Silencing Of X-inactive Specific Transcript Research Articles

Transcription suppression of GABARAP mediated by lncRNA XIST-EZH2 interaction triggers caspase-11-dependent inflammatory injury in ulcerative colitis

BackgroundWe have previously found that enhancer of zeste homolog 2 (EZH2) is correlated with inflammatory infiltration and mucosal cell injury in ulcerative colitis (UC). This study aims to analyze the role of X-inactive specific transcript (XIST), a possible interactive long non-coding RNA of EZH2, in UC and to explore the mechanisms. MethodsC57BL/6N mice were treated with dextran sulfate sodium (DSS), and mouse colonic mucosal epithelial cells were treated with DSS and lipopolysaccharide (LPS) for UC modeling. The UC-related symptoms in mice, and the viability and apoptosis of mucosal epithelial cells were determined. Inflammatory injury in animal and cellular models were assessed through the levels of ACS, occludin, IL-1β, IL-18, TNF-α, caspase-1, and caspase-11. Molecular interactions between XIST, EZH2, and GABA type A receptor-associated protein (GABARAP) were verified by immunoprecipitation assays, and their functions in inflammatory injury were determined by gain- or loss-of-function assays. ResultsXIST was highly expressed in DSS-treated mice and in DSS + LPS-treated mucosal epithelial cells. It recruited EZH2, which mediated gene silencing of GABARAP through H3K27me3 modification. Silencing of XIST alleviated body weight loss, colon shortening, and disease active index of mice and reduced inflammatory injuries in their colon tissues. Meanwhile, it reduced apoptosis and inflammation in mucosal epithelial cells. However, these alleviating effects were blocked by either EZH2 overexpression or GABARAP knockdown. Rescue experiments identified caspase-11 as a key effector mediating the inflammatory injury following GABARAP loss. ConclusionThis study suggests that the XIST-EZH2 interaction-mediated GABARAP inhibition activates caspase-11-dependent inflammatory injury in UC.

METTL14-mediated lncRNA XIST silencing alleviates GDM progression by facilitating trophoblast cell proliferation and migration via the miR-497-5p/FOXO1 axis.

Gestational diabetes mellitus (GDM),a prevalent complication during the gestation period, has been linked to impaired proliferation and migration of trophoblasts causing placental maldevelopment. We previously found that lncRNA X-inactive specific transcript (XIST)played an essential role in GDM progression. Here, we investigated the precise biological functions as well as the upstream and downstream regulatory mechanisms of XIST in GDM. We found that XISTand forkheadbox O1 (FOXO1) were conspicuously upregulated and miR-497-5p and methyltransferase-like 14 (METTL14) were downregulated in the placentas of GDM patients. XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells. METTL14 inhibited XIST expression through m6A methylation modification. XIST overexpression abrogated the positive effect of METTL14 overexpression on HG-cultured HTR8/SVneo cell progression. MiR-497-5p and FOXO1 are downstream regulatory genes of XIST in HTR8/SVneo cells. Reverse experiments illustrated that XIST mediated HTR8/SVneo cell functions by regulating the miR-497-5p/FOXO1 axis. Additionally, XIST silencing augmented glucose tolerance and alleviated fetal detrimental changes in GDM rats. To conclude, METTL14-mediated XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells via the miR-497-5p/FOXO1 axis, thereby alleviating GDM progression in rats.

Long non-coding RNA XIST promotes the malignant features of oral squamous cell carcinoma (OSCC) cells through regulating miR-133a-5p/VEGFB.

Oral squamous cell carcinoma (OSCC) represents a frequently seen oral cavity malignancy, and the mechanisms of its occurrence and development remain unclear. The present work examined the expression and biological function of long non-coding RNA (lncNRA) XIST (X-inactive specific transcript) in OSCC cells and tissues. A total number of 50 OSCC and paired non-carcinoma tissue samples were collected in this study. Gene expression levels in cancer tissues and cells were quantified by RT-qPCR. In addition, gain- and loss-of-function experiments were conducted to investigate the biological roles of XIST as well as its downstream targets in OSCC cells. XIST was upregulated in OSCC cells and tissues, which predicted a poorer prognostic outcome in OSCC patients. Silencing XIST inhibited the growth and invasion of OSCC cells and triggered apoptosis. miR-133a-5p was identified as a downstream target of XIST, which was downregulated in OSCC tissues. miR-133a-5p mediated the effect of XIST by targeting VEGFB. VEGFB overexpression rescued the inhibitory effects of XIST silencing on cell growth, invasion and migration. Taken together, the above data indicates that XIST serves as an oncogenic factor to enhance the growth and invasion of OSCC cells by targeting the miR-133a/VEGFB axis.

XIST sponges miR-320d to promote chordoma progression by regulating ARF6

BackgroundLong non-coding RNAs (lncRNAs) have been demonstrated to play important roles in various tumors, including chordoma. The purpose of this study was to investigate the role and mechanism of lncRNA X-inactive specific transcript (XIST) in chordoma. MethodsRNA levels and protein levels were measured by real-time quantitative polymerase chain reaction (RT‑qPCR) and western blot assay, respectively. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2′-deoxyuridine (EdU) assay and colony formation assay. Tanswell assay was used to examine cell migration and invasion. Cellular glycolysis was examined via the measurement of extracellular acidification rate (ECAR) and lactate production. The interaction between microRNA-320d (miR-320d) and XIST or ADP-ribosylation factor 6 (ARF6) was predicted by bioinformatics analysis and verified by a dual-luciferase reporter and RNA-pull down assays. The xenograft tumor model was used to explore the biological function of XIST in vivo. ResultsXIST was overexpressed in chordoma tissues. XIST knockdown suppressed chordoma cell proliferation, migration, invasion, and glycolysis. XIST acted as a sponge of miR-320d. Moreover, miR-320d overexpression inhibited the proliferation, migration, invasion, and glycolysis of chordoma cells. ARF6 was a direct target of miR-320d, and XIST upregulated ARF6 expression via sponging miR-320d. Furthermore, overexpression of ARF6 reversed the inhibitory effects of XIST knockdown on chordoma cell proliferation, migration, invasion, and glycolysis. Importantly, XIST silencing blocked xenograft tumor growth in vivo. ConclusionXIST knockdown inhibited chordoma progression via regulating the miR-320d/ARF6 axis, providing a novel insight into chordoma pathogenesis.

LncRNA XIST promotes adjuvant-induced arthritis by increasing the expression of YY1 via miR-34a-5p.

This study aims to explore the mechanism by which long non-coding ribonucleic acids (lncRNA) X-inactive specific transcript (XIST) affects the progression of adjuvant-induced arthritis (AIA). Freund's complete adjuvant was used to induce arthritis in rats. The polyarthritis, spleen and thymus indexes were calculated to evaluate AIA. Hematoxylin-eosin (H&E) staining was used to reveal the pathological changes in the synovium of AIA rats. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-8 in the synovial fluid of AIA rats. The cell continuing kit (CCK)-8, flow cytometry, and Transwell assays were used to assess the proliferation, apoptosis, migration and invasion of transfected fibroblast-like synoviocytes (FLS) isolated from AIA rats (AIA-FLS). Dual-luciferase reporter assay was performed to verify the binding sites between XIST and miR-34b-5p or between YY1 mRNA and miR-34b-5p. The XIST and YY1 were highly expressed, and miR-34a-5p was lowly expressed in the synovium of AIA rats and in AIA-FLS. Silencing of XIST impaired the function of AIA-FLS in vitro and inhibited the progression of AIA in vivo. The XIST promoted the expression of YY1 by competitively binding to miR-34a-5p. Inhibition of miR-34a-5p strengthened the function of AIA-FLS by upregulating XIST and YY1. The XIST controls the function of AIA-FLS and may promote the progression of rheumatoid arthritis via the miR-34a-5p/YY1 axis.

Long noncoding RNA X-inactive specific transcript regulates NLR family pyrin domain containing 3/caspase-1-mediated pyroptosis in diabetic nephropathy.

BACKGROUNDNLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology. Long noncoding RNAs (lncRNAs) are active participators of diabetic nephropathy (DN). X inactive specific transcript (XIST) expression has been reported to be elevated in the serum of DN patients.AIMTo evaluate the mechanism of lncRNA XIST in renal tubular epithelial cell (RTEC) pyroptosis in DN.METHODSA DN rat model was established through streptozotocin injection, and XIST was knocked down by tail vein injection of the lentivirus LV sh-XIST. Renal metabolic and biochemical indices were detected, and pathological changes in the renal tissue were assessed. The expression of indicators related to inflammation and pyroptosis was also detected. High glucose (HG) was used to treat HK2 cells, and cell viability and lactate dehydrogenase (LDH) activity were detected after silencing XIST. The subcellular localization and downstream mechanism of XIST were investigated. Finally, a rescue experiment was carried out to verify that XIST regulates NLR family pyrin domain containing 3 (NLRP3)/caspase-1-mediated RTEC pyroptosis through the microRNA-15-5p (miR-15b-5p)/Toll-like receptor 4 (TLR4) axis.RESULTSXIST was highly expressed in the DN models. XIST silencing improved renal metabolism and biochemical indices and mitigated renal injury. The expression of inflammation and pyroptosis indicators was significantly increased in DN rats and HG-treated HK2 cells; cell viability was decreased and LDH activity was increased after HG treatment. Silencing XIST inhibited RTEC pyroptosis by inhibiting NLRP3/caspase-1. Mechanistically, XIST sponged miR-15b-5p to regulate TLR4. Silencing XIST inhibited TLR4 by promoting miR-15b-5p. miR-15b-5p inhibition or TLR4 overexpression averted the inhibitory effect of silencing XIST on HG-induced RTEC pyroptosis.CONCLUSIONSilencing XIST inhibits TLR4 by upregulating miR-15b-5p and ultimately inhibits renal injury in DN by inhibiting NLRP3/caspase-1-mediated RTEC pyroptosis.

LncRNA XIST promotes carboplatin resistance of ovarian cancer through activating autophagy via targeting miR-506-3p/FOXP1 axis.

Resistance to chemotherapy drugs makes ovarian cancer (OC) difficult to treat and ultimately kills patients. Long non-coding RNAs are closely related to carboplatin resistance in OC. In present study, we explored the role of lncRNA X-inactive specific transcript (XIST) on carboplatin resistance in OC. Cell viability, proliferation, and apoptosis were assessed through 2,5-diphenyl-2H-tetrazolium bromide, colony formation, and flow cytometry assays, respectively. Microtubule-associated protein 1A/1B-light chain 3 expression was evaluated by immunofluorescence assay to analyze the cell autophagy. The interaction of XIST/miR-506-3p or miR-506-3p/forkhead box protein P1 (FOXP1) was analyzed using RNA immunoprecipitation (RIP) and dual-luciferases reporter assays. The function of XIST/miR-506-3p/FOXP1 axis in vivo was further confirmed by tumor xenograft study and immunohistochemistry. The expression of XIST and FOXP1 increased while miR-506-3p decreased in OC and carboplatin resistance cells. XIST silencing repressed the proliferative and autophagic capacities of carboplatin resistance cells while promoted the apoptosis. XIST overexpression led to the opposite results. XIST targeted miR-506-3p and downregulated its expression. MiR-506-3p inhibition facilitated the proliferative and autophagic capacities while suppressed the apoptosis of cells, XIST knockdown reversed these effects. MiR-506-3p bound to FOXP1. XIST knockdown or miR-506-3p overexpression reversed the increase of cell proliferative and autophagic abilities and the decrease of apoptosis rate induced by FOXP1 overexpression. XIST affected autophagy and carboplatin resistance in vivo via regulating the miR-506-3p/FOXP1 axis. XIST knockdown inhibited autophagy and carboplatin resistance of OC through FOXP1/protein kinase B (AKT)/mammalian target of rapamycin pathway by targeting miR-506-3p.

Cucurbitacin B regulates lung cancer cell proliferation and apoptosis via inhibiting the IL-6/STAT3 pathway through the lncRNA XIST/miR-let-7c axis

Context Lung cancer, the most common type of cancer, has a high mortality rate. Cucurbitacin B (CuB), a natural compound extracted from Cucurbitaceae plants, has antitumor effects. Objective We investigated the role of CuB on lung cancer and its potential mechanisms. Materials and methods A549 cells were treated with 0.1, 0.3, 0.6, and 0.9 μM CuB for 12, 24, and 48 h or untreated. Gene and protein levels were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Enzyme-linked immunosorbent assay (ELISA) detected inflammatory factors levels (TNF-α and IL-10). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and colony formation assays measured cell viability, apoptosis, and proliferation. The interaction between miR-let-7c and long non-coding RNA X inactive-specific transcript (XIST) or interleukin-6 (IL-6) was verified by dual-luciferase reporter assays. Results CuB treatment inhibited the proliferation of lung cancer cells and promoted cell apoptosis, and increased the expression of Bax and cleaved caspase3, decreased cyclin B1 and Bcl-2 expression. CuB suppressed XIST and IL-6 expression, and enhanced miR-let-7c expression. XIST silencing enhanced the inhibitory effect of CuB on cell proliferation and the promotion effect on apoptosis via upregulating miR-let-7c. Moreover, XIST targeted miR-let-7c to activate the IL-6/STAT axis. MiR-let-7c overexpression enhanced the regulatory effect of CuB on proliferation and apoptosis via suppressing the IL-6/STAT3 pathway. Discussion and conclusion CuB regulated cell proliferation and apoptosis by inhibiting the XIST/miR-let-7c/IL-6/STAT3 axis in lung cancer. These findings indicate CuB may have the possibility of clinical application in lung cancer treatment.

Long noncoding RNA XIST modulates microRNA-135/CREB1 axis to influence osteogenic differentiation of osteoblast-like cells in mice with tibial fracture healing.

Fracture healing is a complex event with the involvement of many cell systems, cytokines, as well as mRNAs. Herein, we report the interactions among long noncoding RNA X-inactive specific transcript (XIST)/microRNA-135 (miR-135)/cAMP response element-binding protein 1 (CREB1) axis during fracture healing. We observed increased expression of XIST in patients with long-term unhealed fracture by microarray analysis. Subsequently, a mouse model with tibial fracture and a cell model using osteoblast-like MC3T3-E1 cells were generated. The XIST overexpression during fracture healing decreased proliferation and differentiation of MC3T3-E1 cells, while silencing of XIST facilitated MC3T3-E1 cell growth. Furthermore, miR-135 targeted CREB1 and negatively regulated its expression. XIST acted as a sponge for miR-135, thereby upregulating CREB1 and promoting the activity of the TNF-α/RANKL pathway. Transfection of miR-135 inhibitor or CREB1 overexpression blocked the stimulating effects of XIST knockdown on MC3T3-E1 cell growth. Besides, specific inhibitors of the TNF-α/RANKL pathway reversed the repressive role of XIST in cell osteogenic differentiation. All in all, these findings suggest that XIST knockdown induces the differentiation of osteoblast-like cells via regulation of the miR-135/CREB1/TNF-α/RANKL axis. XIST, as a consequence, represents an attractive therapeutic strategy to accelerate fracture healing.

The lncRNA XIST/miR-150-5p/c-Fos axis regulates sepsis-induced myocardial injury via TXNIP-modulated pyroptosis

Myocardial injury is a severe complication of sepsis and contributes substantially to the death of critically ill patients. Long non-coding RNAs (lncRNAs) participate in the pathogenesis of sepsis-induced myocardial injury. In this study, we investigated the role of lncRNA X-inactive specific transcript (XIST) in septic myocardial injury and explored its mechanism. Lipopolysaccharide (LPS)-stimulated H9C2 cells and rats subjected to cecal ligation and puncture (CLP) were used as the in vitro and in vivo models. After exposure to LPS, XIST and c-Fos levels were upregulated, but miR-150-5p was downregulated in H9C2 cardiomyocytes and myocardial tissues. XIST affected viability, apoptosis, and pyroptosis in LPS-challenged H9C2 cells. Moreover, XIST knockdown attenuated LPS-induced injury in H9C2 cells by targeting the miR-150-5p/c-Fos axis. c-Fos could bound to the promoter of the TXNIP/XIST gene and enhanced TXNIP/XIST expression. Silencing of XIST improved cardiac function and survival rate and reduced apoptosis and pyroptosis by regulating the miR-150-5p/c-Fos axis in septic rats in vivo. Taken together, our data show that XIST/miR-150-5p/c-Fos axis affected septic myocardial injury, which may indicate a novel therapeutic strategy for sepsis-induced myocardial injury.The authors show that the long non-coding RNA XIST, together with the microRNA miR-150-5p and c-Fos, regulate sepsis-induced myocardial injury via the TXNIP pathway. XIST affects pyroptosis, and XIST knockdown attenuates lipopolysaccharide-induced injury. These results suggest that the XIST/miR-150-5p/c-Fos axis may be a novel therapeutic strategy for sepsis-induced myocardial injury.

Long noncoding RNA XIST knockdown relieves the injury of microglia cells after spinal cord injury by sponging miR-219-5p.

Long noncoding RNAs have been demonstrated to play crucial roles in the pathogenesis of spinal cord injury (SCI). In this study, we aimed to explore the roles and underlying mechanisms of lncRNA X-inactive specific transcript (XIST) in SCI progression. SCI mice model was constructed and evaluated by the Basso–Beattie–Bresnahan method. The SCI cell model was constructed by treating BV2 cells with lipopolysaccharide (LPS). The levels of XIST and miR-219-5p were determined by the reverse transcription quantitative polymerase chain reaction. The concentrations of inflammatory cytokines were measured by enzyme-linked immunosorbent assay. Protein levels were measured via western blot assay. Cell viability and apoptosis were evaluated by cell counting kit-8 assay and flow cytometry analysis, respectively. The relationship between XIST and miR-219-5p was analyzed by online tool starBase, dual-luciferase reporter assay, and RNA immunoprecipitation assay. As a result, the XIST level was enhanced and the miR-219-5p level was declined in the SCI mice model. XIST was also upregulated in LPS-induced BV2 cells. LPS treatment restrained BV2 cell viability and accelerated apoptosis and inflammatory response. XIST knockdown effectively weakened LPS-induced BV2 cell injury. miR-219-5p was identified as a target of XIST. Moreover, inhibition of miR-219-5p restored the impacts of XIST knockdown on cell viability, apoptosis, and inflammation in LPS-treated BV2 cells. In addition, LPS-induced XIST promoted the activation of the nuclear factor-κB (NF-κB) pathway by sponging miR-219-5p. In conclusion, XIST silencing promoted microglial cell viability and repressed apoptosis and inflammation by sponging miR-219-5p, thus promoting the recovery of SCI.

The crosstalk network of XIST/miR-424-5p/OGT mediates RAF1 glycosylation and participates in the progression of liver cancer.

Liver cancer is a major public health concern, but the mechanistic actions of biomarkers contributing to liver cancer remain to be determined. In this study, we aimed to investigate the regulatory cascade of microRNA-424-5p (miR-424-5p), X-inactive-specific transcript (XIST) and O-GlcNAc transferase (OGT) in liver cancer. Differentially expressed miRNAs and target genes related to liver cancer were predicted by bioinformatics analyses, and their expression was determined in liver tissues of patients with liver cancer and liver cancer cells. The RNA immunoprecipitation (RIP), RNA pull-down and dual luciferase reporter assay were used to examine the binding affinity among XIST and miR-424-5p and OGT. Then, gain- and loss-of-function assays were conducted to evaluate the effects of the XIST/miR-424-5p/OGT axis on malignant phenotypes. A nude mouse model of liver cancer was further established for in vivo substantiation. XIST and OGT were up-regulated in liver cancer tissues and cells, responsible for poor prognosis in patients with liver cancer, while miR-424-5p was down-regulated. XIST competitively bound to miR-424-5p to increase OGT expression. XIST silencing inhibited malignant phenotypes of liver cancer cells, while miR-424-5p down-regulation negated its effect. miR-424-5p suppressed RAF1 glycosylation by negatively regulating OGT expression and promoted its ubiquitination/degradation. Furthermore, XIST knockdown inhibited tumour growth and metastasis in nude mice, while ectopic OGT reversed its effect. These results reveal a novel mechanism by which the interaction of XIST/miR-424-5p/OGT participates in the malignancy and metastasis of liver cancer.

Long non‑coding RNA XIST inhibits osteoblast differentiation and promotes osteoporosis via Nrf2 hyperactivation by targeting CUL3.

Osteoporosis (OP) is a common skeletal disorder characterized by a low bone mass and the deterioration of bone structure. Long non-coding (lnc)RNA X inactive-specific transcript (XIST) is highly expressed in the serum and monocytes of patients with OP. Thus, the purpose of the present study was to explore the mechanisms underlying the role of XIST in the progression of OP. To establish animal models of OP, female rats underwent a bilateral ovariectomy. The bone mineral density of individual rats was measured using dual-energy X-ray absorptiometry. The combination of XIST and cullin-3 (CUL3) was analyzed using a dual-luciferase reporter assay. Bone histopathological changes were assessed by hematoxylin and eosin staining. Alkaline phosphatase activity was examined by ALP staining. Finally, a series of functional experiments were performed to examine the effects of XIST on cellular behaviors. In the present study, XIST promoted OP and inhibited bone formation by regulating the expression levels of CUL3 and nuclear factor erythroid 2-related factor 2 (Nrf2) in the rats with OP. Moreover, XIST directly targeted CUL3 and negatively regulated its expression. Of note, CUL3 downregulation reversed the effects of XIST silencing on cell viability, differentiation and mineralization, as well as the expression of Nrf2 and CUL3 in MC3T3-E1 cells. Collectively, XIST was demonstrated to inhibit the differentiation of osteoblasts and promote OP by inhibiting the degradation of Nrf2 via targeting CUL3.

Long non-coding RNA XIST promotes the progression of esophageal squamous cell carcinoma through sponging miR-129-5p and upregulating CCND1 expression

ABSTRACT Long non-coding RNA (lncRNA) X inactive specific transcript (XIST) has been identified as an oncogenic lncRNA in a series of human cancers, including esophageal squamous cell carcinoma (ESCC). In this study, we aimed to further explore the underlying mechanism of XIST on ESCC progression. qRT-PCR assay was used to determine the levels of XIST and miR-129-5p. Western blot analysis was performed to assess cyclin D1 (CCND1) expression. Bioinformatic analysis was performed using starBase v2.0 software. Dual-luciferase reporter and RNA immunoprecipitation assays were employed to confirm the interaction between XIST and miR-129-5p or miR-129-5p and CCND1. Cell cycle progression and apoptosis were measured by flow cytometric analysis, and cell migration and invasion were detected by transwell assay. Mouse studies were used to observe the effect of XIST silencing on tumor growth in vivo. Our results indicated that XIST was upregulated and miR-129-5p was downregulated in ESCC. XIST silencing or miR-129-5p overexpression repressed cell cycle progression, proliferation, migration, invasion, and promoted the apoptosis in ESCC cells. Moreover, XIST directly interacted with miR-129-5p and repressed miR-129-5p expression. MiR-129-5p mediated the regulatory effect of XIST on ESCC cell progression in vitro, and XIST promoted CCND1 expression by sponging miR-129-5p. Additionally, XIST silencing inhibited tumor growth in vivo. Our findings suggested that XIST silencing repressed the progression of ESCC at least partly through regulating the miR-129-5p/CCND1 axis. Targeting XIST might be a potential therapeutic strategy for ESCC treatment.

Silencing of long non-coding RNA XIST represses gastric cancer progression through blocking NFκB pathway via inhibiting HNF4A-mediated transcription of EPHA1.

Gastric cancer (GC) is a common cancer and a leading cause of cancer-related deaths worldwide. Recent studies have supported the important role of long non-coding RNAs (lncRNAs) in GC progression. This study identified functional significance of X inactive specific transcript (XIST) in GC. The expression of XIST and EPHA1 in GC tissues and cells was measured. Then, dual luciferase reporter gene assay, RNA immunoprecipitation (RIP) assay and Chromatin Immunoprecipitation (ChIP) assay were performed to explore the interaction among XIST, EPHA1 and HNF4A. The effects of XIST on GG progression were evaluated by determining expression of proliferation- and invasion-related proteins (Ki67, PCNA, MMP-2, and MMP-9). Further, the functional role of XIST in GC with the involvement of NFκB pathway was also analyzed. Subsequently, the tumor growth in nude mice was evaluated. High expression of XIST and EPHA1 was observed in GC. XIST elevated EPHA1 expression by recruiting HNF4A. In addition, silencing of XIST inhibited GC progression in vitro and in vivo. Overexpressed XIST and EPHA1 yielded a reversed effect on cell proliferation and invasion. SN50 treatment (inhibitor of NFκB pathway) counteracted the promotive effect on GC cell proliferation and invasion mediated by XIST. The present study unveils that XIST increases the enrichment of HNF4A in the promoter region of EPHA1, thus promoting the deterioration of GC.

LncRNA XIST Promotes Growth of Human Chordoma Cells by Regulating miR-124-3p/iASPP Pathway.

IntroductionChordoma is a malignant primary bone tumor that is found in the spine and skull. X-inactive specific transcript (XIST) is a long non-coding RNA (lncRNA) is known to be involved in the development of various cancers, but its precise function and mechanism in human chordoma have not been elucidated. Here, we investigated the role of lncRNA XIST in chordoma progression.MethodsQuantitative real time-polymerase chain reaction (qRT-PCR) was performed to determine lncRNA XIST expression in human chordoma tissues and matched-noncancerous tissues. Western blot was used to determine protein expression. Silencing and overexpression of lncRNA XIST were carried out by RNA interference (RNAi) and lentiviral transduction, respectively. Cell Counting Kit-8 (CCK-8) assay and flow cytometry were employed to examine the effects of lncRNA XIST on growth of human chordoma cells. Lastly, the role of lncRNA XIST in vivo was explored using a xenograft model.ResultsWe found that lncRNA XIST expression was upregulated in chordoma and strongly correlated with poor patient prognosis. Moreover, lncRNA XIST promoted proliferation and inhibited apoptosis of chordoma cells. Mechanistically, upregulation of lncRNA XIST led to a decrease in miR-124-3p expression, thereby promoting the expression of the miR-124-3p target gene, inhibitor of apoptosis-stimulating protein of p53 (iASPP). Addition of miR-124-3p inhibitor or mimic reversed the effects induced by lncRNA XIST silencing or overexpression on chordoma cell proliferation. Lastly, using a xenograft mouse model, we found that silencing of lncRNA XIST decreased tumorigenicity in vivo, as shown by increased tumor cell apoptosis.ConclusionOur findings demonstrate a key role for lncRNA XIST in chordoma progression by regulating miR124-3p/iAPSS pathway.

Silencing of lncRNA XIST inhibits non-small cell lung cancer growth and promotes chemosensitivity to cisplatin.

Long noncoding RNAs (lncRNAs) play critical roles in tumour progression and metastasis. Emerging evidence indicates that the lncRNA X inactive-specific transcript (XIST) is dysregulated in several tumor types, including non-small cell lung cancer (NSCLC). However, in NSCLC and other cancers the oncogenic mechanism of XIST remains incompletely understood. Here, we confirmed that XIST is upregulated in human NSCLC specimens, and is especially overexpressed in tumors previously treated with cisplatin (cis-diamminedichloroplatinum(II); DDP). In vitro, XIST knockdown inhibited NSCLC cell growth and promoted DDP chemosensitivity by stimulating apoptosis and pyroptosis. Moreover, XIST’s oncogenic effects and ability to promote DDP chemoresistance were largely related to its binding to the TGF-β effector SMAD2, which inhibited its translocation to the nucleus and prevented the transcription of p53 and NLRP3, crucial regulators of apoptosis and pyroptosis, respectively. Using DDP-resistant NSCLC cells, mouse xenograft studies verified the oncogenic function of XIST and its ability to inhibit programmed cell death, thereby mediating DDP chemoresistance. These findings suggest that XIST expression may serve as a novel biomarker to predict DDP treatment efficacy, and may help in the design of new therapies to circumvent DDP chemoresistance in NSCLC and other tumor types.

Long Noncoding RNA XIST Contributes to Cervical Cancer Development Through Targeting miR-889-3p/SIX1 Axis.

Background: Cervical cancer (CC) is one of the most common cancers among women in the world. Long noncoding RNAs and microRNAs were identified as important regulators in many physiological processes. The objective of this study was to illuminate the mechanism of X-inactive-specific transcript (XIST)/miR-889-3p/Sine oculis homeobox 1 (SIX1) axis in CC. Methods: The expression levels of XIST, miR-889-3p, and SIX1 were detected by quantitative real-time polymerase chain reaction. Cell proliferation was assessed by cell counting Kit 8 assay. Cell migration and invasion were evaluated by transwell assay. Cell apoptosis was detected by flow cytometry assay. Murine model was established using transfected Me180 cell. The interaction among XIST, miR-889-3p, and SIX1 was tested by dual-luciferase reporter and RNA immunoprecipitation assays. Protein level of SIX1 was measured by Western blot. Results: XIST was highly expressed in CC tissues and cells. Silenced XIST inhibited proliferation, migration, and invasion and induced apoptosis. Moreover, XIST silencing blocked tumor growth in vivo. XIST directly bound to miR-889-3p, and XIST promoted proliferation, migration, and invasion and hindered apoptosis by suppressing miR-889-3p expression. MiR-889-3p targeted SIX1 and negatively regulated SIX1 expression. Furthermore, miR-889-3p had a low expression and SIX1 had a high expression in CC tissues and cells. XIST knockdown reduced SIX1 level by targeting miR-889-3p. In addition, miR-889-3p inhibition abolished the effects of SIX silencing on proliferation, migration, invasion, and apoptosis. Conclusion: XIST knockdown restrained cell proliferation, migration, and invasion and promoted apoptosis by regulating miR-889-3p/SIX1 axis.

Overexpression of XIST facilitates cell proliferation, invasion and suppresses cell apoptosis by reducing radio-sensitivity of glioma cells via miR-329-3p/CREB1 axis.

Glioma is a malignant brain cancer capable of spreading to the microenvironment. Long non-coding RNA (lncRNA) X inactive specific transcript (XIST) was recognized as a significant regulator in many cancers. However, the molecular mechanism of XIST in glioma cell radio-sensitivity requires further exploration. The expression of XIST, microRNA (miR)-329-3p and cyclic AMP response element-binding protein 1 (CREB1) was evaluated by quantitative Real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry, respectively. Transwell assay was performed to detect cell invasion. Protein expression of gamma-H2AX (γ-H2AX) and CREB1 was determined by Western blot. The correlation between miR-329-3p and XIST or CREB1 was determined by dual-luciferase reporter assay. Animal models were established by subcutaneously injecting U251 cells transfected with sh-XIST and sh-NC. XIST and CREB1 were overexpressed whereas miR-329-3p was low-expressed in glioma tumors and cells compared with the normal counterparts. XIST knockdown inhibited cell proliferation, invasion and induced cell apoptosis by enhancing cell sensitivity to X-ray radiation in glioma. Then, we discovered that miR-329-3p directly interacted with XIST or CREB1 in glioma. In addition, miR-329-3p inhibitor abolished XIST silencing-induced regulatory effects on cell proliferation, apoptosis, invasion, and radio-sensitivity. Meanwhile, miR-329-3p inhibitor counteracted CREB1 silencing-induced inhibition on cell progression and facilitation on radio-sensitivity in glioma. Moreover, we found that XIST could increase CREB1 expression by sponging miR-329-3p. Animal experiments revealed that XIST silencing restrained tumor growth in vivo. XIST accelerates cell proliferation, invasion and inhibits cell apoptosis by repressing radio-sensitivity of glioma via enhancing CREB1 expression through sponging miR-329-3p, representing prospective methods for glioma treatment.

Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism.

BackgroundPrimary graft dysfunction (PGD) is a known acute lung injury (ALI) and a major cause of fatality post-lung transplantation. Though some long non-coding RNAs (lncRNAs) have been studied in ALI through regulation of microRNAs (miRNAs), their effects on PGD remain undefined. The present study aims to explore the underlying mechanism of lncRNA X-inactive specific transcript (XIST) in PGD after lung transplantation.MethodsInitially, the expression of miR-21, IL-12A and XIST was determined by RT-qPCR and western blot analysis. The dual luciferase reporter assay, RNA pull-down and RIP assay were performed to identify the targeting relationship between miR-21 and IL-12A and the binding relationship between miR-21 and XIST. Loss- and gain-of-function investigations were conducted in rats treated with prolonged cold ischemia and polymorphonuclear neutrophils (PMNs).FindingsmiR-21 was decreased, whilst XIST and IL-12A were increased in the bronchoalveolar lavage fluid of PGD patients after lung transplantation. Enhanced miR-21 expression in rats and PMNs resulted in downregulated expression of pro-inflammatory factors and chemokines, and enhanced the apoptosis of PMNs. XIST was found to upregulate IL-12A expression in a miR-21-dependent manner. Additionally, XIST silencing enhanced the apoptosis of PMNs and inhibited the neutrophil extracellular trap (NET) formation through upregulation of miR-21 but downregulation of IL-12A in vivo.InterpretationIn summary, lncRNA XIST upregulates IL-12A by binding to miR-21, thereby inducing NET formation and accelerating PGD after lung transplantation. This suggests that inhibition of XIST and NET may be beneficial for the treatment of PGD.