X-inactive Specific Transcript Research Articles

Long non‑coding RNA GAS5 increases the radiosensitivity of A549 cells through interaction with the miR‑21/PTEN/Akt axis.

Radioresistance hinders the therapeutic outcomes of radiotherapy in non-small cell lung cancer (NSCLC). Although long non-coding RNAs (lncRNAs) have been demonstrated to participate in the regulation of multiple cell behaviors, whether they can modulate the radiosensitivity of NSCLC and the underlying molecular mechanisms have not been well investigated. In the present study, it was revealed that NSCLC NCI-H460 cells were more sensitive to ionizing radiation (IR) than A549 cells. Using the RNA-Seq method, four highly differentially expressed lncRNAs were identified, including the growth arrest-specific transcript 5 (GAS5), syntaxin binding protein 5 antisense RNA 1 (STXBP5-AS1), metastasis associated lung adenocarcinoma transcript 1 (MALAT1) and X-inactive specific transcript (XIST), which were predicted to play roles in the acquisition of radiosensitivity. Using real-time quantitative PCR (qPCR), it was demonstrated that lncRNA GAS5 was significantly upregulated in NCI-H460 cells but not in A549 cells during IR. Mechanistically, it was demonstrated that overexpression of lncRNA GAS5 decreased the level of microRNA-21 (miR-21). Overexpression of lncRNA GAS5 or suppression of miR-21 markedly increased the IR-induced cell apoptosis of A549 cells. It was also demonstrated that overexpression of lncRNA GAS5 increased PTEN expression and suppressed Akt phosphorylation through the modulation of miR-21. Notably, it was revealed that IR enhanced the interaction between lncRNA GAS5 and the miR-21/PTEN/Akt axis. In summary, the present findings revealed that lncRNA GAS5 has a radiosensitization effect on NSCLC, indicating the potential application of lncRNA GAS5 in NSCLC radiotherapy.

RETRACTED ARTICLE: LncRNA XIST inhibits ovarian cancer cell growth and metastasis via regulating miR-150-5p/PDCD4 signaling pathway.

The aim of this study was to explore the role of X-inactive specific transcript (XIST), miR-150-5p, and programmed cell death protein 4 (PDCD4) in the pathogenesis of ovarian cancer (OC). The expression levels of XIST, miR-150-5p, and PDCD4 were examined by qRT-PCR or Western blotting as appropriate. The proliferation, apoptosis, migration, and invasiveness of human OC cell lines in vitro were respectively evaluated by CCK-8, TUNEL, wound-healing assay, and transwell assay. A xenograft model of OC was also established to determine the effect of XIST knockdown on tumor growth in vivo. Finally, direct binding between XIST and miR-150-5p and between miR-150-5p and PDCD4 were verified by dual-luciferase reported assay. XIST was significantly downregulated in OC tissues and cell lines compared with the normal ovarian epithelial tissues and cells. XIST knockdown in OC cells significantly facilitated cell growth, migration, and invasion and inhibited apoptosis. In addition, silencing XIST also initiated epithelial-mesenchymal transition (EMT) and conferred cancer stem cell (CSC) survival in vitro. Consistent with the in vitro findings, knockdown of XIST promoted tumorigenesis, suppressed PDCD4, and upregulated miR-150-5p in vivo. XIST directly bound to and downregulated miR-150-5p in OC tissues and cell lines. Furthermore, PDCD4 was identified as the target gene of miR-150-5p, and XIST knockdown downregulated PDCD4 by relieving miR-150-5p inhibition. Finally, blocking miR-150-5p partly abolished the effects of XIST knockdown in vitro. Taken together, XIST inhibits OC growth and metastasis by upregulating PDCD4 via miR-150-5p suppression.

LncRNA XIST promotes glioma proliferation and metastasis through miR-133a/SOX4.

Glioma is the most common malignant brain tumour in adults, and the aetiology and mechanism of this tumour remain largely unknown. Previous studies have demonstrated that the long non-coding RNA X-inactive specific transcript (XIST) is upregulated in many cancers, and a high expression level of XIST is associated with poor clinical outcome. In the present study, the expression and function of XIST were investigated in the glioma cell line U251. XIST and microRNA (miR)-133a levels in glioma cell lines were detected by reverse transcription-quantitative polymerase chain reaction. Small hairpin RNA XIST (sh-XIST) and mimics/inhibitor of miR-133a were transfected in glioma cell lines and cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) were examined. Luciferase assays were used to verify the associations among XIST, miR-133a and SRY-box (SOX)4. When XIST was knocked down, the proliferation, metastasis and EMT of glioma cells decreased. Notably, downstream genes of SOX4 were also upregulated or downregulated upon sh-XIST treatment. Overexpression of miR-133a inhibited glioma proliferation, metastasis and EMT via reducing the expression of SOX4; in contrast, knockdown of miR-133a exhibited the opposite effect, which revealed that miR-133a negatively regulates glioma progression. Furthermore, using luciferase assays, it was demonstrated that XIST and SOX4 could bind miR-133a in the predicted binding site; XIST competed with SOX4 for miR-133a binding. In conclusion, a XIST/miR-133a/SOX4 axis and a mechanism of XIST glioma in promoting cell proliferation and metastasis were revealed. These findings revealed that XIST has an oncogenic role in the tumourigenesis of glioma and may serve as a potential therapeutic target for glioma.

Knockdown of the long noncoding RNA XIST suppresses glioma progression by upregulating miR-204-5p.

Background: Gliomas are the most prevalent primary malignant tumors of the central nervous system. Our previous study showed that miR-204-5p is a tumor suppressor gene in glioma. Bioinformatic analyses suggest that long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) is a potential target gene of miR-204-5p.Methods: We analyzed the expression of XIST and miR-204-5p in glioma tissues and the correlation with glioma grade. A series of in vitro experiments were carried out to elucidate the role of XIST in glioma progression. A mouse xenograft model was established to detect whether knockdown of XIST can inhibit glioma growth. A luciferase assay was performed to determine whether XIST can bind to miR-204-5p and the binding specificity. Cells stably expressing shXIST or shNC were transfected with anti-miR-204-5p or anti-miR-204-5p-NC to evaluate whether XIST mediates the tumor-suppressive effects of miR-204-5p.Results: XIST was upregulated in glioma tissues compared with normal brain tissues (NBTs), while miR-204-5p expression was significantly decreased in glioma tissues compared with NBTs. Both XIST and miR-204-5p expression levels were clearly related to glioma grade, and the expression of XIST was obviously negatively correlated with miR-204-5p expression. Knockdown of XIST inhibited glioma cell proliferation, migration, and invasion, promoted apoptosis of glioma cells, inhibited tumor growth and increased the survival time in nude mice. miR-204-5p could directly bind to XIST and negatively regulate XIST expression. XIST mediated glioma progression by targeting miR-204-5p in glioma cells. XIST crosstalk with miR-204-5p regulated Bcl-2 expression to promote apoptosis.Conclusion: Our results provide evidence that XIST, miR-204-5p and Bcl-2 form a regulatory axis that controls glioma progression and can serve as a potential therapeutic target for glioma.

Down-regulation of Xist and Mir-7a-5p improves LPS-induced myocardial injury.

Background: X-inactive specific transcript (Xist) is a lncRNA, which plays a significant role in X-chromosome inactivation, regulates cell proliferation in tumor cells, and inhibits apoptosis in acute myocardial infarction. On the other hand, miR-7a-5p is involved in cardiomyocytes injury in myocardial ischemia/reperfusion. However, their roles in LPS-induced damage remain unclear.Objectives: This study aimed at using siRNA transfection and lentivirus infection to regulate the expression of xist and miR-7a-5p, and to evaluate their effects on LPS-induced myocardial damage.Method: Mice cardiomyocytes (MCM) cells were divided into six groups, namely the control group, the LPS group, the LPS + lncRNA- group, the LPS + lncRNA+ group, the LPS + miRNA- group, and the LPS + miRNA+ group. Quantitative real-time PCR (qRT-PCR) was performed to assay for the RNA expressions of xist, miR-7a-5p, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and recombinant mitochondrial transcription factor A (Tfam) in all the groups. The ATP level was determined using the adenosine triphosphate (ATP) assay kit according to the manufacturer's instructions. Flow cytometry was performed to estimate the level of apoptosis and proliferation in cells in each group.Results: The level of xist in the myocardial cells was markedly higher in the LPS group compared with the control group; however, it was reduced in the LPS+ lncRNA- group. There was no significant difference in the expression of xist among the LPS+miRNA-, LPS+miRNA+, and LPS groups. Moreover, the expression of mir-7a-5p was significantly reduced in myocardial cells in the LPS group, and moderately reduced in the LPS+ miRNA- group, but remarkably elevated in the LPS+ miRNA+ group (P<0.05). The expression of mir-7a-5p was comparably similar in the LPS+ lncRNA- group, LPS+ lncRNA+ group, and LPS groups. Further, the levels of PGC-1a, and Tfam were determined. In the LPS group, the expression of PGC-1α was significantly reduced but elevated in the LPS+lncRNA- and LPS+ miRNA- groups (P<0.05). There was no significant difference in the level of PGC-1α among the LPS, LPS+ lncRNA+, and LPS+ miRNA+ groups. The expression of Tfam was markedly reduced in the LPS group (P < 0.05), but elevated after the suppression of xist and mir-7a-5p. The expression of Tfam was not significantly different among the LPS group, LPS+ lncRNA+ and LPS+ miRNA+ groups. Notably, overexpression of mir-7a-5p had a mild effect on the expression of Tfam in the LPS+ miRNA+ group compared with the control group. Besides, ATP expression in the LPS group was markedly reduced, but elevated after the inhibition of xist and mir-7a-5p. Suppressing the expression of xist or mir-7a-5p resulted in reduced cell apoptosis and increased cell proliferation.Conclusions: In this study, we established that down-regulation of xist and mir-7a-5p reduces apoptosis in response to LPS.

Bioinformatic analysis of a microRNA regulatory network in Huntington's disease.

Huntington's disease is an autosomal dominant hereditary neurodegenerative disease characterized by progressive dystonia, chorea and cognitive or psychiatric disturbances. The leading cause is the Huntington gene mutation on the patient's chromosome 4 that produces a mutated protein. Recently, attention has focused on the relationship between microRNAs and Huntington's disease's pathogenesis. In Huntington's disease, microRNAs can interact with various transcriptionfactors; dysregulated microRNAs may be associated with the Cytosine deoxynucleotide-Adenine ribonucleotides-Guanine ribonucleotide length and Huntington's disease's progression and severity. This study explores the role of microRNAs in the pathogenesis of Huntington's disease through bioinformatics analysis. By analyzing data from the Gene Expression Omnibus database, we identified a total of 9 differentially expressed microRNA. Subsequently, target genes and long non-coding RNAs were predicted, and a comprehensive regulatory network centered on microRNA was constructed. The microRNA integrated regulatory network, Homo sapiens (hsa)-miR-144-3p, interacted with the largest number of long non-coding RNAs, including X-inactive specific transcriptand taurine upregulated gene 1. The miRNAs, hsa-miR-10b-5p and hsa-miR-196a-5p, regulated most of the target genes, including class I homeobox and brain-derived neurotrophic factorgenes. Additionally, 59 Gene Ontology terms and eight enrichment pathways were identified by analyzing the target genes of hsa-miR-196a-5p and hsa-microRNA-10b-5p. In conclusion, hsa-miR-10b-5p and hsa-miR-196a-5p were significantly and differentially expressed in Huntington's disease, the long non-coding RNAs X-inactive specific transcript, taurine upregulated gene 1, and target genes such as homeobox or brain-derived neurotrophic factor may play critical roles in the pathogenesis of Huntington's disease.

LncRNA XIST promotes the progression of laryngeal squamous cell carcinoma by sponging miR‑144 to regulate IRS1 expression.

The initiation and development of several types of cancer have been linked to long non-coding RNA (lncRNA) X inactive-specific transcript (XIST). Yet, the pattern of expression, function, as well as the molecular mechanism underlying XIST in laryngeal squamous cell carcinoma (LSCC) lack characterization. Therefore, the present study aimed to determine the function and putative mechanism of XIST in the development of LSCC. It was revealed that the level of XIST was significantly higher in LSCC tissues that were associated with advanced Tumor-Node-Metastasis (TNM) stage and the presence of lymph node metastasis. Furthermore, the ability of human LSCC TU212 cells to proliferate, form colonies, migrate and invade was significantly suppressed, while cell apoptosis was significantly increased following knockdown of XIST. Further investigation revealed that XIST knockdown increased the expression of microRNA-144 (miR-144) by acting as an endogenous sponge of miR-144. Inhibition of miR-144 caused a partial reversal of the inhibitory effects mediated following depletion of XIST in LSCC cells. Moreover, an miR-144 target called insulin receptor substrate 1 (IRS1) was significantly decreased by XIST depletion in LSCC cells. IRS1 expression was positively correlated with XIST expression in LSCC tissues. In addition, knockdown of XIST impaired tumor growth in vivo by regulating the miR-144/IRS1 axis. The present study demonstrated that the progression of LSCC is promoted by XIST sponging miR-144 to regulate IRS1 expression, suggesting that XIST can serve as a putative target in the therapy of LSCC.

Long Noncoding RNA FTX Reduces Hypertrophy of Neonatal Mouse Cardiac Myocytes and Regulates the PTEN/PI3K/Akt Signaling Pathway by Sponging MicroRNA-22.

BackgroundCardiac myocyte hypertrophy results from clinical conditions that include hypertension and valvular heart disease, and can result in heart failure. This study aimed to investigate the expression and role of the long noncoding RNA FTX (lnc-FTX), an X-inactive-specific transcript (XIST) regulator transcribed from the X chromosome, in hypertrophy of neonatal mouse cardiac myocytes induced by angiotensin II (Ang II) in vitro.Material/MethodCardiac myocytes were isolated from neonatal mice and cultured with and without Ang II. Immunofluorescence, with localization of an antibody to alpha-smooth muscle actin (α-SMA), was used to identify the neonatal mouse cardiac myocytes. Quantitative real-time polymerase chain reaction (qRT-PCR) measured gene expression levels. The cell counting kit-8 (CCK-8) assay was used to determine cell viability, and Western blot measured protein expression. StarBase v2.0 bioinformatics software was used for target gene prediction and was confirmed with the luciferase reporter assay.ResultsThe expression of lnc-FTX was reduced in mouse cardiac myocytes treated with Ang II. Overexpression of lnc-FTX reduced cell apoptosis, cardiomyocyte contractility, and the expression of c-Jun, A-type natriuretic peptide (ANP), and B-type natriuretic peptide (BNP) induced by Ang II. The target of lnc-FTX was micro-RNA 22 (miRNA-22). The mechanism of action of lnc-FTX in neonatal mouse cardiac myocytes was through suppression of the PI3K/Akt signaling pathway by promoting the release of PTEN by sponging miRNA-22.ConclusionsThe expression of lnc-FTX was associated with reduced hypertrophy of neonatal mouse cardiac myocytes and regulated the PTEN/PI3K/Akt signaling pathway by sponging miRNA-22.

Long non-coding XIST raises methylation of TIMP-3 promoter to regulate collagen degradation in osteoarthritic chondrocytes after tibial plateau fracture

BackgroundHypermethylation of gene promoters has been regarded as an epigenetic regulator for gene inactivation in the development of several diseases. In the current study, we aimed to explore how long noncoding RNA X-inactive specific transcript (lncRNA XIST) function in collagen degradation in chondrocytes of osteoarthritis (OA) after tibial plateau fracture by regulating tissue inhibitor of metalloproteinase-3 (TIMP-3) promoter methylation.MethodsIn silico analysis was used to screen differentially expressed lncRNAs in cartilage tissues of OA. Chondrocytes were then successfully isolated from normal and OA cartilage tissues and identified, with the expressions of lncRNA XIST and TIMP-3 examined. The methylation levels of TIMP-3 promoter were determined by MS-PCR. The binding of lncRNA XIST to DNA methyltransferase and the binding of TIMP-3 promoter to DNA methyltransferase were determined by a series of experiments, including RIP, RNA pull-down, and ChIP assays.ResultsThe differentially expressed lncRNA XIST was determined in OA. In addition, cartilage tissues of OA showed upregulation of lncRNA XIST and downregulation of TIMP-3. LncRNA XIST was primarily localized in the nucleus and was capable of binding to the promoter of TIMP-3. The silencing of lncRNA XIST decreased the methylation levels of TIMP-3 promoter and increased the expressions of TIMP-3, which consequently inhibited collagen degradation in OA chondrocytes. Furthermore, TIMP-3 over-expression reversed the effect of lncRNA XIST on collagen degradation in OA chondrocytes.ConclusionCollectively, lncRNA XIST raises collagen degradation in OA chondrocytes after tibial plateau fracture by accelerating the methylation of TIMP-3 promoter by recruiting DNA methyltransferase.

Silencing of long noncoding RNA XIST attenuated Alzheimer's disease-related BACE1 alteration through miR-124.

Alzheimer's disease (AD) is a chronic progressive neurodegenerative disorder. However, its pathogeneticmechanism is still poorly understood. An increasing number of studies have evidenced the important role of long noncoding RNAs (lncRNAs) in AD. The aim of the current study was to investigate the effect and molecular mechanism of the lncRNA X-inactive specific transcript (XIST) in AD. Bilateral common carotid arteryocclusion (2VO) was used to induce an AD model in mice. Hydrogen peroxide (H2 O2 ) was used to induce an AD model in N2a cells. The lncRNA XIST, miR-124, and BACE1 messenger RNA expression levels were detected by a real-time polymerase chain reaction. The BACE1 protein expression level was detected by western blot and immunofluorescence assay. The Aβ1-42 expression level was detected using an enzyme-linked immunosorbent assay kit. The expression level of lncRNA XIST was significantly upregulated in AD models, both in vivo and in vitro. Silencing of lncRNA XIST negatively regulated miR-124 and positively regulated BACE1 expression in N2a cells, which is attenuated by cotransfection of anti-miR-124 oligodeoxyribonucleotide (AMO-124). Silencing of lncRNA XIST reversed the effect of H2 O2 on miR-124, BACE1, and Aβ1-42 expression in N2a cells, which was reversed by cotransfection of AMO-124. Silencing of lncRNA XIST attenuated AD-related BACE1 alteration through miR-124. LncRNA XIST may be a new potential target for the treatment of AD.

Knockdown of long noncoding RNA XIST mitigates the apoptosis and inflammatory injury of microglia cells after spinal cord injury through miR-27a/Smurf1 axis

Spinal cord injury (SCI) is a devastating neuropathological condition. Long noncoding RNA X-inactive specific transcript (XIST) is an acknowledged cancer-related gene and participates in the development of SCI. However, role of XIST in SCI remains to be well revealed. Expression of XIST, miRNA-27a-3p (miR-27a) and smad ubiquitination regulatory factor 1 (Smurf1) was detected using RT-qPCR and western blotting. Cell apoptosis and inflammatory injury were assessed by sulforhodamine B (SRB) assay, flow cytometry, western blotting and enzyme-linked immunosorbent assay. The relationship among miR-27a, XIST and Smurf1 was confirmed by dual-luciferase reporter assay, RNA immunoprecipitation and RNA pull-down assay. As a result, we observed higher level of XIST and Smurf1, but lower level of miR-27a in SCI rats and lipopolysaccharide (LPS)-induced primary microglial cells. in vitro, LPS induced SCI microglia cells as described by decreased cell viability and B cell lymphoma 2 (Bcl-2) expression, and increased cell apoptosis rate, Bax and cleaved caspase 3 levels, and tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) secretions. in vivo, a T10 laminectomy caused SCI rats as evidenced by decreased Basso-Beattie-Bresnahan Locomotor Rating Scale (BBB) score and induced expression of Bax, cleaved caspase 3, TNF-α and IL-6. However, silencing of XIST could mitigate the apoptosis and inflammatory injury in LPS-induced microglia and SCI rats. Mechanically, miR-27a interacted with XIST and Smurf1 via target binding. Either miR-27a downregulation or Smurf1 overexpression partially reversed the role of XIST deletion in LPS-treated microglial cells. Collectively, knockdown of XIST could alleviate the apoptosis and inflammatory injury of SCI models in vitro and in vivo through directly modulating miR-27a/Smurf1 axis.

LncRNA-XIST/microRNA-126 sponge mediates cell proliferation and glucose metabolism through the IRS1/PI3K/Akt pathway in glioma.

Abnormal glucose metabolism may contribute to cancer progression. Glioma represents a cancer resulting from an imbalance between glucose metabolism and tumor growth. However, the molecular mechanisms responsible for dysregulated brain glucose metabolism and lactate accumulation in glioma remain to be elucidated. The present study identified a long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) as a candidate to mediate glucose metabolism in glioma. Cell viability, migration, invasion, and resistance to apoptosis were evaluated in lncRNA-XIST-depleted glioblastoma cells by short hairpin RNA. Glucose uptake, lactate production, as well as levels of glucose transporter 1 (GLUT1) and GLUT3, were measured. Luciferase assay, RNA pull-down, and RNA immunoprecipitation were performed to validate the interactions among lncRNA-XIST, microRNA-126 (miR-126), and insulin receptor substrate 1 (IRS1). An in vivo analysis was carried out in nude mice bearing glioblastoma cell xenografts. The study found that lncRNA-XIST knockdown inhibited cell viability, migration, invasion, resistance to apoptosis, and glucose metabolism of glioblastoma cells. LncRNA-XIST functioned as a competing endogenous RNA of miR-126 and then regulated IRS1/PI3K/Akt pathway in glioblastoma cells. In vivo results demonstrated lncRNA-XIST knockdown reduces the tumorigenicity of glioblastoma cells. Taken together, we demonstrated a novel cellular mechanism that was dependent of the lncRNA-XIST/miR-126/IRS1/PI3K/Akt pathway in enhanced glucose metabolism in glioma.

Human induced pluripotent stem cells from two azoospermic patients with Klinefelter syndrome show similar X chromosome inactivation behavior to female pluripotent stem cells.

STUDY QUESTIONDoes the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype?SUMMARY ANSWEROur results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features.WHAT IS KNOWN ALREADYSo far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture.STUDY DESIGN, SIZE, DURATIONThe study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor.PARTICIPANTS/MATERIALS, SETTING, METHODSIn this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives.MAIN RESULTS AND THE ROLE OF CHANCEApplying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS.LIMITATIONS, REASONS FOR CAUTIONDetailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments.WIDER IMPLICATIONS OF THE FINDINGSAs KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI ‘escapee’ genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2–388/2016–40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program ‘Growsperm’ (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest.

Upregulation of Long Noncoding RNA (lncRNA) X-Inactive Specific Transcript (XIST) is Associated with Cisplatin Resistance in Non-Small Cell Lung Cancer (NSCLC) by Downregulating MicroRNA-144-3p.

BackgroundPatients with advanced non-small cell lung cancer (NSCLC) treated with cisplatin, also termed cis-diamminedichloroplatinum (CDDP) or diamminedichloroplatinum (DDP), may develop chemoresistance. This study aimed to investigate the role of long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) and multidrug resistance-1 (MDR1) in tumor tissue samples and the chemoresistant human NSCLC cell lines, H460/DDP and A549/DDP, and in a murine A549/DDP tumor xenograft.Material/MethodsTissue samples were from patients with NSCLC who responded cisplatin (DDP-sensitive) (n=24), patients with NSCLC unresponsive to cisplatin (DDP-resistant) (n=30), and normal lung tissue (n=25). In H460/DDP and A549/DDP cells, expression of XIST, microRNA (miR)-144-3p, MDR1, and multidrug resistance-associated protein 1 (MRP1) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The MTT assay measured cell survival and proliferation, a transwell assay evaluated cell migration, and flow cytometry measured apoptosis. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays examined the relationship between XIST and miR-144-3p. Tumor xenografts from A549/DDP cells were studied in BALB/c nude mice.ResultsIn tissue from patients with DDP-resistant NSCLC and the mouse A549/DDP tumor xenograft, lncRNA-XIST expression was upregulated and miR-144-3p expression was inhibited. In A549/DDP and H460/DDP cells, down-regulation of lncRNA-XIST and upregulation of miR-144-3p reduced cell survival, proliferation, migration, induced apoptosis and suppressed MDR1 and MRP1 expression.ConclusionsUpregulation of lncRNA-XIST was associated with cisplatin resistance in NSCLC by downregulating miRNA-144-3p in H460/DDP and A549/DDP cells, a murine A549/DDP tumor xenograft, and human tumor tissues from patients with cisplatin-resistant NSCLC.

Silence of lncRNA XIST represses myocardial cell apoptosis in rats with acute myocardial infarction through regulating miR-449.

To study the influences of long non-coding ribonucleic acid (lncRNA) X-inactive specific transcript (XIST) on rats with acute myocardial infarction (AMI), and its regulatory mechanism. A total of 30 Sprague-Dawley rats were randomly assigned into Sham group, Model group, and lncRNA XIST small interfering RNA (XIST siRNA) group. The AMI rat model was prepared through ligating the left anterior descending coronary artery. The left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular systolic diameter (LVDs), and left ventricular diastolic diameter (LVDd) of rats were determined using a color Doppler ultrasound system. Reverse transcription-polymerase chain reaction was performed to measure the expression levels of lncRNA XIST, microRNA (miR)-449, and Notch1 in rat heart tissues in each group. Pathological morphology of rat heart tissues in each group was observed via hematoxylin-eosin (HE) staining. Cell apoptosis in rat heart tissues was evaluated through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Compared with those in Sham group, rats in Model group had significantly increased LVEDV, LVESV, LVDs, and LVDd. After transfection with lncRNA XIST siRNA, XIST level in rat heart tissues was remarkably declined in XIST siRNA group compared with that in Model group. According to HE staining results, the pathological injuries in rat heart tissues were greatly improved in XIST siRNA group compared with those in Model group. TUNEL staining results revealed that the apoptosis rate of cells in rat heart tissues in XIST siRNA group was markedly lower than that in Model group. Higher level of miR-449 and lower level of Notch1 were observed in rats of XIST siRNA group than those of Model group. Knockdown of lncRNA XIST can repress the myocardial cell apoptosis in AMI model rats by downregulating miR-449 level.

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 expression as a prognostic factor in human cancers: A meta-analysis.

A large number of literature has shown that high expression of X inactive-specific transcript (XIST) is associated with poor prognosis and metastasis of cancer in patients. However, most of this literature is limited by the small sample sizes and discrete outcomes. Therefore, a meta-analysis was performed to investigate the relation between XIST expression and tumor node metastasis (TNM) stage, lymph node metastasis, distant metastasis, and overall survival of cancer patients. We searched for literature in PubMed, Embase, and Web of Science. The pooled hazard ratios (HRs) or odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to evaluate the association of XIST expression with prognosis and clinicopathological characteristics of cancer patients. Finally, a total of 14 articles involving 1123 patients were included in this meta-analysis. The results suggested that high expression of XIST has a significant relationship with a relatively poor overall survival for patients with malignant tumors (HR 1.82; 95% CI 1.32, 2.52; P = 0.0003). Moreover, high expression of XIST was significantly associated with poor TNM stage (OR 3.64; 95% CI 2.62, 5.07; P < 0.0001), lymph node metastasis (OR 2.39; 95% CI 1.65, 3.46; P < 0.0001) and distant metastasis (OR 2.84; 95% CI 1.90, 4.23; P < 0.0001). In conclusion, high expression of lncRNA XIST may be a predictive factor of poor prognosis in human cancers.

Silencing of long noncoding RNA XIST protects against renal interstitial fibrosis in diabetic nephropathy via microRNA-93-5p-mediated inhibition of CDKN1A.

Long noncoding RNAs (lncRNAs) have been reported to play an important role in diabetic nephropathy (DN). However, the molecular mechanism involved in this process remains poorly understood. Thus, the present study aimed to explore the function and molecular mechanism of dysregulated lncRNA X-inactive specific transcript (XIST) in DN. DN mouse models were established by streptozotocin treatment, and human renal tubular epithelial HK-2 cells were exposed to high glucose to produce an in vitro model. XIST was highly expressed in renal tissues of patients with DN, mice with DN, and high glucose-exposed HK-2 cells. To identify the interaction among XIST, miR-93-5p, and cyclin-dependent kinase inhibitor 1A (CDKN1A) and to analyze the functional significance of their interaction in renal interstitial fibrosis, we altered endogenous expression of XIST and miR-93-5p and CDKN1A. Dual-luciferase reporter assay results suggested that XIST was highly expressed in the kidney tissue of DN mice and high glucose-exposed HK-2 cells. XIST was identified to be a lncRNA that could bind to miR-93-5p, and CDKN1A was a target of miR-93-5p. Downregulated expression of XIST led to an increase in miR-93-5p expression, thereby decreasing CDKN1A and suppressing renal interstitial fibrosis in DN. Consistently, XIST knockdown reduced the expression of fibrosis markers (fibronectin, collagen type IV, and transforming growth factor-β1). Restoration of CDKN1A or decreasing miR-93-5p yielded a reversed effect on renal interstitial fibrosis. In conclusion, our study demonstrated that silenced XIST inducing miR-93-5p-dependent CDKN1A inhibition was beneficial for preventing renal interstitial fibrosis in DN, which may provide a future strategy to prevent the progression of DN.

LncRNA XIST promotes extracellular matrix synthesis, proliferation and migration by targeting miR-29b-3p/COL1A1 in human skin fibroblasts after thermal injury

BackgroundLong noncoding RNAs (lncRNAs) have been reported to be associated with dermis process during burn wound healing. This study aimed to investigate the role of lncRNA X-inactive specific transcript (XIST) in human skin fibroblasts (HSF) and extracellular matrix (ECM) as well as the regulatory network of XIST/microRNA-29b-3p (miR-29b-3p)/collagen 1 alpha 1 (COL1A1).MethodsThe wound samples were collected from 25 patients with deep partial thickness burn at day 5 after burn. The thermal injured model was established using HSF cells. The expressions of XIST, miR-29b-3p and COL1A1 were measured by quantitative real-time polymerase chain reaction and western blot. ECM synthesis, cell proliferation and migration were detected by western blot, cell counting kit-8 and trans-well assays, respectively. The interaction between miR-29b-3p and XIST or COL1A1 was explored by bioinformatics analysis and luciferase reporter assay.ResultsThe expressions of XIST and COL1A1 were enhanced but miR-29b-3p expression was decreased after thermal injury. XIST overexpression promoted ECM synthesis, cell proliferation and migration in thermal injured HSF cells. However, XIST knockdown played an opposite effect. miR-29b-3p overexpression inhibited ECM synthesis, cell proliferation and migration, which was reversed by XIST. COL1A1 silence suppressed ECM synthesis, cell proliferation and migration by miR-29b-3p targeting. Moreover, COL1A1 up-regulation weakened the effect of XIST silence on ECM synthesis and HSF cell function.ConclusionXIST promoted ECM synthesis, cell proliferation and migration by sponging miR-29b-3p and targeting COL1A1 in HSF cells after thermal injury, indicating the promoting role of XIST in wound healing.

Long Non-coding RNA XIST May Influence Cervical Ossification of the Posterior Longitudinal Ligament Through Regulation of miR-17-5P/AHNAK/BMP2 Signaling Pathway.

Long non-coding RNAs (lncRNAs) play an important role in the development of bone-related diseases. This study was conducted to investigate the role and mechanism of lncRNA X inactive specific transcript (XIST) in the occurrence of cervical ossification of the posterior longitudinal ligament (OPLL). Here, primary human ligament fibroblasts cells (LFCs) were isolated from 30 cases of OPLL and 30 normal cervical posterior longitudinal ligament (non-OPLL) tissues to perform the qPCR and Western blot assay. We found that the mRNA level of lncRNA XIST was significantly increased in OPLL LFCs compared to non-OPLL LFCs. By bioinformatics analysis, we found that lncRNA XIST has four binding sites for miR-17-5p and found that the mRNA level of miR-17-5p was also significantly decreased in OPLL LFCs compared to non-OPLL LFCs. Since AHNAK is the target gene of miR-17-5p, we further found that the expression of AHNAK was significantly reduced in non-OPLL LFCs after being transfected with miR-17-5p mimic. The qPCR results showed that the mRNA expressions of BMP2 and Runx2 were significantly decreased. After being transfected with lncRNA XIST siRNA in the non-OPLL LFCs, the mRNA levels of lncRNA XIST, AHNAK, BMP2, and Runx2 were significantly decreased and the phosphorylated protein of Smad1/5/8 was reduced. After being cultured by mechanical vibration, the mRNA levels of lncRNA XIST, AHNAK, BMP2, Runx2, COL1, OC, OPN, and Phospho1 were significantly increased, but the mRNA expression of miR-17-5p was significantly decreased. The expression of phosphorylated Smad1/5/8 protein was also significantly increased. Together, this study was the first to determine that XIST gene inhibition plays an important role in the occurrence of cervical OPLL, through the mechanism of regulation of miR-17-5P/AHNAK/BMP2 signaling pathway. Thus, XIST may be a potential target that could be modulated for the treatment of cervical OPLL.