Mechanistic study on lncRNA XIST/miR-124-3p/ITGB1 axis in renal fibrosis in obstructive nephropathy.

OBJECTIVE: The purpose of this study was to investigate the role and possible mechanism of lncRNA XIST in renal fibrosis and to provide potential endogenous targets for renal fibrosis in obstructive nephropathy (ON). METHODS: The study included 50 cases of ON with renal fibrosis (samples taken from patients undergoing nephrectomy due to ON) and 50 cases of normal renal tissue (samples taken from patients undergoing total or partial nephrectomy due to accidental injury, congenital malformations, and benign tumors). Treatment of human proximal renal tubular epithelium (HK-2) cells with TGF-ß1 simulated renal fibrosis in vitro. Cell viability and proliferation were measured by CCK-8 and EdU, and cell migration was measured by transwell. XIST, miR-124-3p, ITGB1, and epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, α-SMA, and fibronectin) were detected by PCR and immunoblot. The targeting relationship between miR-124-3p and XIST or ITGB1 was verified by starBase and dual luciferase reporter gene experiments. In addition, The left ureter was ligated in mice as a model of unilateral ureteral obstruction (UUO), and the renal histopathology was observed by HE staining and Masson staining. RESULTS: ON patients with renal fibrosis had elevated XIST and ITGB1 levels and reduced miR-124-3p levels. The administration of TGF-ß1 exhibited a dose-dependent promotion of HK-2 cell viability, proliferation, migration, and EMT. Conversely, depleting XIST or enhancing miR-124-3p hindered HK-2 cell viability, proliferation, migration, and EMT in TGF-ß1-damaged HK-2 cells HK-2 cells. XIST functioned as a miR-124-3p sponge. Additionally, miR-124-3p negatively regulated ITGB1 expression. Elevating ITGB1 weakened the impact of XIST depletion on TGF-ß1-damaged HK-2 cells. Down-regulating XIST improved renal fibrosis in UUO mice. CONCLUSION: XIST promotes renal fibrosis in ON by elevating miR-124-3p and reducing ITGB1 expressions.

Long noncoding RNA XIST inhibition promotes Leydig cell apoptosis by acting as a competing endogenous RNA for microRNA-145a-5p that targets SIRT1 in late-onset hypogonadism.

Leydig cell (LCs) apoptosis is responsible for decreased serum testosterone levels during late-onset hypogonadism (LOH). Our study was designed to illustrate the regulatory effect of lncRNA XIST on LCs and to clarify its molecular mechanism of action in LOH. The Leydig cells (TM3) was treated by 300 µM H2O2 for 8 h to establish Leydig cell oxidative stress model in vitro. The expression levels of lncRNA XIST in the testicular tissues of patients with LOH were measured using fluorescence in situ hybridization (FISH). The interaction between lncRNA XIST/SIRT1 and miR-145a-5p was assessed using starBase and dual-luciferase reporter gene assays. Apoptotic cells and Caspase3 activity were determined by flow cytometry (FCM) assay. Testosterone concentration was determined by ELISA. Moreover, histological assessment of testicles in mice was performed by using HE staining and the TUNEL assay was used to determine apoptosis. We found that the lncRNA XIST was downregulated in the testicular tissues of LOH patients and mice and in H2O2-induced TM3 cells. XIST siRNA significantly promoted apoptosis, enhanced Caspase3 activity and reduced testosterone levels in H2O2-stimulated TM3 cells. Further studies showed that the miR-145a-5p inhibitor reversed the effect of XIST-siRNA on H2O2-induced Leydig cell apoptosis. MiR-145a-5p negatively regulated SIRT1 expression, and SIRT1-siRNA reversed the effects of the miR-145a-5p inhibitor on H2O2 stimulated TM3 cells. The in vivo experiments indicated that silencing of the lncRNA XIST aggravated LOH symptoms in mice. Inhibition of lncRNA XIST induces Leydig cell apoptosis through the miR-145a-5p/SIRT1 axis in the progression of LOH.

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 XIST and forkhead box 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.

LncRNA XIST Protects Against Polycystic Ovary Syndrome via the Regulation of miR-212-3p/RASA1 Axis.

The polycystic ovary syndrome (PCOS), a common endocrine disorder, is mainly related to infertility. Moreover, it is characterized by promoted androgen, suppressed ovulation and insulin resistance. Long non-coding RNA X inactive specific transcript (lncRNA XIST), known as an oncogene or a cancer inhabited factor, is involved in several disease. However, the diagnostic mechanisms of lncRNA XIST in PCOS have not been clarified. Our study aimed to explain whether lncRNA XIST regulates KGN cells proliferation and apoptosis via microRNA (miR)-212-3p/RASA1 axis in PCOS. Levels of lncRNA XIST, miR-212-3p and RASA1 in KGN cells were detected through reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. Fluorescence in situ Hybridization (FISH) was performed to confirm the expression of lncRNA XIST and miR-212-3p in KGN cells. StarBase and dual-luciferase reporter assay were applied for exploring the interaction between miR-212-3p and RASA1. Cell viability, apoptosis, protein expression of Bcl-2 and Bax were assessed by MTT, flow cytometry analysis, RT-qPCR and western blot, respectively. We found that lncRNA XIST was low-expressed, miR-212-3p was over-expressed, and RASA1 was dramatically down-regulated in KGN cells. LncRNA XIST negatively regulated miR-212-3p expression in KGN cells. MiR-212-3p interacted with RASA1 and negatively regulated RASA1 levels in KGN cells. Up-regulation of lncRNA XIST signally decreased cells viability, stimulated more apoptotic cells, enhanced Bax expression, and depressed Bcl-2 level in KGN cells. However, these observations were abolished after miR-212-3p mimic treatment. Furthermore, miR-212-3p inhibitor significantly inhibited cell proliferation, enhanced more apoptotic cells, increased Bax expression, and decreased Bcl-2 level in KGN cells, and these effects were eliminated by RASA1-siRNA transfection. Our observations revealed that lncRNA XIST protects against PCOS through regulating miR-212-3p/RASA1 axis, suggesting that lncRNA XIST may be a promising therapeutic target for PCOS therapy.

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.

lncRNA XIST induces Aß accumulation and neuroinflammation by the epigenetic repression of NEP in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia globally, but effective treatment is lacking. We aimed to explore lncRNA XIST role in AD and the mechanisms involved in the effect of changes in lncRNA XIST on the expression of Aß-degrading enzymes. The mouse model of AD and the cell model induced by Aß were established. LncRNA XIST, IDE, NEP, Plasmin, ACE, EZH2 expressions and distribution of XIST in the nucleus and cytoplasm were detected by qRT-PCR. Inflammatory cytokines IL-6, IL-1ß, TNFα, IL-8, and Aß42 levels were detected by ELISA. TUNEL was used to measure brain tissue damage. Cell proliferation was detected by CCK-8 assay. Flow cytometry detected cell apoptosis. RIP validated the combination of XIST and EZH2. ChIP verified that XIST recruits EZH2 to mediate enrichment of HEK27me3 in the NEP promoter region. The protein expression in brain tissues and cells was detected by Western blot. The expression of lncRNA XIST was increased in AD mice and cell models. Inflammation and injury of nerve cells occurred in AD mice and cell models. The knockdown of lncRNA XIST alleviated Aß-induced neuronal inflammation and damage. LncRNA XIST affected the expression of Aß-degrading enzyme NEP, and lncRNA XIST was negatively correlated with NEP expression in AD mice. LncRNA XIST regulated NEP expression partly through epigenetic regulation by binding with EZH2. LncRNA XIST mediated neuronal inflammation and injury through epigenetic regulation of NEP. Overall, our study found that lncRNA XIST induced Aß accumulation and neuroinflammation by the epigenetic repression of NEP in AD.

LncRNA XIST from the bone marrow mesenchymal stem cell derived exosome promotes osteosarcoma growth and metastasis through miR-655/ACLY signal.

BACKGROUND: Long non-coding RNA X-inactive specific transcript (XIST) regulates the progression of a variety of tumors, including osteosarcoma. Bone marrow mesenchymal stem cells (BMSCs) can be recruited into osteosarcoma tissue and affect the progression by secreting exosomes. However, whether BMSCs derived exosomes transmit XIST to regulate the growth and metastasis of osteosarcoma and the related mechanism are still unclear. METHOD: In this study, BMSCs derived exosomes were used to treat human osteosarcoma cells MG63 and 143B, and the level of XIST in BMSCs was intervened by siRNA. CCK-8, EdU, transwell assays were used to analyze the changes of cell proliferation, migration and invasion. Bioinformatics analysis, RNA pulldown and dual-luciferase reporter gene assays validated the targeted relationship of XIST with miR-655 and the interaction between miR-655 and ACLY 3'-UTR. 143B/LUC cell line was used to establish an animal model of in situ osteosarcoma to verify the found effects of XIST on osteosarcoma. Oil Red O staining, Western blot and so on were used to detect the changes of lipid deposition and protein expression. RESULTS: It was found that BMSCs derived exosomes promoted the proliferation, migration and invasion of osteosarcoma cells, and the down-regulation of XIST inhibited this effect. miR-655 mediated the role of BMSCs derived exosomal XIST in promoting the progression of osteosarcoma and down-regulation of miR-655 could reverse the effects of inhibiting XIST on the proliferation, migration and invasion of osteosarcoma cells. Meanwhile, animal level results confirmed that BMSCs derived exosomal XIST could promote osteosarcoma growth and lung metastasis by combining with miR-655. In-depth mechanism study showed that BMSCs derived exosomal XIST combined with miR-655 to increase the protein level of ACLY, which led to lipid deposition and activate ß-catenin signal to promote the proliferation, migration and invasion of osteosarcoma cells. CONCLUSION: This study showed that BMSCs derived exosomal XIST could enter osteosarcoma cells, bind and down-regulates the level of miR-655, resulting in an increase in the level of ACLY, thus increasing the lipid deposition and the activity of ß-catenin signal to promote the growth and metastasis of osteosarcoma.

Identification and functional analysis of LncRNA-XIST ceRNA network in prostate cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) play a functional role in the progression of prostate cancer (PCa). However, the molecular mechanism, expression, or function of the lncRNA XIST in PCa is not well understood. Therefore, the major goal of this study was to investigate the involvement of XIST in PCa. METHODS: We used the The Cancer Genome Atlas (TCGA) database to conduct a pan-cancer bioinformatics analysis of XIST and identified that it may play an important role in prostate cancer. This finding was verified using clinical samples and in vitro assays. Finally, we constructed an XIST ceRNA network for prostate cancer. RESULTS: Our in vitro and in vivo results showed that the XIST gene expression level was higher in PCa derived cells and tissues compared to that in normal cells and tissues. XIST gene expression level was positively correlated with the invasion and proliferation of tumour cells. Furthermore, the downregulation of XIST inhibited the growth of subcutaneous 22Rv1 xenografts in nude mice. In addition, we constructed a XIST ceRNA network. Consistent with previous studies, we found that the role of XIST is mediated through via sponges, such as miRNA -96-5p, miRNA -153-3p, and miRNA-182-5p. CONCLUSION: High expression level of XIST can lead to enhanced carcinogenicity in PCa. Therefore, XIST has the potential to be used as a prognostic marker and may become a new research focus for the treatment of PCa.

LncRNA XIST facilitates S1P-mediated osteoclast differentiation via interacting with FUS.

INTRODUCTION: The diagnosis and treatment of osteoporosis, a frequent age-related metabolic bone disorder, remain incomprehensive and challenging. The potential regulatory role of lncRNA XIST and sphingosine kinase 1 (SPHK1) pathway need experimental investigations. MATERIALS AND METHODS: RAW264.7 cells and BMMs were obtained for in vitro studies and 30 ng/mL RANKL was implemented for induction of osteoclast differentiation. The suppressing of lncRNA XIST, SPHK1 and fused in sarcoma (FUS) was achieved using small hairpin RNA, while overexpression of XIST and FUS was constructed by pcDNA3.1 vector system. Tartrate-resistant acid phosphatase (TRAP) staining was used for observation of formation of osteoclasts. RNA-pulldown analysis and RNA binding protein immunoprecipitation (RIP) was implemented for measuring mRNA and protein interactions. RT-qPCR was conducted to determining mRNA expression, whereas ELISA and Western blotting assay was performed for monitoring protein expression. RESULTS: RANKL induced osteoclast differentiation and upregulated expression of osteoclastogenesis-related genes that included NFATc1, CTSK, TRAP and SPHK1 and the level of lncRNA XIST in both RAW264.7 cells and BMMs. However, knockdown of lncRNA XIST or suppressing SPHK1 significantly reserved the effects of RANKL. LncRNA XIST was further demonstrated to be interacted with FUS and increased the stability of SPHK1, indicating its ability in promoting osteoclast differentiation through SPHK1/S1P/ERK signaling pathway. CONCLUSION: LncRNA XIST promoted osteoclast differentiation via interacting with FUS and upregulating SPHK1/S1P/ERK pathway.

LncRNA XIST Engages in Psoriasis via Sponging miR-338-5p to Regulate Keratinocyte Proliferation and Inflammation.

INTRODUCTION: Psoriasis is an immune-mediated polygenic inflammatory skin disease in which keratinocyte proliferation is an important mechanism. The study investigated the role and regulatory relationship between lncRNA XIST and miR-338-5p in psoriatic patients and cell models. METHODS: Serum samples were collected from 55 psoriasis patients. HaCaT was recruited for the cell experiments, and induced by M5 cytokines to mimic psoriasis in vitro. XIST and miR-338-5p levels were detected via qRT-PCR. Cell viability under different treatments was evaluated using CCK-8. ELISA was applied to measure the concentration of inflammatory cytokines. The regulatory relationship was confirmed using luciferase reporter gene assay. RESULTS: Serum XIST was elevated in patients with psoriasis and can distinguish the psoriasis patients from healthy controls according to the receiver operating characteristic curve. A high level of XIST was positively correlated with the PASI score and serum tumor necrosis factor-alpha (TNF-α), interleukin-17A [IL-17A], and IL-22 concentrations in psoriasis patients. XIST silencing suppressed M5-induced keratinocyte proliferation and restrained the discharge of inflammatory cytokines (TNF-α, IL-17A, IL-22) and chemokines (CXCL1, CXCL8, CCL20). XIST can sponge miR-338-5p, and miR-338-5p downregulation abolished the inhibitory effect of XIST silencing on cell proliferation and inflammation. miR-338-5p was highly expressed in the clinical serum samples from psoriasis patients. The target relationship between miR-338-5p and IL-6 was proved. CONCLUSION: LncRNA XIST is highly expressed in the serum of patients with psoriasis, and was positively correlated with disease severity and inflammation. XIST may regulate keratinocyte proliferation and inflammation via regulating miR-338-5p/IL-6 axis.

Serum exosomal lncRNA XIST is a potential non-invasive biomarker to diagnose recurrence of triple-negative breast cancer.

Exosomal lncRNAs secreted by cancer cells can serve as potential biomarkers in the diagnosis and prognosis of various tumours. Here, we are committed to explore the diagnostic and prognostic value of serum exosomal XIST secreted by tumour cells to predict recurrence in patients with triple-negative breast cancer (TNBC). Significant increments in XIST and exo-XIST from tumour tissues and blood serum were found in reoccurring TNBC patients by comparison with non-recurrences. Levels of serum exo-XIST were only significantly increased in TNBC recurrence and no association with other clinicopathological parameters. Additionally, serum exo-XIST levels could be served as an assessment of change in the load of triple-negative breast cancer. Expressions of exo-XIST were markedly decreased after resection of the primary breast tumours and obviously elevated at the time of recurrence. Finally, an obvious association was identified between serum exo-XIST levels and a poorer overall survival (OS) in TNBC patients. Levels of serum exo-XIST may serve as a diagnostic and prognostic biomarker to predict the recurrent TNBC-loading status.

Steroid receptor coactivator-1 enhances the stemness of glioblastoma by activating long noncoding RNA XIST/miR-152/KLF4 pathway.

Glioblastoma (GBM) recurrence is attributed to the presence of therapy-resistant glioblastoma stem cells. Steroid receptor coactivator-1 (SRC-1) acts as an oncogenic regulator in many human tumors. The relationship between SRC-1 and GBM has not yet been studied. Herein, we investigate the role of SRC-1 in GBM. In this study, we found that SRC-1 expression is positively correlated with grades of glioma and inversely correlated with glioma patient's prognosis. Steroid receptor coactivator-1 promotes the proliferation, migration, and tumor growth of GBM cells. Notably, SRC-1 knockdown suppresses the stemness of GBM cells. Mechanistically, long noncoding RNA X-inactive specific transcript (XIST) is regulated by SRC-1 at the posttranscriptional level and mediates the function of SRC-1 in promoting stemness-like properties of GBM. Steroid receptor coactivator-1 can promote the expression of Kruppel-like factor 4 (KLF4) through the XIST/microRNA (miR)-152 axis. Additionally, arenobufagin and bufalin, SRC small molecule inhibitors, can reduce the proliferation and stemness of GBM cells. This study reveals SRC-1 promotes the stemness of GBM by activating the long noncoding RNA XIST/miR-152/KLF4 pathway and provides novel markers for diagnosis and therapy of GBM.

LncRNA XIST upregulates TRIM25 via negatively regulating miR-192 in hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: Long non-coding RNA (lncRNA) XIST has been implicated in the progression of a variety of tumor diseases. The purpose of this study was to explore the molecular role of lncRNA XIST in human hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: The expression levels of lncRNA XIST, miR-192 and TRIM25 in HBV-related HCC tissues and HepG2.2.15 cells were detected by qRT-PCR. Biological information and luciferin gene reporter assay were performed to detect the interaction among lncRNA XIST, miR-192 and TRIM25. CCk-8 assay, wound healing assay and colony formation assay were conducted to detect the proliferation and migration ability of HepG2.2.15 cells. RESULTS: qRT-PCR results showed that the expression levels of lncRNA XIST were remarkably increased in HBV-related HCC tissues and HepG2.2.15 cells. In addition, miR-192 was a direct target gene of lncRNA XIST, and the expression of miR-192 and lncRNA XIST were negatively correlated. Moreover, overexpression of miR-192 observably inhibited the proliferation and migration of HCC cells, while overexpression of lncRNA XIST showed an opposite effect. Furthermore, TRIM25 was a direct target of miR-192, and lncRNA XIST could up-regulate the expression of TRIM25 by targeting miR-192. CONCLUSION: LncRNA XIST could up-regulate the expression of TRIM25 by targeting and binding to miR-192, thus accelerating the occurrence and development of HCC.

lncRNA XIST knockdown suppresses hypoxia/reoxygenation (H/R)-induced apoptosis of H9C2 cells by regulating miR-545-3p/G3BP2.

This study was aimed at determining the roles and functions of lncRNA XIST/miR-545-3p/G3BP2 axis during hypoxia/reoxygenation (H/R)-induced H9C2 cell apoptosis. H9C2 cells were distributed into two groups, the H/R injury and control groups. High-throughput lncRNA sequencing was applied in the determination of differentially expressed lncRNAs between H/R-induced H9C2 cells and normal H9C2 cells. Real-time polymerase chain reactions (RT-PCR) were used to confirm the expression levels of lncRNA XIST in H/R-induced H9C2 cells. H9C2 cells were then transfected with lncRNA XIST recombinant plasmid (lncRNA XIST), sh-LINC XIST, agomiR-545-3p, antagomiR-545-3p, pcDNA-G3BP2, sh-G3BP2, and a corresponding negative control (NC). Bioinformatic analyses revealed that MiR-545-3p was a target for lncRNA XIST. This finding was confirmed by dual-luciferase reporter assay. The degree of cell apoptosis was evaluated by a flow cytometer. RT-PCR and western blot were performed to assess the apoptotic-related proteins in each group. A total of 859 differentially expressed lncRNAs (up-regulated = 502, down-regulated = 357) were identified. LncRNA XIST was found to be down-regulated in H/R-induced H9C2 cells while miR-545-3p was distinctly up-regulated. miR-545-3p was established to be a direct target for LncRNA XIST. LncRNA XIST significantly enhanced the apoptotic rate, while its inhibition suppressed the apoptotic rate. AgomiR-545-3p partially blocked the lncRNA XIST and enhanced the apoptosis of H/R-induced H9C2 cells. Moreover, miR-545-3p was shown to be a direct target for G3BP2. The overexpression of G3BP2 partially reversed the apoptotic effects of miR-545-3p on H/R-induced H9C2 cells. lncRNA XIST/miR-545-3p/GBP2 was found to be an apoptotic regulator in H/R-induced H9C2 cells.

Iron accumulation regulates osteoblast apoptosis through lncRNA XIST/miR-758-3p/caspase 3 axis leading to osteoporosis.

Postmenopausal osteoporosis (PMOP) is mainly caused by multiple factors. Recent studies have suggested that iron accumulation (IA) was closely related to PMOP. However, the detailed molecular mechanisms have not been well demonstrated. We constructed the IA mouse model by intraperitoneal injections of ferric ammonium citrate (FAC) and cell model by culturing with the medium containing FAC. Osteoporosis was confirmed in mouse bone tissues using H&E staining, and the level of serum ferritin, alkaline phosphatase (ALP), procollagen-1 N-terminal peptide (P1NP), and osteocalcin in mice was examined by ELISA. The expressions of XIST and miR-758-3p were detected by qRT-PCR. Cell proliferation and apoptosis were measured by CCK-8, TUNEL, and flow cytometry. The expression levels of apoptotic-related proteins were evaluated by western blot. Dual luciferase reporter assay was used to examine the molecular interaction. The expressions of ALP, P1NP, and osteocalcin, and the H&E staining of bone tissues in mice were analyzed to confirm the biological function of XIST and miR-758-3p in vivo. XIST was up-regulated while miR-758-3p was down-regulated in IA mouse and cell models. XIST knockdown significantly reduced FAC-induced osteoblast apoptosis, which was mimicked by transfection with miR-758-3p mimics. XIST acted as a sponge of miR-758-3p, which targeted caspase 3. IA led to the high expression of XIST and promoted osteoblast apoptosis through miR-758-3p/caspase 3. Transfection with shXIST or miR-758-3p mimics alleviated IA-induced mouse osteoporosis. IA regulated osteoblast apoptosis through XIST/miR-758-3p/caspase 3 axis, which might provide alternative targets for the treatment of osteoporosis.

Long non-coding RNA XIST regulates chondrogenic differentiation of synovium-derived mesenchymal stem cells from temporomandibular joint via miR-27b-3p/ADAMTS-5 axis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a common degenerative disease in jaw joint, accompanied by articular cartilage destruction. Differentiation of stem cells to cartilage has important therapeutic implications in TMJ cartilage repair. Previous studies revealed that lncRNA XIST participated in various biological processes. However, the effect of XIST on chondrogenic differentiation of synovium-derived mesenchymal stem cells (SMSCs) remains unclear. Our study aimed to investigate the function of XIST in chondrogenic differentiation of human SMSCs from TMJ. METHODS: Alcian blue staining was performed to determine proteoglycan in SMSCs. qPCR, western blotting and immunofluorescence assays were allowed to assess sex determining region Y-box 9 (SOX9), Collagen type II alpha 1 chain (COL2A1) and Aggrecan (ACAN) expression. The direct interaction between miR-27b-3p and XIST or ADAMTS-5 was confirmed by dual luciferase reporter assay or RNA immunoprecipitation (RIP) assay. RESULTS: XIST was remarkably down-regulated in chondrogenic differentiation of SMSCs. Functional analysis demonstrated that XIST silencing promoted chondrogenic differentiation of SMSCs. Dual luciferase reporter and RIP assays identified that XIST acted as a sponge for miR-27b-3p. Moreover, XIST regulated ADAMTS-5 expression by directly binding miR-27b-3p. More importantly, miR-27b-3p/ADAMTS-5 rescued the effects of XIST on chondrogenic differentiation of SMSCs. CONCLUSION: The results suggest that XIST modulates SMSCs chondrogenic differentiation via the miR-27b-3p/ADAMTS-5 axis, which provides new targets for TMJOA treatment.

GINS2 was regulated by lncRNA XIST/miR-23a-3p to mediate proliferation and apoptosis in A375 cells.

Melanoma ranks second in aggressive tumors, and the occurrence of metastasis in melanoma results in a persistent drop in the survival rate of patients. Therefore, it is very necessary to find a novel therapeutic method for treating melanoma. It has been reported that lncRNA XIST could promote the tumorigenesis of melanoma. However, the mechanism by which lncRNA XIST regulates the progression of melanoma remains unclear. The proliferation of A375 cells was measured by clonal formation. Cell viability was detected by MTT assay. Flow cytometry was performed to detect cell apoptosis and cycle. The level of GINS2, miR-23a-3p, and lncRNA XIST was investigated by qRT-PCR. Protein level was detected by Western blot, and the correctness of prediction results was confirmed by Dual luciferase. In present study, GINS2 and lncRNA XIST were overexpressed in melanoma, while miR-23a-3p was downregulated. Silencing of GINS2 or overexpression of miR-23a-3p reversed cell growth and promoted apoptosis in A375 cells. Mechanically, miR-23a-3p directly targeted GINS2, and XIST regulated GINS2 level though mediated miR-23a-3p. Moreover, XIST exerted its function on cell proliferation, cell viability, and promoted the cell apoptosis of A375 cells though miR-23a-3p/GINS2 axis. LncRNA XIST significantly promoted the tumorigenesis of melanoma via sponging miR-23a-3p and indirectly targeting GINS2, which can be a potential new target for treating melanoma.

Downregulation of lncRNA XIST promotes proliferation and differentiation, limits apoptosis of osteoblasts through regulating miR-203-3p/ZFPM2 axis.

Background: Bone fracture is a common medical condition. Evidence suggested that long noncoding RNAs (lncRNAs) could regulate the bio-function in osteoblast. In this study, we explored the role and mechanism of lncRNA X-inactive specific transcript (XIST) on the proliferation, apoptosis, and differentiation of osteoblasts using MC3T3-E1 cells. Methods: Expression of XIST, microRNA-203-3p (miR-203-3p), and zinc finger protein multitype 2 (ZFPM2) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis of MC3T3-E1 cells were measured using the Cell Counting Kit-8 (CCK-8) and the flow cytometry. Western blot was used to measure the expression of cell cycle-related proteins, apoptosis-related proteins, and ZFPM2. Levels of differentiation-related factors were measured by qRT-PCR, western blot, and alkaline phosphatase (ALP) kit. Target interaction between miR-203-3p and XIST or ZFPM2 was predicted through bioinformatics analysis and verified by dual-luciferase reporter, RNA immunoprecipitation (RIP) assay, or RNA pull-down assay. Results: The expression of XIST and ZFPM2 was increased while miR-203-3p was decreased in plasmas and MC3T3-E1 cells. Knockdown of XIST promoted the proliferation, differentiation, but limited apoptosis in MC3T3-E1 cells. . Mechanically, overexpression of XIST could reverse the bio-function of miR-203-3p transfection. Additionally, miR-203-3p inverted a series of bio-functional effects of ZFPM2. Furthermore, anti-miR-203-3p rescued si-XIST-induced downregulation of ZFPM2. Conclusion: Downregulation of lncRNA XIST promoted osteoblast proliferation and differentiation, but limited apoptosis by miR-203-3p/ZFPM2 axis.

Platycodin D (PD) regulates LncRNA-XIST/miR-335 axis to slow down bladder cancer progression in vitro and in vivo.

Recently, increasing evidences indicated that Platycodin D (PD) served as an effective anti-tumor drug for cancer treatment in clinic. However, the molecular mechanisms are still unclear. In the present study, we proved that PD regulated LncRNA-XIST/miR-335 axis to hamper the development of bladder cancer in vitro and in vivo. Mechanistically, PD inhibited malignant phenotypes, including cell proliferation, invasion, migration and epithelial-mesenchymal transition (EMT), and promoted cell apoptosis in bladder cancer cells in a time- and dose-dependent manner. In addition, the following experiments validated that PD inhibited LncRNA-XIST expressions, while increased miR-335 expression levels in bladder cancer cells. Next, by conducting the dual-luciferase reporter gene system assay and RNA pull-down assay, we validated that LncRNA-XIST inhibited miR-335 expressions through acting as RNA sponges, and the promoting effects of PD stimulation on miR-335 levels were abrogated by upregulating LncRNA-XIST. Interestingly, both silencing LncRNA-XIST and miR-335 overexpression enhanced the inhibiting effects of PD on the malignant phenotypes in bladder cancer cells. Consistently, the xenograft tumor-bearing mice models were established, and the data indicated that PD slowed down tumor growth and inhibited tumorigenesis in vivo, which were also aggravated by downregulating LncRNA-XIST. In general, analysis of data proved that targeting LncRNA-XIST/miR-335 axis was novel to enhance the anti-tumor effects of PD in bladder cancer in vitro and in vivo, and this study provided alternative therapeutic strategies for bladder cancer treatment in clinic.

LncRNA XIST Promotes Migration and Invasion of Papillary Thyroid Cancer Cell by Modulating MiR-101-3p/CLDN1 Axis.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy in the worlds. Long non-coding RNA X-inactive specific transcript (XIST) was found to upregulate in PTC tissues and cell lines. However, the molecular mechanism underlying PTC metastasis and whether XIST plays regulatory role in PTC are still largely unknown. qRT-PCR was performed to detect the expression of lncRNA XIST and mRNAs. Western blotting was carried out to detect CLDN1, MMP2, and MMP9. Transwell assay was used to detect migration and invasion. Starbase bioinformatics prediction and luciferase assay were used to validate the relationship of miR-101-3p and XIST or CLDN1. LncRNA XIST was upregulated in PTC tissues and cells. XIST knockdown suppressed migration and invasion of PTC cells. XIST could directly bind with miR-101-3p. Overexpression of miR-101-3p suppressed migration and invasion of PTC cells. CLDN1 was the target of miR-101-3p, and overexpression of CLDN1 can reverse the inhibition of cell migration and invasion by miR-101-3p, What's more, miR-101-3p inhibition and CLDN1 overexpression can reverse the affection of sh-XIST on migration and invasion of PTC cells inhibition. XIST promotes migration and invasion of papillary thyroid cancer cell via directly regulating miR-101-3p/CLDN1 axis, which is a novel mechanistic of XIST in the regulation of PTC.