Effect of Phellodendri amurense extract on hippocampal nerve cells induced by Aβ / 中国临床药理学与治疗学

AIM: To investigate the effect and molecular mechanism of Phellodendron amurense egtract on hippocampal nerve cells induced be Aβ

The effect and mechanism of lncRNA XIST targeting miR-150 on LPS-induced apoptosis and secretion of inflammatory factors in mouse lung epithelial MLE-12 cells / 西安交通大学学报(医学版)

【Objective】 To study the effect and mechanism of lncRNA XIST targeting miR-150 on LPS-induced apoptosis and secretion of inflammatory factors in MLE-12 cells. 【Methods】 Mouse lung epithelial MLE-12 cells were divided into control, LPS (LPS treatment), sh-NC+LPS (shRNA control transfection, LPS treatment), and sh-XIST+LPS (XIST shRNA transfection, LPS treatment) groups. We used qRT-PCR method to analyze and detect XIST expression level, Annexin V-FITC/PI double staining method to detect cell apoptosis, Western blotting method to analyze and detect the protein expressions of Bax and Bcl-2 in cells, TBA method to detect MDA content, Xanthine oxidation method to detect SOD activity, DCFH-DA method to detect ROS level, and ELISA method to analyze the levels of IL-1β and TNF-α in the culture supernatant. We used the bioinformatics software Starbase to analyze the possible target genes of XIST (miR-150), and then the luciferase reporter system to identify the target relationship between the two. In MLE-12 cells, XIST shRNA and miR-150 inhibitor were co-transfected, and then treated with LPS. We also measured apoptosis, oxidative damage indicators, and inflammatory factor secretion changes. 【Results】 Compared with control group, XIST level in MLE-12 cells of LPS group increased, apoptosis rate and Bax protein expression level increased, Bcl-2 protein expression level decreased, ROS and MDA level increased, SOD level decreased, and the secretion of IL-1β and TNF-α increased. Compared with the sh-NC+LPS group, the sh-XIST+LPSMLE-12 group had decreased XIST level, decreased apoptosis rate and Bax protein expression level, and increased Bcl-2 protein expression level, decreased ROS and MDA levels, increased SOD levels, and decreased IL-1β and TNF-α secreted by cells. Down-regulating XIST targeting promoted miR-150 expression. miR-150 inhibitor could reverse the effects of XIST shRNA on LPS-induced MLE-12 cell apoptosis, oxidative damage indicators, and inflammatory factor secretion. 【Conclusion】 Down-regulation of lncRNA XIST targeting miR-150 inhibits LPS-induced apoptosis and secretion of inflammatory factors in mouse lung epithelial MLE-12 cells.

Xist noncoding RNA could act as a tumor suppressor gene in patients with classical Hodgkin's disease

Background: Xist is a long noncoding RNA involved in the X chromosome inactivation in females. It may act as an onco-suppressor gene in hematologic malignancies, and its activity is strongly dependent from SATB1 gene expression. However, its potential role in Hodgkin's disease (HD) onset and progression is unknown. Materials and Methods: Three gene expression microarray datasets were analyzed for the expression of Xist and SATB1 in patients with classical HD, namely, GDS4222 (130 patients and 54,000 gene features), GSE39134 (29 patients and 54,000 features), and E-MEXP-507 (29 patients and 27,648 probes). The first two were oligonucleotide arrays (platform: Affymetrix gene chip HG-U133-Plus2), whereas the latter was a cDNA two-channel array (platform: OncoChip. v2). Summary and time-dependent receiver operating characteristic (ROC) analysis were applied to obtain a summary measure (summary area under the ROC curve [sAUC]) of association between gene expression and unfavorable patient outcome in each probe set. Results: Xist was overexpressed among females in each data set. A slight overexpression was associated with a good prognosis both in males (sAUC = 0.75, 95% confidence interval [CI]: 0.70–0.80) and at a lesser extent, in females (sAUC = 0.64, 95% CI: 0.59–0.69). However, this finding was limited to the analysis of the biggest database (GDS4222). No association was found between Xist and SATB1 expression. Conclusions: A reactivation of Xist might act as an onco-suppressor gene in male patients with HD, which seems independent from SATB1 expression. The possibility that Xist could contribute to the better survival of female patients should also be investigated

LncRNA XIST knockdown ameliorates oxidative low-density lipoprotein-induced endothelial cells injury by targeting miR-204-5p/TLR4

Oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury is a key contributor toatherosclerosis development. However, the role and mechanism of long noncoding RNA X-inactive specifictranscript (XIST) in atherosclerosis remain largely unknown. The ox-LDL-induced human umbilical veinendothelial cells (HUVECs) injury was analyzed by cell viability, apoptosis, inflammatory cytokines secretionand oxidative stress. The expression levels of XIST, microRNA-204-5p (miR-204-5p) and toll-like receptor 4(TLR4) were detected by quantitative real-time polymerase chain reaction and western blot, respectively. Thetarget interaction between miR-204-5p and XIST or TLR4 was explored by bioinformatics analysis, luciferaseassay and RNA immunoprecipitation. The expression of XIST was enhanced in ox-LDL-treatedHUVECs. Knockdown of XIST attenuated ox-LDL-induced viability inhibition, apoptosis production,inflammatory response and oxidative stress in HUVECs. XIST was validated as a sponge of miR-204-5pand TLR4 acted as a target of miR-204-5p. Knockdown of miR-204-5p reversed silence of XISTmediated suppressive role in ox-LDL-induced injury. TLR4 alleviated miR-204-5p-mediated inhibitiveeffect on ox-LDL-induced injury. Moreover, XIST could regulate TLR4 expression by spongingmiR-204-5p. In conclusion, silence of XIST displayed a protective role in ox-LDL-induced injury inHUVECs by regulating miR-204-5p/TLR4 axis, providing a novel mechanism for understanding thepathogenesis of atherosclerosis.

LncRNA XIST interacts with miR-454 to inhibit cells proliferation, epithelial mesenchymal transition and induces apoptosis in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a one of the subtypes of breast cancer which accounts for approximately 10–20% of all breast cancers. LncRNA XIST (XIST) is reported to be dysfunctional in numerous tumortypes and is involved in the key pathways of cancer initiation, progression and metastasis. Thus, in the presentstudy, we explored the detailed molecular mechanism of XIST in TNBC. XIST was down-regulated in TNBCtissues and cell lines. Overexpressed XIST inhibited cell proliferation, epithelial mesenchymal transition(EMT) and induced apoptosis in vitro as well as suppressed TNBC tumor growth in vivo. MicroRNA (miR)-454 was up-regulated in TNBC tissues and cell lines. Knockdown of miR-454 inhibited TNBC progression bysuppressing cell proliferation, EMT and inducing cell apoptosis. Moreover, miR-454 was predicted andconfirmed to be a target of XIST, and rescue assay indicated that overexpressed miR-454 could reverse XISTrestoration mediated-anti-tumor effects on TNBC cells. In conclusion, XIST interacts with miR-454 to inhibitcells proliferation, EMT and induce apoptosis in TNBC, indicating a promising treatment strategy for TNBCpatients.

Research progress on the role of lncRNA XIST mediated ceRNA regulatory net‐ work in various malignant tumors / 中国肿瘤生物治疗杂志

@#[摘 要] lncRNA XIST是在哺乳动物中发现最早的lncRNAs之一，尤其是在X染色体失活相关疾病中起着重要作用，在胃癌、肝细胞癌、结直肠癌、胰腺癌、膀胱癌、骨肉瘤、鼻咽癌、食管鳞状细胞癌等非生殖系统相关恶性肿瘤中，其作为癌基因通过介导ceRNA调控网络，促进肿瘤细胞的增殖、迁移、侵袭和耐药等生物学行为。本文就 lncRNA XIST的分子生物学属性及介导的ceRNA调控网络在多种恶性肿瘤中作用的研究进展作一综述，以期为后续的肿瘤防治研究提供新的方向。

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

Background: Long 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). Methods: The 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. Results: The 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. Conclusion: XIST 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.

Upregulation of long non-coding RNA XIST has anticancer effects on epithelial ovarian cancer cells through inverse downregulation of hsa-miR-214-3p / 부인종양

OBJECTIVE: The present study is to evaluate the biological functions of long non-coding RNA (lncRNA), X-inactive specific transcript, X-inactive specific transcript (XIST) in human epithelial ovarian cancer (EOC). METHODS: XIST was upregulated in EOC cell lines, CAOV3 and OVCAR3 cells by lentiviral transduction. The effects of XIST overexpression on cancer cell proliferation, invasion, chemosensitivity and in vivo tumor growth were investigated, respectively. Possible sponging interaction between XIST and human microRNA hsa-miR-214-3p was further evaluated. Furthermore, hsa-miR-214-3p was overexpressed in XIST-upregulated CAOV3 and OVCAR3 cells to evaluate its effect on XIST-mediated EOC regulation. RESULTS: Lentivirus-mediated XIST upregulation had significant anticancer effects in CAOV3 and OVCAR3 cells by suppressing cancer cell proliferation, invasion, increasing cisplatin chemosensitivity and inhibiting in vivo tumor growth. Hsa-miR-214-3p was confirmed to directly bind XIST, and inversely downregulated in XIST-upregulated EOC cells. In EOC cells with XIST upregulation, secondary lentiviral transduction successfully upregulated hsa-miR-214-3p expression. Subsequently, hsa-miR-214-3p upregulation functionally reversed the anticancer effects of XIST-upregulation in EOC. CONCLUSION: Upregulation of lncRNA XIST may suppress EOC development, possibly through sponging effect to induce hsa-miR-214-3p downregulation.

XIST Induced by JPX Suppresses Hepatocellular Carcinoma by Sponging miR-155-5p

PURPOSE: The influence of X-inactive specific transcript (XIST) and X-chromosome inactivation associated long non-coding RNAs (lncRNAs) just proximal to XIST (JPX) on hepatocellular carcinoma (HCC) remains controversial in light of previous reports, which the present study aimed to verify. MATERIALS AND METHODS: The DIANA lncRNA-microRNA (miRNA) interaction database was used to explore miRNA interactions with JPX or XIST. JPX, XIST, and miR-155-5p expression levels in paired HCC specimens and adjacent normal tissue were analyzed by RT-qPCR. Interaction between XIST and miR-155-5p was verified by dual luciferase reporter assay. Expression levels of miR-155-5p and its known target genes, SOX6 and PTEN, were verified by RT-qPCR and Western blot in HepG2 cells with or without XIST knock-in. The potential suppressive role of XIST and JPX on HCC was verified by cell functional assays and tumor formation assay using a xenograft model. RESULTS: JPX and XIST expression was significantly decreased in HCC pathologic specimens, compared to adjacent tissue, which correlated with HCC progression and increased miR-155-5p expression. Dual luciferase reporter assay revealed XIST as a direct target of miR-155-5p. XIST knock-in significantly reduced miR-155-5p expression level and increased that of SOX6 and PTEN, while significantly inhibiting HepG2 cell growth in vitro, which was partially reversed by miR-155-5p mimic transfection. JPX knock-in significantly increased XIST expression and inhibited HepG2 cell growth in vitro or tumor formation in vivo in a XIST dependent manner. CONCLUSION: JPX and XIST play a suppressive role in HCC. JPX increases expression levels of XIST in HCC cells, which suppresses HCC development by sponging the cancer promoting miR-155-5p.

Analysis of C43G mutation in the promoter region of the XIST gene in patients with idiopathic primary ovarian insufficiency / 대한생식의학회지

OBJECTIVE: The XIST gene is considered to be an attractive candidate gene for skewed X-chromosome inactivation and a possible cause of primary ovarian insufficiency (POI). The purpose of this study was to investigate whether the XIST gene promoter mutation is associated with idiopathic POI in a sample of the Korean population. METHODS: Subjects consisted of 102 idiopathic POI patients and 113 healthy controls with normal menstrual cycles. Patients with the following known causes of POI were excluded in advance: cytogenetic abnormalities, prior chemo- or radiotherapy, or prior bilateral oophorectomy. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: The mean age of onset of ovarian insufficiency was 28.7+/-8.5 years and the mean values of serum luteinizing and follicle-stimulating hormones and estradiol in the POI group were 31.4+/-18.2 mIU/mL, 74.5+/-41.1 mIU/mL, and 30.5+/-36.7 pg/mL, respectively. We found no cytosine to guanine (C43G) variation in the XIST gene in both POI patients and controls. CONCLUSION: The C43G mutation in the promoter region of the XIST gene was not present in the Korean patients with idiopathic POI in our study, in contrast to our expectation, suggesting that the role of XIST in the pathogenesis of POI is not yet clear.

Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.

The Study of X Chromosome Inactivation Mechanism in Klinefelter's Syndrome by cDNA Microarray Experiment

To investigate the XIST gene expression and its effect in a Klinefelter''s patient, we used Klinefelter''s syndrome (XXY) patient with azoospermia and also used a normal male (XY) and a normal female (XX) as the control, We were performed cytogenetic analysis, Y chromosomal microdeletion assay (Yq), semi-quantitative RT-PCR, and the Northern blot for Klinefelter''s syndrome (KS) patient, a female and a male control, We extracted total RNA from the KS patient, and from the normal cells of the female and male control subjects using the RNA prep kit (Qiagen), cDNA microarray contained 218 human X chromosome-specific genes was fabricated. Each total RNA was reverse transcribed to the first strand cDNA and was labeled with Cy-3 and Cy-5 fluorescein, The microarray was scanned by ScanArray 4000XL system. XIST transcripts were detected from the Klinefelters patient and the female by RT-PCR and Northern blot analysis, but not from the normal male, In the cDNA microarray experiment, we found 24 genes and 14 genes are highly expressed in KS more than the normal male and females, respectively. We concluded that highly expressed genes in KS may be a resulted of the abnormal X inactivation mechanism.

Inactivation of mammalian X-chromosome during spermatogenesis: Temporal expression of genes in the laboratory mouse.

At zygotene/pachytene stage of meiosis in mammalian testis, the X—Y heterobivalent is sequesterd into a heterochromatinized body whose genetic inactivity is shown by lack of uridine incorporation. For the genic level evaluation of the X-inactivation, activities of three X-linked genes were assayed in testicular cell types in the laboratory mouse. While hypoxanthine phosphoribosyl transferase is functional at least up to pachytene stage of primary spermatocytes, glucose-6-phosphate dehydrogenase appears to be active only in the Sertoli cells. No transcript of the muscle specific muscular dystrophin gene was obtained from its regular 5' promoter. Thus, inactivation of X-linked genes in testis occurs at different stages during spermatogenesis, independently of heterochrornatinizati on of the XY-body. We propose that Xist transcript, the putative regulator of X-inactivation in female soma, is also the regulator in testis. However, due to its extremely low level in testis the transcript may regulate by "spreading" in a gradient and affect the genes in a temporal order. Thus, besides other factors, physical proximity of the genes to Xist may determine the stage of their inactivation.