Comprehensive analysis of transcriptome-wide m6A methylome in intermediate-stage Esophageal squamous cell carcinoma.

N6-Methyladenosine (m6A) RNA methylation is the most prevalent post-transcriptional modification mechanism in humans and plays an important role in the pathogenesis of various tumors. However, the investigation of m6A modification in Esophageal squamous cell carcinoma (ESCC) remains in its infancy. Transcriptome-wide m6A changes were profiled using human m6A epitranscriptomic microarrays in ESCC tissues and adjacent normal tissues. Differentially m6A-methylated mRNAs, lncRNAs and Mid-size ncRNAs were identified, followed by functional enrichment analysis. Moreover, we also establish a competing endogenous RNA (ceRNA) network based on the m6A-related lncRNA growth arrest specific 5 (GAS5) specific transcript（NR_152533）. The results suggested that RALYL may be a target gene for miR-3912-5p, and that GAS5 regulated RALYL expression by binding to miR-3912-5p.

LncRNA-GAS5/miR-382-3p axis inhibits pulmonary artery remodeling and promotes autophagy in chronic thromboembolic pulmonary hypertension.

BACKGROUND: We have clarified the role of miR-382-3p in chronic thromboembolic pulmonary hypertension (CTEPH), but what is less clear lies in its upstream regulatory mechanism. OBJECTIVE: To explore the regulation mechanism of GAS5/miR-382-3p axis on CTEPH. METHODS: In vitro, we constructed cell models by treating Pulmonary Artery Smooth Muscle Cells (PASMCs) with platelet-derived growth factor-BB (PDGF-BB). The effects of different concentrations of PDGF-BB on the activity of PASMCs were tested by cell counting kit-8 (CCK-8). The upstream lncRNA of miR-382-3p was screened and confirmed through bioinformatics analysis, RNA pull-down, quantitative reverse transcription polymerase chain reaction (qRT-PCR), dual luciferase reporter gene and RNA immunoprecipitation assays. The effects of GAS5/miR-382-3p axis on the viability, migration, and expressions of autophagy- and angiogenesis-related proteins were confirmed by rescue experiments (CCK-8, wound healing and western blot). In vivo, animal models by perfusing autologous blood vessels, the effects of GAS5 overexpression or silencing on the expressions of miR-382-3p, angiogenesis- and autophagy-related genes, mean pulmonary arterial pressure (mPAP) and pulmonary artery wall were determined by biological signal acquisition system, hematoxylin-eosin staining, qRT-PCR and western blot. RESULTS: PDGF-BB dose-dependently promoted PASMCs viability. XIST and GAS5 expressions in PASMCs were affected by the concentration of PDGF-BB, but only GAS5 can be pulled down by miR-382-3p probe. GAS5 targeted miR-382-3p to inhibit the viability and migration of PAMSCs, mPAP in CTEPH rats, pulmonary artery wall thickening and angiogenesis, and promote autophagy. CONCLUSIONS: GAS5/miR-382-3p axis is involved in the regulation of pulmonary artery remodeling and autophagy in CTEPH.

LncRNA-GAS5 promotes spinal cord repair and the inhibition of neuronal apoptosis via the transplantation of 3D printed scaffold loaded with induced pluripotent stem cell-derived neural stem cells.

BACKGROUND: Stem cell transplantation has been increasingly used for spinal cord repair, and some achievements have been made. However, limited stem cell sources as well as immune rejection and ethical issues have restricted its wide application. Therefore, to achieve further breakthroughs regarding the application of stem cell transplantation to treat spinal cord injury (SCI), it is important to develop a stem cell line that can effectively avoid immune rejection and ethical issues. METHODS: Urine cells (UCs) were induced to differentiate into induced pluripotent stem cells (iPSCs), which then further differentiated into neural stem cells (NSCs). Relevant tests were performed, and three-dimensional (3D) printed scaffolds were prepared. Thirty C57BL/6 mice were divided into 5 groups based on a random number table: a sham group, an SCI group, an SCI + control group, an SCI + siNC group, and an SCI + siGAS5 group (n=6). The latter 4 groups replicated SCI models. Mice in the SCI + control group were transplanted with 3D scaffolds loaded with iPSC-derived NSCs (iPSd-NSCs). Mice in the SCI + siNC group and the SCI + siGAS5 group were transplanted with scaffolds loaded with iPSd-NSCs-siNC and 3D scaffolds loaded with iPSd-NSCs-siGAS5, respectively. Mice in the other groups were injected with the same amount of normal saline. Hematoxylin-eosin staining was used to observe the histopathology of the injured spinal cord, the Basso-Mouse Scale was used to assess the motor function of the hind limbs of the mice, and Western blot was used to detect the expression of apoptosis-related proteins after SCI. RESULTS: iPSd-NSCs were successfully induced and differentiated, and 3D printed heparin sulfate-collagen scaffolds were prepared, inside which a 3D loose porous structure was shown by electron microscopy. Morphological observations showed that iPSd-NSC transplantation improved SCI in mice, while GAS5 silencing inhibited the reparative effect of iPSd-NSC transplantation on SCI in mice. Western blot results indicated that iPSd-NSC transplantation significantly increased the expression level of B cell lymphoma/leukemia-2 (Bcl-2) (P<0.01) but decreased the expression levels of Bcl-2 associated X protein, cytochrome C, and cleaved caspase-3 (P<0.001). CONCLUSIONS: The overexpression of lncRNA-GAS5 can promote spinal cord repair and inhibit neural apoptosis via the transplantation of 3D printed scaffolds loaded with iPSd-NSCs.

The interplay between HIF-1α and long noncoding GAS5 regulates the JAK1/STAT3 signalling pathway in hypoxia-induced injury in myocardial cells.

BACKGROUND: Long non-coding RNA (lncRNA) GAS5 is associated with hypoxia-induced diseases whereas hypoxia-inducible factor-1α (HIF-1α) plays an important role in hypoxic injury of cells. The current study explores the regulatory functions of GAS5/HIF-1α which co-play in anoxic injury among rat cardiomyocytes H9C2 cells. METHODS: Hypoxia in vitro model was established through anoxic incubation while normal culture of H9C2 cells was considered as control. The expression levels of GAS5 and HIF-1α were quantified through RT-qPCR. CCK-8 was applied to determine cell viability. Cell apoptosis rate was calculated using flow cytometry whereas inflammatory cytokines were detected using ELISA method. The impact of downregulating GAS5 or HIF-1α or both upon hypoxic cells was assessed on the basis of changes in cell viability, apoptosis, and inflammatory response. The activity of JAK1/STAT3 signaling was evaluated through RT-qPCR for mRNA expression. AG490 was introduced to inactivate JAK1/STAT3 pathway and to unveil the impact of JAK1/STAT3 signaling on GAS5/HIF-1α and cell viability, apoptosis and inflammation in hypoxic cells. RESULTS: The results infer that hypoxia suppressed cell viability, promoted inflammation and apoptosis among H9C2 cells. GAS5 or HIF-1α recorded higher expression in hypoxia-induced cells whereas the cell viability got restored with reduction in inflammation and apoptosis. The downregulation of HIF-1α enhanced the protective effect of knocking down GAS5 in hypoxia H9C2 cells. JAK1/STAT3 signaling pathway got activated in hypoxic cells and was regulated by GAS5 and HIF-1α. The inhibition of signaling pathway increased the cell viability but it decreased both inflammation and apoptosis. CONCLUSIONS: GAS5 and HIF-1α could regulate hypoxic injury in H9C2 cells through JAK1/STAT3 signaling pathway. This scenario suggests that the inhibitors of GAS5 and HIF-1α may synergize with AG-490 to protect myocardial cells from hypoxic injury.

Long non­coding RNA GAS5 protects against Mycoplasma pneumoniae pneumonia by regulating the microRNA­222­3p/TIMP3 axis.

Mycoplasma pneumoniae pneumonia (MPP) is a type of pneumonia induced by M. pneumoniae (MP) infection. The present study investigated the effect of long non­coding RNA growth arrest­specific 5 (GAS5) in MPP and the underlying molecular mechanism of this. The expression of GAS5, microRNA­222­3p, (miR­222­3p) and tissue inhibitor of metalloproteinases­3 (TIMP3) in MPP was investigated using reverse transcription­quantitative PCR. Lipid­associated membrane protein (LAMP)­induced THP­1 cells were used to model MPP. The viability of LAMP­induced THP­1 cells was analyzed using an MTT assay. Expression levels of interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α (TNF­α) pro­inflammatory cytokines, and the anti­inflammatory cytokine heme oxygenase­1 (HO­1) in LAMP­induced THP­1 cells were measured by ELISA. A dual­luciferase reporter assay assessed the associations among GAS5, miR­222­3p and TIMP3. The expression of GAS5 and TIMP3 was downregulated in MPP. Expression of miR­222­3p was upregulated. GAS5­overexpression increased the viability of LAMP­induced THP­1 cells. GAS5 upregulation decreased the levels of IL­1ß, IL­6, TNF­α and HO­1 levels in LAMP­induced THP­1 cells. GAS5 directly interacted with miR­222­3p. TIMP3 was a target of miR­222­3p. miR­222­3p upregulation or TIMP3­knockdown reversed the promotion effect on cell viability as well as the inhibitory effect on inflammation caused by GAS5­overexpression in LAMP­induced THP­1 cells. GAS5­overexpression increased the viability and decreased the inflammation of LAMP­induced THP­1 cells by regulating the miR­222­3p/TIMP3 axis. These results demonstrated a potential therapeutic target for MPP treatment.

Quercetin reverses adriamycin resistance by activating GSK-3β/β-catenin pathways through GAS5 in breast cancer cells / 中国临床药理学与治疗学

AIM: To investigate the ability of quercetin to reverse acquire adriamycin (ADR) resistance and explored its probably mechanism. METHODS: The CCK-8 assay was used to detect the cytotoxicity of quercetin in MCF-7/ADR cells and the reversal effect of ADR. The colony formation assay and Hoechst 332582 staining were used to detect the cell proliferation, cell apoptosis and the accumulation of rhodamine 123 (Rh123) respectively. The RNA expression levels of GAS5 and ABCB1 were detected by qRT-PCR. The protein expression levels of GSK-3β, β-catenin, c-MYC, cyclin D1, and ABCB1 were detected by Western blot. RESULTS: Quercetin (10, 20, 40 μmol/L) significantly enhanced the sensitivity of MCF-7/ADR to ADR, inhibitd cell proliferation, and increased the intracellular accumulation of Rh123. Treatment with quercetin in MCF-7/ADR cells, the expression levels of GAS5 and GSK-3β were increased, whereas the expression levels of β-catenin, c-myc, cyclin D1 and ABCB1 were decreased. Further research revealed that reduction of GAS5 by RNA interference (si-GAS5) induced inhibitory effects on the expressions of GAS5 and GSK-3β, and enhanced the expressions of β-catenin, c-myc, cyclin D1, and ABCB1. Furthermore, treatment by quercetin combined with si-GAS5 in MCF-7/ADR cells, the expressions of these proteins were effectively reversed in comparison to quercetin combined with siRNA negative control (sncRNA). CONCLUSION: Quercetin increases the expression of GAS5by GSK-3β/β-catenin signaling pathway, which inhibits the expression of ABCB1, ultimately reversing ADR resistance in the MCF-7/ADR cells.

Quercetin promotes apoptosis of breast cancer cells by targeting GAS5/Notchl signaling pathway / 中国药理学通报

Aim To investigate the possible mechanism of quercetin in promoting apoptosis of breast cancer cells. Methods Breast cancer cells MCF-7 were treated with quercetin. Cell viability was detected by MTT assay, cell proliferation and cloning ability were measured by plate colony formation assay, and cell apoptosis was examined by Annexin V-FITC/PI double staining assay. Meanwhile, MCF-7 cells were treated with quercetin (40, 80, 160 μmol · L

Long non-coding RNA GAS5 accelerates oxidative stress in melanoma cells by rescuing EZH2-mediated CDKN1C downregulation.

BACKGROUND: The significance of long non-coding RNAs (lncRNAs) in mediating oxidative stress of cancers has been implicated recently. This study proposed a potential therapeutic target lncRNA growth arrest-specific transcript 5 (GAS5) for melanoma, due to its crucial role in oxidative stress and apoptosis of melanoma cells by regulating the enhancer of zeste homolog 2 (EZH2)-mediated CDKN1C expression. METHODS: The lncRNA GAS5 expression pattern was examined in melanoma tissues and cells. The correlation of lncRNA GAS5, EZH2, and CDKN1C with survival rate of melanoma patients was analyzed. In melanoma cell lines, lncRNA GAS5 expression was overexpressed or knocked down to clarify its effects on cell viability, apoptosis, and oxidative stress. The interaction between lncRNA GAS5 and EZH2 was examined by RIP and RNA pull-down assays followed by verification of the target relationship between EZH2 and CDKN1C. RESULTS: High expression of EZH2 and poor expression of lncRNA GAS5 and CDKN1C was observed in melanoma tissues and found to be correlated with the reduction in survival expectancy of melanoma patients. Overexpression of lncRNA GAS5 or CDKN1C or EZH2 knockdown could inhibit cell viability but enhance melanoma cell apoptosis and oxidative stress. Importantly, lncRNA GAS5 attenuated EZH2 expression by recruiting E2F4 to the EZH2 promoter region and knockdown of EZH2 upregulated CDKN1C expression by inhibiting the H3K27me3. CONCLUSION: The evidence provided by our study highlighted the involvement of lncRNA GAS5 in the translational suppression of EZH2 as well as the upregulation of CDKN1C, resulting in the promotion of melanoma cell apoptosis and oxidative stress.

Elevation of long non-coding RNA GAS5 and knockdown of microRNA-21 up-regulate RECK expression to enhance esophageal squamous cell carcinoma cell radio-sensitivity after radiotherapy.

OBJECTIVE: Lately, lncRNAs have been proposed to function in the radio-sensitivity of tumor cells, yet the role of lncRNA GAS5 in that of esophageal squamous cell carcinoma (ESCC) has scarcely been studied. This study aims to examine GAS5's effects on ESCC cell radio-sensitivity. METHODS: GAS5, miR-21 and RECK expression in radiation-sensitive and radiation-resistant ESCC tissues, and TE-1 and TE-1-R cells was determined. TE-1 and TE-1-R cells were treated with pcDNA-GAS5 or miR-21 inhibitors to figure out their roles in ESCC cell proliferation, radio-sensitivity, and apoptosis via gain- and loss-of-function experiments. RESULTS: We found underexpressed GAS5 and RECK, and overexpressed miR-21 in ESCC. GAS5 elevation and miR-21 inhibition reduced viability and the colony formation ability, and enhanced the apoptosis of ESCC cells under radiation. CONCLUSION: Our study reveals that GAS5 elevation up-regulates RECK expression by down-regulating miR-21 to increase ESCC cell apoptosis after radiation therapy, thus enhancing cell radio-sensitivity.

Long non-coding RNA GAS5 promotes osteogenic differentiation of bone marrow mesenchymal stem cells by regulating the miR-135a-5p/FOXO1 pathway.

Studies have shown that promoting the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts could protect against osteoporosis. Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) participate in BMSC osteogenic differentiation. This study aimed to investigate the role and underlying mechanism of growth arrest-specific transcript 5 (GAS5) in osteogenic differentiation. The mechanism was mainly focused on miR-135a-5p/FOXO1 pathway by gain- and loss-of function tests. GAS5 and FOXO1 expression was decreased, whereas miR-135a-5p expression was increased, in the BMSCs from osteoporotic mice. Levels of GAS5 and FOXO1 were increased and miR-135a-5p expression was decreased during osteogenic differentiation of BMSCs. Overexpression of GAS5 promoted, whereas knockdown of GAS5 suppressed, BMSC osteogenic differentiation. As for the mechanism, GAS5 functioned as a competing endogenous RNA for miR-135a-5p to regulate FOXO1 expression. In conclusion, GAS5 promoted osteogenesis of BMSCs by regulating the miR-135a-5p/FOXO1 axis. This finding suggests that targeting GAS5 may be a useful therapy for treating postmenopausal osteoporosis.

Overexpression of lncRNA GAS5 suppresses prostatic epithelial cell proliferation by regulating COX-2 in chronic non-bacterial prostatitis.

Chronic non-bacterial prostatitis (CNP) is a common urologic disease that is linked to the development of prostate cancer. Long non-coding RNA (lncRNA) GAS5 has been identified to mediate cell proliferation in prostate cancer, although its role in CNP is still unclear. Human prostate epithelial cell line RWPE-1 was induced by lipopolysaccharide (LPS) to mimic CNP model in vitro. Real-time PCR was performed to determine the expression of GAS5 and COX-2, while western blotting was used to evaluate the protein expression of COX-2. The interaction between GAS5 and COX-2 was determined using RNA pull-down and RNA immunoprecipitation (RIP). Cell proliferation was determined using MTT assay. The expression of GAS5 was decreased, while COX-2 was increased in prostatitis tissues and in LPS-induced RWPE-1 cells. The overexpression of GAS5 suppressed the protein level of COX-2, and inhibited cell proliferation of LPS-induced RWPE-1 cells and HPECs, which was rescued by the co-transfection with pcDNA-GAS5 and pcDNA-COX-2. GAS5 was confirmed to promote the ubiquitination of COX-2, and the in vivo GAS5-overexpressed CNP rat model decreased the motor scores, the volume of prostate tissues, the average number of inflammatory cells, prostatic proliferation, and COX-2 expression. Our findings revealed that overexpression of GAS5 inhibited cell proliferation via negatively regulating the expression of COX-2, thus alleviating the progression of CNP.

Decreased Expression of Long Non-Coding RNA GAS5 in Ovarian Cancer Patients and Its Clinical Significance / 中山大学学报(医学科学版)

[Objective]Dysregulated long noncoding RNAs(lncRNAs)have been found involved in human diseases,including cancers. Long non-coding RNA growth arrest-specific 5(GAS5)was reported to be dysregulated in different types of cancers. Howev-er,the role of GAS5 in ovarian cancer remains elusive.[Methods]In the present study,the expression of GAS5 was detected in 108 ovarian cancer tissues and compared adjacent normal tissues by quantitative real-time PCR(qRT-PCR).[Results]The results showed that the expression levels of lncRNA GAS5 were significantly decreased in cancer tissues(P=0.0004),and it was negatively correlated with tumor size(5 cm,P<0.0001),invasion depth(T1-T2 vs. T3-T4,P=0.0021),and tumor grade(Ⅰ~Ⅱgrades vsⅢ~Ⅳgrades,P=0.0086)in ovarian cancer patients. Kaplan-Meier analysis demonstrated that decreased lncRNA GAS5 expression contributed to poor disease-free survival and overall survival.[Conclusion]In conclusion ,our study suggested that decreased lncRNA GAS5 expression may beidentified as a potential poor prognostic biomarker in ovarian cancer.