Non-coding RNA Xist Research Articles

Abstract 2452: Evidence of decreased X chromosome inactivation in primary ovarian tumors

Abstract Copy number alterations and loss of heterozygosity (LOH) are common in ovarian tumors, including those on the X chromosome. In females, one of the two copies of the X chromosome is transcriptionally silenced during embryonic development in the epigenetic process of X chromosome inactivation. Specifically, the long non-coding RNA XIST is expressed from the inactive X (Xi) and triggers chromosome-wide DNA methylation. In breast and ovarian cancer cell line studies, Xi deletion with concomitant duplication of the active X (Xa) has been observed. We sought to examine which copy of the X chromosome, Xi or Xa, was gained or lost in a set of primary ovarian tumors, using levels of XIST expression as a marker of X chromosome inactivation. To determine which copy of the X chromosome harbors the alterations, we compared local copy number and LOH in Xq13.2 to the levels of XIST expression in a set of N=134 primary tumors from Mayo Clinic ovarian cancer patients. Associations with XIST expression were examined using ANOVA, with copy number of two and absence of LOH as the reference level. Associations between expression of XIST and expression of other genes were examined with a mixed effects model, to account for the repeated observations across patients, after adjusting for local copy number and LOH. Associations with XIST expression and overall survival were performed with Cox regression. Additional analyses were performed in a subset of 24 patient tumors with X chromosome-wide alterations to simplify interpretation, including a visual examination of chromosome-wide DNA methylation patterns. Analyses were replicated in N=106 ovarian tumors from TCGA with X chromosome-wide alterations. Across N=134 ovarian tumors from Mayo Clinic, abnormal copy number (not equal to two) was associated with reduced XIST expression (p=0.0016). Levels of XIST expression were similar for tumors with one copy and tumors with three or more copies. Similarly, levels of XIST expression were also lower for tumors with LOH (p=5.2E-5). Reduced XIST expression was also associated with poorer survival, after adjusting for age (p=0.02). Results were consistent in the subset of tumors with whole chromosome alterations (pCN=0.0013, pLOH=0.0019). For these tumors, when LOH was present, XIST expression was low and the DNA methylation distribution is bi-modal, suggesting that the inactive X was lost and the active X was gained. Furthermore, after adjusting for copy number and LOH, XIST expression was weakly positively correlated with the median expression of other genes (R2=0.04, p=0.026). In N=106 TCGA tumors with whole X chromosome alterations, XIST expression was also reduced in tumors with abnormal copy number (p=0.006) and with LOH (p=1.5E-10). Taken together, because XIST is expressed from Xi and not Xa, associations of reduced XIST expression with abnormal copy number and presence of LOH suggest that Xi is frequently lost and Xa is gained in ovarian tumors, consistent with cell line studies. These results should be further validated in larger sample sets and examined for association with clinical outcome. Citation Format: Yilin Song, Bryan M. McCauley, Melissa C. Larson, Sebastian M. Armasu, Kate Lawrenson, Ellen L. Goode, Nicholas B. Larson, Stacey J. Winham. Evidence of decreased X chromosome inactivation in primary ovarian tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2452.

LncRNA XIST promotes chemoresistance of breast cancer cells to doxorubicin by sponging miR-200c-3p to upregulate ANLN.

The resistance of breast cancer cells to drugs is a major obstacle to effective cancer chemotherapy. Here, we study the function mechanisms of long non-coding RNA XIST in chemoresistance of breast cancer to doxorubicin. We examined the 50% inhibitive concentration of doxorubicin to MDA-MB-231 and MDA-MB-231/ADM cells, showing that the doxorubicin resistance of MDA-MB-231/ADM cells was much higher than MDA-MB-231 cells. The gene or protein expression of XIST and ANLN were also higher in MDA-MB-231/ADM cells than that in MDA-MB-231 cells. Moreover, XIST overexpression promoted cell proliferation and inhibited apoptosis of doxorubicin-treated MDA-MB-231 cells by promoting ANLN expression. XIST silencing inhibited cell proliferation and promoted apoptosis of doxorubicin-treated MDA-MB-231/ADM cells by inhibiting ANLN expression. Luciferase reporter assay showed that XIST functioned as a competing endogenous RNA to repress miR-200c-3p, which controlled its downstream target ANLN. In conclusion, these data reveal that XIST promotes chemoresistance of breast cancer cells to doxorubicin by sponging miR-200c-3p to upregulate ANLN. This work explores the relationship between lncRNA XIST and doxorubicin resistance in breast cancer cells and highlights a novel therapeutic target for the treatment of breast cancer.

Investigating nuclear organization of the inactive X in female lymphocytes

Abstract Autoimmune diseases are devastating, incurable disorders that affect over 20 million people in the US, and many of these disorders have a striking female bias. The genetic basis for this sex-bias arises from the X chromosome. Female mammals, with two X chromosomes, utilize the epigenetic process of X Chromosome Inactivation (XCI), which transcriptionally silences one X. Expression of the long noncoding RNA Xist from the inactive X (Xi) reorganizes the Xi into a compact, bipartite structure, and targets the Xi to the nuclear periphery in somatic cells. The paradigm of XCI maintenance in somatic cells is stable association of Xist RNA and heterochromatic marks on the Xi, which function to maintain the Xi structure. Recently, we found a dynamic mechanism of XCI maintenance in female lymphocytes. In naïve female B cells, Xist RNA is not localized to the Xi, but after stimulation Xist RNA and heterochromatin marks return to the Xi. How this dynamic XCI process impacts Xi structure in lymphocytes is unknown, including which factors regulate Xi nuclear organization in this system. Using a novel allele-specific imaging system, we will present new findings on the compaction and organization of the Xi in lymphocytes. Additionally, we are investigating the regulatory roles of chromatin organization and nuclear structure proteins for lymphocyte-specific XCI maintenance, and how these factors regulate X-linked gene expression. Together, these results underscore the importance of Xi structure for the fidelity of XCI in female lymphocytes.

Sex differences in the proliferation of pulmonary artery endothelial cells: implications for plexiform arteriopathy.

The sex-biased disease pulmonary arterial hypertension (PAH) is characterized by the proliferation and overgrowth of dysfunctional pulmonary artery endothelial cells (PAECs). During inflammation associated with PAH, granzyme B cleaves intersectin-1 to produce N-terminal (EHITSN) and C-terminal (SH3A-EITSN) protein fragments. In a murine model of PAH, EHITSN triggers plexiform arteriopathy via p38-ELK1-c-Fos signaling. The SH3A-EITSN fragment also influences signaling, having dominant-negative effects on ERK1 and ERK2 (also known as MAPK3 and MAPK1, respectively). Using PAECs engineered to express tagged versions of EHITSN and SH3A-EITSN, we demonstrate that the two ITSN fragments increase both p38-ELK1 activation and the ratio of p38 to ERK1 and ERK2 activity, leading to PAEC proliferation, with female cells being more responsive than male cells. Furthermore, expression of EHITSN substantially upregulates the expression and activity of the long non-coding RNA Xist in female PAECs, which in turn upregulates the X-linked gene ELK1 and represses expression of krüppel-like factor 2 (KLF2). These events are recapitulated by the PAECs of female idiopathic PAH patients, and may account for their proliferative phenotype. Thus, upregulation of Xist could be an important factor in explaining sexual dimorphism in the proliferative response of PAECs and the imbalanced sex ratio of PAH.

Downregulation of XIST ameliorates acute kidney injury by sponging miR-142-5p and targeting PDCD4.

Acute kidney injury (AKI) is a common kidney disease that markedly affects public health. To date, the roles of long noncoding RNA XIST in AKI are poorly understood. Here, we investigated the biological functions of XIST in AKI. We observed that XIST expression increased in patients with AKI and HK-2 cells stimulated by CoCl2 . In addition, a rat AKI model induced by ischemia-reperfusion was established. Tumor necrosis factor-α, interleukin-6, and cyclooxygenase-2 messenger RNA expression were induced in vivo; moreover, XIST expression was upregulated. Knockdown of XIST significantly repressed CoCl2 -triggered injury in HK-2 cells. However, microRNA (miR)-142-5p, a downstream target of XIST, was downregulated in AKI. miR-142-5p was repressed by XIST and miR-142-5p could inhibit CoCl2 -induced injury in HK-2 cells. Moreover, PDCD4 expression was significantly increased in AKI. PDCD4 was predicted to be the target of miR-142-5p. Subsequently, loss of PDCD4 was able to retard injury in HK-2 cells exposed to CoCl2. Thus, we suggest that XIST regulates miR-142-5p and PDCD4, and it has the potential to function as a biomarker in therapeutic strategies for AKI.

METTL14 suppresses proliferation and metastasis of colorectal cancer by down-regulating oncogenic long non-coding RNA XIST

BackgroundN6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulation in eukaryotic cells. However, understanding of m6A in colorectal cancer (CRC) is very limited. We designed this study to investigate the role of m6A in CRC.MethodsExpression level of METTL14 was extracted from public database and tissue array to investigate the clinical relevance of METTL14 in CRC. Next, gain/loss of function experiment was used to define the role of METTL14 in the progression of CRC. Moreover, transcriptomic sequencing (RNA-seq) was applied to screen the potential targets of METTL14. The specific binding between METTL14 and presumed target was verified by RNA pull-down and RNA immunoprecipitation (RIP) assay. Furthermore, rescue experiment and methylated RNA immunoprecipitation (Me-RIP) were performed to uncover the mechanism.ResultsClinically, loss of METTL14 correlated with unfavorable prognosis of CRC patients. Functionally, knockdown of METTL14 drastically enhanced proliferative and invasive ability of CRC cells in vitro and promoted tumorigenicity and metastasis in vivo. Mechanically, RNA-seq and Me-RIP identified lncRNA XIST as the downstream target of METTL14. Knockdown of METTL14 substantially abolished m6A level of XIST and augmented XIST expression. Moreover, we found that m6A-methylated XIST was recognized by YTHDF2, a m6A reader protein, to mediate the degradation of XIST. Consistently, XIST expression negatively correlated with METTL14 and YTHDF2 in CRC tissues.ConclusionOur findings highlight the function and prognostic value of METTL14 in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.

LncRNA XIST modulates HIF-1A/AXL signaling pathway by inhibiting miR-93-5p in colorectal cancer.

BackgroundLong noncoding RNA (LncRNA) XIST is one of the genes that exists in different types of cancers. Earlier researches showed that XIST can advance the progression of colorectal cancer. Nevertheless, the potential molecular mechanism of XIST in combination with miR‐93‐5p has not been explored in colorectal cancer.MethodsWe performed qRT‐PCR to explore the level of XIST. And a serious experiments in vitro and in vivo were performed to explore the function of XIST. The relationship between XIST/HIF‐1A and miR‐93‐5p was confirmed by RIP and dual‐luciferase assays.ResultsIn the present research, our team demonstrated the upregulation of XIST expression, which was related to tumor progression, and the downregulation of miR‐93‐5p in cells and tissues of colorectal cancer. XIST is the competitive endogenous RNA of miR‐93‐5p to promote HIF‐1A, and then the upregulated AXL level facilitates the EMT process, migration, and proliferation of colorectal cancer. At last, we proved that XIST enhanced the in vivo and in vitro activities of colorectal cancer by regulating AXL signaling.ConclusionIn summary, the above results indicate that XIST promotes colorectal cancer tumorigenesis by regulating miR‐93‐5p/HIF‐1A/AXL signaling pathway, which will supply a novel perspective to diagnose and treat colorectal cancer disease.

Long non-coding RNA XIST is down-regulated and correlated to better prognosis in ovarian cancer.

Ovarian cancer (OC) is one of the most common gynecological malignant tumors. Understanding the molecular mechanisms involved in the development of ovarian cancer is helpful for the diagnosis and treatment of OC. In this study, we found that lncRNA XIST was significantly overexpressed in normal ovarian tissues and down-regulated in OC. Moreover, we showed XIST was associated with the development of OC and down-regulated in advanced stage OC compared to early-stage OC samples. Overexpression of XIST was significantly associated with longer survival in patients with OC. Also, our analysis also showed that lncRNA XIST was closely related to biological processes such as transcription, protein phosphorylation, transport, protein ubiquitination, and DNA repair. To further reveal the function and role of XIST in OC, we constructed a protein-protein interaction network and an endogenous competitive RNA network. The present study provided a theoretical basis for the diagnosis and treatment of OC.

Genetic Ablation of lncRNA <i>XACT</i> Does Not Change Expression Dosage of X-Linked 1 Genes But Impacts Differentiation Potential in Female Human Pluripotent Stem Cells

Female human pluripotent stem cells (hPSCs) regularly show erosion of X-chromosome inactivation (XCI) featured by loss of the long non-coding (lnc) RNA XIST and accumulation of XACT. Although both lncRNAs are expressed from X-chromosome, XACT is mainly expressed in pluripotent cells, and the expression pattern is reciprocal to XIST, suggesting an important role in dosage compensation and differentiation. In this study, we aimed to dissect the role of XACT in the erosion of dosage compensation and differentiation potential in female hPSCs. We found that XACT RNA accumulation on inactive X-chromosomes is hPSC line dependent. XIST RNA overlapped with H3K27me3 but not H3K9me3, and XIST/H3K27me3 loss occurred during erosion in all female hPSC lines analyzed. These results indicate that a common mechanism for initiation of erosion depends on XIST loss but not XACT accumulation on inactive X-chromosomes. We further demonstrated that XACT deletion does not affect X-linked gene dosage in eroded hPSCs. Additionally, aberrant XIST RNA diffusion induced by the CRISPR activation system was independent of the presence of XACT RNA. In contrast, genetic ablation of XACT in female hPSCs resulted in the up-regulation of neuron-related genes to facilitate neural differentiation. Finally, we confirmed that XACT repression by the CRISPR inhibition system leads to dysregulation of the genes and affects neural differentiation, indicating that XACT RNA is involved in gene regulation affecting neural differentiation. Our study demonstrates that XACT is dispensable for the maintenance of dosage of X-linked genes, but impacts neural differentiation in female hPSCs, implying that XACT might play important roles in the regulation of spatiotemporal development rather than X-chromosome dosage compensation in human pluripotent cells.

Long noncoding RNA XIST acts as a competing endogenous RNA to promote malignant melanoma growth and metastasis by sponging miR-217.

Long noncoding RNA XIST acts as a competing endogenous RNA to promote malignant melanoma growth and metastasis by sponging miR-217.

Localized accumulation of Xist RNA in X chromosome inactivation.

The non-coding RNA Xist regulates the process of X chromosome inactivation, in which one of the two X chromosomes present in cells of early female mammalian embryos is selectively and coordinately shut down. Remarkably Xist RNA functions in cis, affecting only the chromosome from which it is transcribed. This feature is attributable to the unique propensity of Xist RNA to accumulate over the territory of the chromosome on which it is synthesized, contrasting with the majority of RNAs that are rapidly exported out of the cell nucleus. In this review I provide an overview of the progress that has been made towards understanding localized accumulation of Xist RNA, drawing attention to evidence that some other non-coding RNAs probably function in a highly analogous manner. I describe a simple model for localized accumulation of Xist RNA and discuss key unresolved questions that need to be addressed in future studies.

Long non-coding RNA XIST promotes hepatocellular carcinoma progression by sponging miR-200b-3p.

Recent studies have proved that long noncoding RNAs (lncRNAs) act as an important role in many diseases. In this research, lncRNA XIST was explored to identify how it functions in the development of hepatocellular carcinoma (HCC). Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was utilized to detect XIST expression in HCC patients. Then, we conducted Cell Counting Kit-8 (CCK-8) assay and colony formation assays in vitro. Furthermore, mechanism assays and the interaction between XIST and miR-200b-3p were conducted. By comparing with XIST expression in adjacent tissues, the XIST expression level was significantly higher in HCC samples. Moreover, functional assays showed that the cell growth ability of HCC cells was inhibited after XIST was silenced in vitro, and tumor formation was inhibited after XIST was silenced in vivo. Further experiments showed that miR-200b-3p was directly targeted by XIST. Above results suggest that XIST could enhance the cell growth ability of HCC by targeting miR-200b-3p, which suggest that XIST may be a potential therapeutic target in HCC.

Evaluation of transcriptional levels of the natriuretic peptides, endothelin-1, adrenomedullin, their receptors and long non-coding RNAs in rat cardiac tissue as cardiovascular biomarkers of aging

Chronological age is considered one of the major risk factors for cardiovascular disease and mortality. The study aimed to evaluate the transcriptional levels of the natriuretic peptides (NP), endothelin (ET)-1, adrenomedullin (ADM), their receptors and long non-coding (Lnc) RNA MIAT, MALAT-1, CARMEN and XIST in rat cardiac tissue as cardiovascular biomarkers of aging. Three groups of male Wistar rats were studied: A (n = 6; young), B (n = 13; adult), C (n = 10; old). Total RNA was extracted from left ventricle and analyzed by Real-Time PCR. Echocardiographic and histological analyses were performed. A significant increase of Atrial NP (ANP) and Brain NP (BNP) mRNA was observed in C while C-type NP (CNP) remained in a steady-state in B and C; ET-1 mRNA increased significantly as a function of age. Any difference was observed for NP receptors. ETA expression was statistically lower in B than A while ETB were similar in all the three groups. The ADM showed an opposite trend to that of the other peptides decreasing significantly as a function of age and presenting a counter-regulation of calcitonin receptor-like receptor (CLR) and receptor activity modifying protein (RAMP)-2. LncRNA transcripts decreased significantly as a function of age except for XIST. ADM and LncRNA trend suggest that the animals are subjected to “successful aging” as also confirmed by histological analysis. Applying a multivariate logistic regression analysis, only LnANP (p = 0.003) and LnADM (p = 0.023) resulted significantly associated with aging identifying them, for the first time, as independent markers of aging. The study underlining the importance of a multi-label biomolecular approach in the evaluation of aging.

Downregulation of long non-coding RNA XIST inhibits cell proliferation, migration, invasion and EMT by regulating miR-212-3p/CBLL1 axis in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors in the world and its 5-year survival rate is very low. Long non-coding RNA X-inactive specific transcript (lncRNA XIST) has been demonstrated to play vital roles in NSCLC, but the exact molecular mechanisms underlying NSCLC still need to be further explored. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed to detect the expression of XIST, miR-212-3p and Casitas B-lineage proto-oncogene like 1 (CBLL1). Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to analyze the relationship among XIST, miR-212-3p and CBLL1. Cell Counting Kit-8 (CCK-8) assay and transwell invasion assay were carried out to evaluate cell proliferation, migration and invasion, respectively. Western blot analysis was conducted to examine the protein expression of CBLL1, E-cadherin, N-cadherin and Vimentin. Murine xenograft assay was conducted to explore the role of XIST in vivo. Expression levels of XIST and CBLL1 were markedly upregulated, while the miR-212-3p level was markedly downregulated in NSCLC tissues and cells. MiR-212-3p was identified as a direct target of XIST, and miR-212-3p was predicted to target CBLL1. XIST knockdown repressed NSCLC cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in vitro, and suppressed tumor growth in vivo, while miR-212-3p inhibition restored the effects. Furthermore, CBLL1 overexpression could abolish the effects of miR-212-3p overexpression on NSCLC cell proliferation, migration, invasion and EMT in vitro. XIST was significantly decreased in NSCLC tissues and cells, and XIST knockdown suppressed the proliferation, migration, invasion and EMT of NSCLC cells by miR-212-3p/CBLL1 axis. These findings facilitated our understanding of lncRNA regulation in NSCLC.

Long non-coding RNA XIST promotes malignant behavior of epithelial ovarian cancer.

PurposeThis study aims to investigate the functional role of long non-coding RNA XIST in epithelial ovarian cancer (EOC).MethodsDetection of XIST expression levels in EOC tissues and cell lines was done using qRT-PCR. The relationship between XIST expression and clinicopathological features of EOC patients was compared and analyzed. The cumulative survival rates were calculated using Kaplan-Meier. A Cox hazard model was used to identify risk factors for survival. Lastly, the effects of XIST on EOC cell were assessed in vitro.ResultsXIST was up-regulated in EOC tissues and cell lines. The expression of XIST was closely related to the tumor grade, distant metastasis, and FIGO stage in the EOC patients. The Cox regression analysis showed that high XIST expression was an independent predictor of prognosis in patients with EOC. In in vitro experiments, reducing XIST expression significantly suppressed cell proliferation, migration and invasion in EOC cells.ConclusionXIST highly expressed in the EOC and plays a role in tumor promotion, which may be a potential target for the treatment of EOC.

CROSSalive: a web server for predicting the in vivo structure of RNA molecules.

RNA structure is difficult to predict in vivo due to interactions with enzymes and other molecules. Here we introduce CROSSalive, an algorithm to predict the single- and double-stranded regions of RNAs in vivo using predictions of protein interactions. Trained on icSHAPE data in presence (m6a+) and absence of N6 methyladenosine modification (m6a-), CROSSalive achieves cross-validation accuracies between 0.70 and 0.88 in identifying high-confidence single- and double-stranded regions. The algorithm was applied to the long non-coding RNA Xist (17 900 nt, not present in the training) and shows an Area under the ROC curve of 0.83 in predicting structured regions. CROSSalive webserver is freely accessible at http://service.tartaglialab.com/new_submission/crossalive. Supplementary data are available at Bioinformatics online.

Long noncoding RNA XIST enhances ethanol-induced hepatic stellate cells autophagy and activation via miR-29b/HMGB1 axis.

Activation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrogenesis, including alcoholic liver fibrosis (ALF). Furthermore, autophagy contributes to HSCs activation. This study aims to investigate the role and the mechanisms of long noncoding RNA XIST in regulating HSCs autophagy and activation. Human HSC cells (LX-2) were treated with 100 mmol/L ethanol to mimic HSCs activation. The HSCs activation was evaluated by determining cell viability and protein levels of fibrosis markers α-smooth muscle actin (α-SMA) and collagen type 1 α1 (CoL1A1). The autophagy was evaluated by measuring autophagy markers Beclin-1 and LC3-II. The interaction among XIST, miR-29b, and high-mobility group box-1 (HMGB1) were analyzed using luciferase reporter assay, qRT-PCR, and western blot. Lentiviruses targeting sh-XIST (LV-sh-XIST) were injected into ALF model mice via tail vein to elucidate the in vivo role of XIST in ALF injury. XIST was upregulated in ethanol-activated LX-2 cells. Furthermore, XIST served as a competitive endogenous RNA of miR-29b to facilitate HMGB1 expression, and thus enhanced ethanol-induced HSCs autophagy and activation. Further in vivo assay showed that downregulation of XIST by LV-sh-XIST alleviated ALF injury in ALF model mice. Collectively, XIST enhances ethanol-induced HSCs autophagy and activation via miR-29b/HMGB1 axis.

Long non-coding RNA XIST predicting advanced clinical parameters in cancer: A Meta-Analysis and case series study in a single institution.

Dysregulated expression of long non-coding RNA X-inactive specific transcript (lncRNA-XIST) has been indicated in various cancer types. In the present study, a meta-analysis was conducted to evaluate the potential role of lncRNA-XIST in predicting the clinicopathological parameters of patients with cancer. Eligible studies were obtained through a systematic search of PubMed, Web of Science, Embase and the Cochrane Library, of articles published prior to January 2019. The combined odds ratio and 95% confidence interval were calculated to determine the association between lncRNA-XIST expression and patient outcome. In addition, 45 pairs of osteosarcoma (OS) tissues and adjacent healthy tissues from a single institution were analyzed for the expression of lncRNA-XIST, and its association with clinicopathological features; ultimately, a total of 1,869 cancer patients from 25 studies were assessed. The results demonstrated that high expression levels of lncRNA-XIST were significantly associated with lymphatic metastasis, larger tumor size, advanced cancer stage and distant metastasis. However, sex was not associated with lncRNA-XIST expression level. In the OS patient cohort, it was demonstrated that lncRNA-XIST was highly expressed in OS tissues, which negatively correlated with patient prognosis. The present study indicated that lncRNA-XIST may serve as a potential biomarker for advanced clinical parameters in human cancer.

BRCa1 participates in XIST RNa localization on inactive x chromosome

Introduction . Inactive X chromosome (Xi) is associated with noncoding XIST RNA, series of proteins and contains multiple epigenetic modifications that altogether determine a silence of the most of X-linked genes. Recently the data were obtained that tumor suppressor BRCA1 is also associated with Xi. The purpose of this study was to reveal the colocalization of BRCA1 and XIST RNA and precise spatial organization on Xi with the high resolution of confocal microscopy.Materials and methods . The object of the study is IMR90hTERT diploid immortalized fibroblast cell line. For BRCA1 and XIST RNA colocalization analysis on Xi the method of fluorescent hybridization in situ associated with immunofluorescent cell staining (immunoFISH) and confocal microscopy were used. For BRCA1 and heterochromatin protein-1 colocalization study the method of double immunofluorescent staining and common fluorescent microscopy were applied. Results . The study using confocal fluorescent microscopy with higher resolution has demonstrated at first the colocalization of BRCA1 with XIST RNA region of Xi revealed with XIST RNA probes and with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. Altogether, the data obtained suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the regulation of XIST RNA association with Xi. Moreover, according to the results of confocal microscopy, BRCA1 also colocalizes with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. This indicates a possible involvement of this protein in the replication of pericentromeric repeats in cellular chromosomes. Colocalization of BRCA1 with heterochromatin protein-1α presented in pericentromeric regions of all chromosomes supports this suggestion.Conclusions . Altogether, the data obtained in this study suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the association with noncoding inhibiting XIST RNA and in replication of heterochromatin regions.

The functional role and clinical significance of long non-coding RNA Xist in cervical cancer

The functional role and clinical significance of long non-coding RNA Xist in cervical cancer