Abstract
Objective:
To determine the expression and functional roles of a long non-coding RNA (IncRNA) X-inactive specific transcript (XIST) in leiomyoma.
Design:
Experimental study
Setting:
Academic research laboratory
Patients:
Women undergoing hysterectomy for leiomyoma.
Intervention:
Over and under expression of XIST; blockade of SP1.
Main Outcome Measure:
Expression of XIST in leiomyoma and its effects on miR-29c, miR-200c and their targets.
Results:
Leiomyoma expressed significantly more XIST as compared with matched myometrium which was independent of race/ethnicity and menstrual cycle phase. Using three-dimensional spheroid culture system, XIST levels were induced in leiomyoma smooth muscle cells (LSMC) following treatment with 17β-Estradiol, progesterone and their combination. The expression of XIST was down-regulated by treatment with the SP1 inhibitor mithramycin A and SP1 siRNA. Knockdown of XIST resulted in inhibition of cell proliferation, up-regulation of miR-29c and miR-200c and a concomitant inhibition of target genes of these miRNAs namely, collagen type I (COL1A1), collagen type III (COL3A1) and fibronectin (FN1). In contrast, overexpression of XIST in myometrium smooth muscle cells (MSMC) repressed miR-29c and miR-200c, and induced COL1A1, COL3A1 and FN1 levels. Using RNA immunoprecipitation analysis we confirmed XIST has sponge activity over miR-29c and miR-200c which is more pronounced in leiomyoma as compared with myometrium.
Conclusion:
Our data demonstrate that increased expression of XIST in leiomyoma results in reduced expression of miR-29c and miR-200c with a consequent up-regulation of genes targeted by these microRNAs including COL1A1, COL3A1 and FN1 which play key roles in extracellular matrix accumulation associated with fibroids.
Keywords: Leiomyoma, LncRNA, XIST, miR-29, miR-200
Capsule:
XIST plays a role in leiomyoma pathogenesis through its regulation of expression of miR-29c and miR-200c
Introduction
Leiomyomas are benign tumors and the most common indication for all hysterectomies performed (1). The growth of these tumors is dependent on ovarian steroids and they are characterized by excess accumulation of the extracellular matrix (ECM), inflammation, and increased angiogenesis (2, 3). The pathogenic mechanisms for leiomyoma initiation and progression have been under intense investigation (4-6). Our laboratory has focused on the mechanism underlying aberrant expression of protein coding genes such as those involved in ECM accumulation and inflammation with special emphasis on the role of non-coding or regulatory RNAs. As such we have identified two microRNAs (miRNAs) that are crucial to the pathogenesis of these tumors namely miR-29c and miR-200c (7-10). The levels of those two miRNAs are suppressed in fibroids compared with myometrium, and are under ovarian steroid control. Other groups have also reported reduced expression of other members of the miR-29 family namely miR-29a and miR-29b in fibroids as compared with myometrium (11, 12). The correction of aberrant expression of miR-29b through its overexpression resulted in shrinkage of tumor size in an animal model of fibroid (11). MiR-29c has a number of targets including collagens (7, 13-15), elastin (13, 14, 16) and matrix metalloproteinases (MMPs) (14, 17), while miR-200c targets genes critical in cellular transition, inflammation, angiogenesis, cell cycle regulation and matrix remodeling examples of which are inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), zinc finger E-Box binding homeobox 1 (ZEB1), zinc finger E-Box binding homeobox 2 (ZEB2), vascular endothelial growth factor A (VEGFA), tissue inhibitor of metalloproteinases 2 (TIMP2), cyclin-dependent Kinase 2 (CDK2) and fibronectin (FN1) (8-10, 18). Previous reports indicated many of the genes targeted by miR-29c and miR-200c are dysregulated in fibroids and thus relevant to the pathogenesis of these tumors (2). In a recent study we profiled the expression of long non-coding RNAs (lncRNAs) defined as having greater than 200bp in leiomyomas and demonstrated differential expression of a number of these non-coding RNAs (19). One mechanism by which lncRNAs play a regulatory function in protein coding gene expression is through their ability to sponge and thereby sequester miRNAs (20, 21). Based on our previous reports showing reduced expression of miR-29c and miR-200c in fibroids we postulated that the lncRNA X-inactive specific transcript (XIST), which has been shown in other tissues to sponge these miRNAs (22, 23) might also play a role in fibroids to modulate the expression of miR-29c and miR-200c. As such we hypothesized that the expression of XIST in fibroids is increased and this would result in sequestering miR-29c and miR-200c thereby lowering their levels and upregulating their target genes such as collagens and fibronectin. We tested this hypothesis in vitro using primary leiomyoma smooth muscle cells (LSMC) and myometrium smooth muscle cells (MSMC) in a three-dimensional (3D) culture system.
Materials and Methods
Tissue Collection and Primary Cell Isolation
Leiomyomas and paired myometrium were obtained from 61 patients undergoing hysterectomy at Harbor-UCLA Medical Center. These patients were not on hormonal treatments for at least 3 months prior to surgery. The protocol was approved by the Institutional Review Board at the Lundquist Institute at Harbor-UCLA Medical Center (#036247). Informed consent was obtained from all the patients participating in the study before surgery. The paired tissues were obtained from Caucasians (N=13 with 8 pairs kindly provided by Dr. Al Hendy at University of Illinois), African Americans (N=18), Hispanics (N=23) and Asians (N=7). The mean age of patients was 45 ± 5.6 years with a range of 30–56 years. The menstrual cycle phase was determined by histologic analysis of hematoxylin and eosin stained sections (24) and identified 31 patients in follicular phase and 15 patients in luteal phase among the total 61 patients. The tissues were either snap frozen and stored in liquid nitrogen for further analysis, or used for isolation of MSMC and LSMC as previously described (25). Briefly, MSMC and LSMC were cultured in DMEM supplemented with 10% fetal bovine serum until reaching confluence with a change of media every 2–3 days. Cells at passages p1 to p3 were used for all experiments. Cell culture experiments were performed at least three times using LSMC or MSMC obtained from different patients as indicated in the figure legends. Overall, 17 LSMC and 5 MSMC were used in this study. All supplies for isolation and cell culture were purchased from Sigma-Aldrich (St. Louis, MO), Invitrogen (Carlsbad, CA) and Fisher Scientific (Atlanta, GA).
Spheroid Cell Culture
Isolated LSMC or MSMC were plated in 6-well (1 x 105 cells/well) or 96-well (5 x 103 cells/well) plates which were coated with 0.5% agarose gel and incubated 48 hours for spheroids formation (26).
Ovarian Steroids Treatment
Primary LSMC were plated in 6-well plates as above for spheroids formation in phenol red-free media with charcoal-stripped fetal bovine serum for 48 hours and then treated with 17β-estradiol (E2), progesterone (P4), or E2 plus P4 (Sigma-Aldrich) at 10−8 M concentration for 48 hours. The dose of E2 or P4 selected is within physiological range and has been used in prior publications (27, 28). Total RNA was isolated and subjected to quantitative PCR analysis.
siRNA Transfection
Primary LSMC prior to the formation of spheroids were transfected with 50 nM of siRNA negative control (siNC) or siRNA against SP1 (siSP1; Santa Cruz Biotechnology, Dallas, TX) or XIST (siXIST; 5’- GGAAGUACCCGCUCCAUAA-3’) using PureFection transfection reagent (System Biosciences, Inc., Mountain View, CA) according to the manufacturer’s protocol.
RNA Isolation and qPCR Analysis
Total RNA was extracted from MSMC and LSMC using Trizol (Thermo Fisher Scientific, Waltham, MA) and their quantity and quality was determined (ND-1000 Spectrophotometer, NanoDrop Technologies, Wilmington, DE) as previously described (13, 29). Subsequently, RNA sample of 1 μg each was reverse transcribed using random primers for XIST. Primer design for miR-29c and miR-200c and PCR conditions have been described previously (30). Quantitative PCR was carried out using SYBR gene expression master mixes (Applied Biosystems, Carlsbad, CA). Reactions were incubated for 10 min at 95°C followed by 40 cycles for 15 seconds at 95°C and 1 min at 60°C. Levels of mRNA and miRNA were quantified using the Invitrogen StepOne System and normalized to FBXW2 (31) and RNU6B, respectively. All reactions were run in triplicate and relative expression was determined using the comparative cycle threshold method (2−ΔΔCq), as recommended by the supplier (Applied Biosystems). Abundance values were expressed as fold changes compared to the corresponding control group. The primer sequences used were as follows: XIST (sense, 5’-TTGCCCTACTAGCTCCTCGGAC -3’; antisense, 5’- TTCTCCAGATAGCTGGCAACC-3’); SP1 (sense, 5’-GCTGCCGCTCCCAACTT-3’; antisense, 5’- CTGAATATTAGGCATCACTCCAGGTA-3’); FBXW2 (sense, 5’-CCTCGTCTCTAAACAGTGGAATAA-3’; antisense, 5’- GCGTCCTGAACAGAATCATCTA-3’); MiR-29c (sense, 5'-GCAGTAGCACCATTTGAAATC-3'; antisense, 5'-GGTCCAGTTTTTTTTTTTTTTTAACC-3'); MiR-200c (sense, 5'-AGTAATACTGCCGGGTAATGA-3'; antisense, 5'-GGTCCAGTTTTTTTTTTTTTTTCCA-3'); and RNU6B (sense, 5'-ATTGGAACGATACAGAGAAGATTAG-3'; antisense, 5'-AATATGGAACGCTTCACGAAT-3').
Cell Proliferation Assay
The LSMC spheroids were transfected with 50 nM siNC or siRNA against XIST as described above and cell proliferation was determined using the CellTiter-Glo 3D Cell Viability Assay (Promega Madison, WI) after 96 hours transfection following the manufacturer’s protocol. The assay was performed in six replicates per condition and repeated three times using cells isolated from three different patients.
Immunoblotting
Total protein isolated from paired tissue samples and LSMC following treatment conditions was subjected to immunoblotting as previously described (32, 33). Briefly, samples were suspended in RIPA buffer containing 1 mM EDTA and EGTA (Boston BioProducts, Ashland, MA) supplemented with 1 mM PMSF and a complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN), sonicated, and centrifuged at 4°C for 10 min at 14,000 rpm. The concentration of protein was determined using the BCA™ Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL). Equal aliquots (thirty micrograms) of total protein for each sample were denatured with SDS-PAGE sample buffer, and separated by electrophoresis on a SDS polyacrylamide gel. After transferring the samples to a nitrocellulose membrane, the membrane was blocked with TBS-Tween + 5% milk, and probed with the following primary antibodies: COL1A1 (Fitzgerald Industries Intl, Acton, MA), COL3A1 and FN1 (Proteintech Group, Inc, Chicago, IL). The membranes were washed with TBS containing 0.1%Tween-20 wash buffer after each antibody incubation cycle. SuperSignal West Pico Chemiluminescent Substrate™ (Thermo Scientific Pierce, Rockford, IL) was used for detection, and photographic emulsion was used to identify the protein bands, which were subsequently quantified by densitometry. The membranes were also stripped and probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology, Dallas, TX) serving as the loading control. The densities of the specific protein bands were determined using image J program (http://imagej.nih.gov/ij/), normalized to GAPDH or a band obtained from staining the membrane with Ponceau S. Results were expressed as means ± SEM as a ratio relative to the control group (siNC) designated as 1.
Generation of Lentivirus Driven XIST Knockdown Cells
Lentivirus plasmid constructs (piLenti-siXIST-GFP) which contain XIST specific siRNA sequences or scrambled control siRNA (piLenti-siRNA-GFP) were purchased from Applied Biological Materials Inc. (Richmond, BC, CA). These plasmid constructs contain a puromycin selection marker gene and express green fluorescence protein. Lentivirus was propagated by co-transfection of human embryonic kidney cells (293T) with psPAX2 (a gift from Didier Trono; Addgene plasmid # 12260), pMD2.G (a gift from Didier Trono; Addgene plasmid # 12259), piLenti-siXIST-GFP or piLenti-siRNA-GFP (as control) using PureFection transfection reagent according to the manufacturer’s instruction (System Biosciences). Supernatant media containing lentiviruses were collected every day for 72h and used for transduction of primary LSMC with addition of polybrene at the final concentration of 8μg/ml. Stable populations with expression of scrambled control or XIST knockdown LSMC were selected with puromycin (0.5ug/ml) after 48h transduction.
Generation of Lentivirus Driven XIST Overexpression Cells
Two XIST overexpression lentiviral plasmids pHIV-XIST#1 and pHIV-XIST#2 were constructed for this experiment. pHIV-XIST#1 was made by insertion of a EcoRI/BamHI-digested PCR amplified fragment of XIST (NR_001564; +12403/+13439) covering two miR-200c binding sites into the lentiviral plasmid pHIV-Luc which was a gift from Bryan Welm (Addgene plasmid # 21375). The fragment of XIST was amplified using primers with the following sequences: forward primer 5'-CGGAATTCCTGCCTCTCTTGGGCTATTC-3', reverse primer 5'-CGGGATCCAGACTGGCCCAGGCATAATA-3'. pHIV-XIST#2 was made by insertion of a EcoRI/BamHI-digested PCR amplified fragment of XIST (NR_001564; +14651/+15579) covering one miR-29c and one miR-200c binding sites into the lentiviral plasmid pHIV-Luc. The fragment of XIST was amplified using primers with the following sequences:forward primer 5'-CGGAATTCTGAGGCCAATTTGTGTTTGC-3',reverse primer 5'-CGGGATCCACCTAGTACCCAGCACCAA-3'. Lentivirus was propagated as described above. Two lentiviral particles made from pHIV-XIST#1 and pHIV-XIST#2 were used for co-transduction of primary MSMC with addition of polybrene at the final concentration of 8μg/ml. Cells transduced with lentiviral particle made from pHIV-Luc were used as control. All cells were transduced again after 48h and harvested at 72h or 96h following initial transduction.
RNA Immunoprecipitation
RNA immunoprecipitation (RIP) assay was performed using an EZ-Magna RIP RNA Binding Protein Immunoprecipitation Kit according to the protocol of manufacturer (Millipore, Burlington, MA). Briefly, fresh specimens of leiomyoma and matched myometrium were lysed in RIP lysis buffer, followed by incubation with RIP buffer containing magnetic beads-bound human anti-argonaute 2 (Ago2) antibody (Millipore) or negative control normal mouse immunoglobulin G (IgG; Millipore). Next, the samples were incubated with proteinase K to digest protein and the immunoprecipitated RNA was isolated. The precipitated RNA was subjected to qPCR analysis to detect the level of target sequences. The assay was performed three times using specimens collected from three different patients.
Statistical analysis
Throughout the text, all data are presented as mean ± SEM and analyzed by PRISM software (Graph-Pad, San Diego, CA). Dataset normality was determined by the Kolmogrove-Smirnoff test. Comparisons involving two groups were analyzed using un-paired Student’s t-tests as appropriate. One-way ANOVA was used for comparisons involving multiple groups. Statistical significance was established at P<0.05.
Results
We initially determined XIST levels in leiomyoma and matched myometrium (N=61) and found a significant increase in expression of XIST in leiomyoma (Fig.1A). The analysis further indicated no significant differences based on racial/ethnic and menstrual cycle phase in expression of XIST (Fig. 1B and 1C). In the next series of in vitro experiments we used 3D culture with spheroids size ranging from 50 μm to 250 μm in diameter (Fig. 1D). Initially we examined the effect of ovarian steroids in regulating the expression of XIST. As shown in Fig. 1E 17β-estradiol (E2; 10−8 M) and progesterone (P4; 10−8 M) significantly stimulated the expression of XIST mRNA. Combination of E2 with P4 further augmented the expression of XIST (Fig. 1E). Since we had previously demonstrated that fibroids expressed higher levels of activated SP1 (7), we sought to determine if SP1 regulated XIST expression. We blocked the expression of SP1 with mithramycin A and by siRNA transfection. As shown in Fig. 1F both mithramycin A and SP1 siRNA significantly reduced the expression of XIST in LSMC spheroids, indicating that SP1 regulates the transcription of XIST. The functional relevance of XIST in LSMC proliferation was demonstrated in in vitro experiments where XIST was knocked down through transfection with siRNA and cell proliferation was determined by CellTiter-Glo 3D Cell Viability Assay (Fig. 2A and 2B). As shown in Fig. 2A the siRNA effectively knocked down XIST and this resulted in a significant reduction of cell proliferation (Fig. 2B).
Figure 1.
(A) The expression of XIST in 61 paired myometrium (Myo) and leiomyoma (Lyo). *: p<0.05. (B) Relative (mean ± SEM) expression of XIST in Lyo and matched Myo based on ethnicity in Caucasians (N=13), African Americans (N=18), Hispanics (N=23) and Asians (N=7). (C) Relative (mean ± SEM) expression of XIST in Lyo and matched Myo based on menstrual cycle phase in follicular phase (N=31) and luteal phase (N=15). (D) A representative image of primary leiomyoma spheroids in culture. Scale bar = 100 μm. (E) The effect of DMSO (control), E2 (10−8 M), P4 (10−8 M), and E2+P4 after 48 hours of culture on the expression of XIST in LSMC spheroids (N=3). (F) Relative expression of XIST after 24 hours treatment of LSMC spheroids with mithramycin A (1 μM; N=4) or 96 hours after transfection with siRNA against SP1 on levels of SP1 and XIST in LSMC spheroids (N=3). The results are presented as mean ± SEM of independent experiments as mentioned above using cells isolated from different patients in each set. P values (*: p<0.05) are indicated by corresponding lines.
Figure 2.
The effect of XIST knockdown through transfection of LSMC spheroids with siRNA against XIST for 96 hours on XIST expression (A; N=7), cell proliferation (B; N=3), expression of miR-29c and miR-200c (C; N=7), and protein levels of COL1A1, COL3A1 and FN1 determined by western blot (D; N=6) along with the relative band density analysis (E). The results shown in (D) are representative of six sets of independent experiments and presented as mean ± SEM using cells isolated from different patients in each set. P values (*: p<0.05) are indicated by the corresponding lines.
Review of XIST and miR-29c and miR-200c sequences suggested putative binding sites in XIST for miR-29c and miR-200c (Supplemental table 1). To establish if XIST could sponge miR-29c and miR-200c we transfected LSMC spheroids with siRNA against XIST and as shown in Fig. 2C in response to the XIST knockdown the expression of miR-29c and miR-200c was increased. We next determined the expression of targets of miR-29c (COL1A1and COL3A1) and as shown in Fig. 2D and 2E the protein abundance of both targets of miR-29c were decreased in response of XIST knockdown. Similarly, the expression of FN1 which is a target of miR-200c was decreased in response to XIST knockdown (Fig. 2D and 2E). We further confirmed these results by stable knockdown of XIST in fibroid cells through lentivirus transduction and puromycin selection (Fig. 3A). As shown in Fig. 3B and 3C in response to stable knockdown of XIST in spheroids there was a significant increase in the expression of miR-29c and miR-200c, while the protein levels of their targets COL1A1, COL3A1 and FN1 were significantly repressed (Fig. 3D and 3E). Moreover, we overexpressed two fragments of XIST which contain sequences complementary to miR-29c and miR-200c in primary MSMC spheroids via lentivirus transduction. After 96h transduction the fragment 1 (+12403/+13439) was increased an average fold of 27.76 (± 5.753) and the fragment 2 (+14651/+15579) was increased an average fold of 15.3 (± 3.079). In response to XIST overexpression the levels of these miRNAs were down-regulated significantly (Fig. 4A), along with up-regulation of their target gene (COL1A1, COL3A1 and FN1) protein expression (Fig. 4B and 4C). These experiments thus establish the presence of a lncRNA-miRNA network in leiomyoma involving the long non-coding RNA XIST and miR-29c and miR-200c.
Figure 3.
(A) The representative images of green fluorescence derived from GFP in puromycin-selected primary leiomyoma cells after transduction with lentivirus expressing scrambled oligonucleotides (a; siNC) or siRNA against XIST (b; siXIST). (B-E) Shows the expression of XIST (B), miR-29c and miR-200c (C), and protein levels of COL1A1, COL3A1 and FN1 (D) along with the relative band densities analysis (E) in stable XIST knockdown primary leiomyoma spheroids (N=3). The results shown in (D) are representative of three sets of independent experiments and presented as mean ± SEM using cells isolated from different patients in each set. P values (*: p<0.05) are indicated by the corresponding lines.
Figure 4.
(A-C) The effects of overexpression of two fragments of XIST through lentivirus transduction of primary MSMC spheroids on the expression of miR-29c and miR-200c (A; 72h; N=3), and representative protein levels of COL1A1, COL3A1 and FN1 (B; 96h; N=3) along with the relative band density analysis (C). The results are presented as mean ± SEM using cells isolated from different patients in each set. P values (*: p<0.05) are indicated by the corresponding lines. (D-E) Shows the results of RNA immunoprecipitation assay with Ago2 antibody to determine the association between XIST and miR-29c and miR-200c using lysates from fresh specimens of leiomyoma and matched myometrium (N=3). The expression of XIST (D) and miR-29c and miR-200c (E) from purified RNA was determined by qPCR. The results are presented as mean ± SEM. *: p<0.05 vs IgG; #: p<0.05 vs Ago2 of the Myo group.
In order to further establish an interaction between XIST and miR-29c and miR-200c we carried out RNA immunoprecipitation study using a specific antibody to Ago2 protein. As shown in Fig. 4D and 4E the results indicated that both miR-29c and miR-200c co-precipitated with XIST in myometrium and matched leiomyoma with greater binding of the antibody to these miRNAs in leiomyoma as compared with myometrium.
Discussion
The results of this study provide the first evidence for a functional role for XIST in leiomyoma pathogenesis and establish the presence of lncRNA-miRNA network involving XIST and miR-29c/miR-200c. This long non-coding RNA is expressed at higher levels in fibroids compared with normal myometrium which is independent of race/ethnicity and menstrual cycle phase. Estradiol, progesterone and the combination of estradiol and progesterone stimulated the expression of XIST. Our results indicated that XIST is also regulated by the transcription factor SP1 which we previously demonstrated to be upregulated in its active phosphorylated form in fibroids (7). The functional relevance of XIST to fibroid pathology was further demonstrated by our data in which knockdown of XIST decreased LSMC spheroids proliferation. Knockdown of XIST in LSMC spheroids resulted in overexpression of miR-29c and miR-200c and downregulation of their respective target proteins including COL1A1, COL3A1 and FN1. RNA immunoprecipitation showed direct interaction between XIST and miR-29c and miR-200c, confirming its role as a sponge for miR-29c and miR-200c, and a potential mechanism for down-regulation of these key miRNAs in fibroids.
XIST is transcribed from the inactive X chromosome. The XIST RNA directly binds to the inactive X chromosome and recruits polycomb repressive complex 2 (PRC2) which catalyzes the tri-methylation of histone H3 on lysine 27 resulting in chromatin repression and transcriptional silencing (34, 35). The TSIX antisense gene is a transcript of the XIST gene which acts in cis to repress the transcription of XIST through modification of chromatin structure (36). With the exception of one study (37) the role of XIST and TSIX in fibroid pathogenesis has not been investigated. In the study by Sato et al. XIST expression was downregulated in 36% of leiomyoma specimens and the authors proposed downregulation of XIST as a potential mechanism of aberrant DNA hypomethylation on the X chromosome in leiomyomas. In our study the majority of leiomyomas had overexpression of XIST. This discrepancy could be due to racial differences in the study population which was predominantly Hispanic in our study versus Asian population in the study by Sato et. al (37), or due to differences in the internal control used for normalization of PCR data. In this study we used FBXW2 which we have found to vary the least in expression in fibroids among 5 different internal controls tested as opposed to GAPDH used by Sato et al which was previously shown to vary in expression in fibroids (31). Several of our findings provide support for the functional relevance of XIST in fibroid pathogenesis. Firstly, our results indicate that ovarian steroids have a stimulatory effect on XIST expression. This is in contrast to another setting where sex steroids were shown to have a negative effect on XIST regulation (38); in that study treatment of boys with cryptorchidism with the GNRH analogue resulted in reduced expression of testicular XIST (38). Secondly, our data demonstrates that XIST regulates LSMC cell proliferation. Thirdly, knockdown of XIST resulted in downregulation of genes known to be aberrantly expressed in fibroid such as collagens and fibronectin. Fourthly, our data indicates that the transcription factor SP1 which in its active phosphorylated form is increased in fibroids (7) also regulates the expression of XIST in fibroids by increasing its transcription. To our knowledge this is the first demonstration that SP1 plays a role in XIST regulation.
XIST is widely involved in tumorigenesis both as an oncogene and as a tumor suppressor (39). These effects are exerted by sponging different miRNAs in different types of tumors. Similar to leiomyoma cells, XIST has been shown to facilitate cell proliferation in colorectal cancer cells by targeting miR-486-5p (40), and hepatocellular carcinoma by targeting miR-139-5p/PDK1/Akt axis (41). It acts as an oncogene in osteosarcoma by sponging miR-137 (42), in esophageal cancer by sponging miR-494 to regulate CDK6 (43), in retinoblastoma by sponging miR-101 (44), in papillary thyroid carcinoma by targeting miR-141 (45), and in colon cancer by sponging miR-34a via Wnt/B-catenin signaling pathway (46). XIST also has anticancer effects such as in ovarian cancer cells by targeting miR-214-3p (47) and hepatocellular carcinoma by sponging miR-155-5p (48). Our data in leiomyoma indicates that XIST is a molecular sponge for both miR-29c and miR-200c which we have previously demonstrated to be downregulated in fibroids (7, 8). Through this sponging effect and lowering the levels of these miRNAs their target genes like collagen subtype and FN1 are upregulated. The interaction between XIST and miR-29 and miR-200 family has been demonstrated in other cell types. XIST was shown to target miR-29a in skin fibroblasts following thermal injury (49), and to target miR-29c in nasopharyngeal carcinoma cells which similar to fibroids have elevated expression of XIST and lower miR-29c expression (22). XIST accelerated cervical cancer progression by competitively binding miR-200a and upregulating Fus (50) and by targeting miR-200c to regulate the stemness properties and tumorigenicity of bladder cancer cells (23).
In summary, our data demonstrate a functional role for XIST in fibroid pathogenesis and establishes a network involving XIST, miR-29c and miR-200c. The expression of XIST is upregulated in fibroids, and is regulated by ovarian steroids and the transcription factor SP1. XIST interacts directly with miR-29c and miR-200c both of which are known to be downregulated in fibroids. Therapeutic targeting of XIST and normalizing its expression in fibroid could be a strategy to also normalize the expression of miR-29c and miR-200c and their target genes, which regulate ECM and cell proliferation.
Supplementary Material
Supplementary Table 1: Sequence alignment demonstrating putative binding sites of XIST with miR-29c and miR-200c.
Acknowledgements
This study was supported by NIH (HD088868).
Footnotes
All authors have declared that no conflict of interests existed.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplementary Table 1: Sequence alignment demonstrating putative binding sites of XIST with miR-29c and miR-200c.