deleted genomic region of the MACC1 gene.

RNA purification and qRT-PCR analysis
Total RNA from breast cancer cells was isolated with TRIzol (Invitrogen) following the manufacturer's instructions.Concentration of isolated RNA was determined with a Nanodrop (Agilent).One microgram of total RNA was reverse transcribed into cDNA using a Reverse Transcription System (Tiangeng, China) according to the manufacturer's protocol.Quantitative real-time PCR (RT-qPCR) was performed by SYBR Green Master Mix (Tiangen, China) using a Real Time PCR System (Applied Biosystems).Primers used in the PCR assay are described in Tables S1 and S2.To amplify primary MACC1 RNA, the primers were designed within the region between the fifth exon and the fifth intron.GAPDH mRNA was used as an internal control and for normalization.All experiments were replicated at least three times.

MS2-tagged RNA pulldown assays
Pulldown assays of MS2-tagged MACC1-AS1 were performed as previously described using a recombinant fusion protein (MBP-MCP) that contains a maltose-binding domain (MBP) and a domain (MCP) that recognizes the MS2 hairpins. 24Briefly, MBP-MCP-conjugated amylose resin (NEB, USA) was incubated with nuclear extracts prepared from cells expressing MS2-tagged MACC1-AS1 variants at 4 C for 4 h in the presence of RNase and protease inhibitors.After extensive washing, bound MACC1-AS1-MS2 RNP complexes were eluted with 100 ml

Figure 1 .
Figure 1.MACC1-AS1 up-regulates MACC1 mRNA expression in breast cancer cells (A) A schematic representation of the genomic structure of lncRNA MACC1-AS1 and the MACC1 gene.Human MACC1-AS1 is the sixth intron of the opposite strand of the MACC1 gene on chromosome 7. Exons of the MACC1 gene and lncRNA MACC1-AS1 are depicted as red or green boxes, respectively, and introns are shown in dark.The arrowhead represents the orientation of transcripts.(B and C) Silencing endogenous MACC1-AS1 in BT-549 cells by siRNA or by ASO (antisense oligonucleotide) decreased MACC1 mRNA expression.**p < 0.01.(D and E) MACC1-AS1 induced cell proliferation can be rescued by knocking down MACC1 mRNA expression.**p < 0.01.(F and G) MDA-MB-231 cells stably expressing MACC1-AS1 were treated with actinomycin D (5mg/ml) for 24 h.Relative levels of MACC1 mRNA were determined by qRT-PCR.Data are normalized to GAPDH mRNA and presented as means G SD from three independent experiments.**p < 0.01.

Figure 2 .
Figure 2. MACC1-AS1 interacts with the distal promoter of the MACC1 gene (A) Upper: a schematic luciferase reporter in which the À450 bp or À2,000 bp of the MACC1 promoter (MACC1p) was fused into the 5 0 -region of the Renilla luciferase gene of the psiCHECK-2 construct.Lower: BT-549 cells with or without MACC1-AS1 siRNA treatment were transfected with the luciferase reporters.Luciferase activities were determined after 36 h transfection using a dual-luciferase assay system.Renilla luciferase activity was normalized to the activity of firefly luciferase.**p < 0.01.(B) Left: a schematic representation of luciferase reporters driven by different MACC1 promoters.Right: luciferase activity was determined in MDA-MB-231 cells after transfecting with the individual reporters.***p < 0.001.(C) ChIRP experiments were performed to detect potential interaction of MACC1-AS1 with the MACC1 promoter in MBA-MB-231 cells.Upper: locations of the two set of primers for amplifying the distal region and the core region of the MACC1 promoter are indicated with arrows.Lower left: Enrichment of MACC1-AS1 in the ChIRP precipitates were analyzed by RT-qPCR in MACC1-AS1-expressing and control cells.Lower right: PCR indicated that MACC1-AS1 was preferentially interacted with the distal region of the MACC1 promoter.(D) Upper: PCR and agarose gel electrophoresis showed that in CRISPR edited BT-549 cells (knocking out [KO]), the distal region of the MACC1 promoter was knocked out.Lower: qRT-PCR indicates the expression of MACC1 mRNA in WT and KO cell.MACC1-AS1 was not able to increase MACC1 expression when the distal region of the MACC1 promoter was deleted.**p < 0.01 as determined by Student's t test.(E) Proliferation assays indicated that either KO of the MACC1 distal promoter or knocking down MACC1 mRNA decreased cell proliferation.Data are presented as means G SD from three independent experiments.***p < 0.001.

Figure 5 .
Figure 5. MACC1-AS1 mediates the binding of DDX5 to the MACC1 core promoter (A) Protein A beads conjugated with DDX5 antibodies were used for ChIP assays in MDA-MB-231 cells with or without MACC1-AS1 expression.Upper penal: Western blots showing immunoprecipitated DDX5.Normal IgG was used as a negative control.Lower two panels: PCR and agarose gel electrophoresis indicated that MACC1-AS1 increased binding of DDX5 to the core promoter of the MACC1 gene, but not the internal control of the MMP2 promoter.(B) Left upper panel: DDX5 was immunoprecipitated in BT-549 cells in which MACC1-AS1 was silenced by siRNA.Left lower panel: agarose gel electrophoresis showing that MACC1-AS1 knocking down decreased binding ability of DDX5 to the MACC1 core promoter.Right panel: qPCR confirmed that silencing of MACC1-AS1 significantly reduced the potential of DDX5 to bind to the core promoter of the MACC1 gene.(C) Upper: DDX5 was immunoprecipitated in BT-549 cells where the endogenous MACC1 distal promoter was knocked out by CRISPR editing.Lower: PCR and agarose gel electrophoresis showing that knocking out the distal promoter reduced binding of DDX5 to the MACC1 core promoter.(D) ChIP experiments were performed using DDX5 antibody and cell lysates expressing full-length (1-639) and truncated (1-325 and 310-639) MACC1-AS1 fragments.qPCR indicated that the enrichment of the core promoter in DDX5 precipitates was only appeared in the cells expressing full length of MACC1-AS1.(E)RT-qPCR assays indicated that the enrichment of the core promoter in DDX5 precipitates was higher in the cells expressing full-length MACC1-AS1 than the cells expressing truncated MACC1-AS1.

Figure 6 .
Figure 6.DDX5 elevates recruitment of SP-1 to the MACC1 core promoter (A) ChIP experiments were performed using SP-1 and normal IgG antibodies.PCR and agarose gel electrophoresis indicated that the core promoter of the MACC1 gene was enriched in the SP-1 precipitates.(B) ChIP assays were performed to detect the binding ability of SP-1 to the MACC1 core promoter in cells expressing full-length (1-639) and truncated MACC1-AS1 (150-639).Upper: Western blots show precipitated SP-1.Lower: relative levels of the amplicon corresponding to the core promoter in SP-1 precipitates were analyzed by qPCR.Data are presented as means G SD from three independent experiments.**p < 0.01 as determined by Student's t test.(C) Analyzing the binding potential of SP-1 to the MACC1 core promoter in the MACC1 distal region knockout or MACC1-AS1 silenced BT-549 cells by ChIP assays.The MMP-2 promoter was used as an internal control.Upper: Western blots indicate the precipitated SP-1 in tested cells.Lower: amplicon corresponding to the core promoter of the MACC1 gene was amplified by qPCR.Relative levels of the core promoter are presented as means G SD from three independent experiments.**p < 0.01.(D) Upper: a schematic representation of a luciferase reporter driven by the À2000 bp MACC1 promoter.Black box indicates the SP-1 binding site.WT: wild-type promoter.Mutant: SP-1 site mutated promoter.Luciferase activity was determined after transfecting the reporter into MDA-MB-231 cells with or without MACC1-AS1 expression.Data are presented from three independent experiments.**p < 0.01 as determined by Student's t test.