Growth Arrest-specific Transcript Research Articles

Long noncoding RNA GAS5 alleviates the inflammatory response of human periodontal ligament stem cells by regulating the NF-κB signalling pathway.

This study investigated the role of lncRNA growth arrest-specific transcript 5 (GAS5) in the inflammatory response of periodontal ligament stem cells (PDLSCs) during periodontitis with attempts to its possible mechanisms. Gingiva samples were collected from healthy people and patients with periodontitis. The ligature-induced periodontitis model was established in mice. Cell transfection was utilized to knock down and overexpress GAS5 in PDLSCs. Quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization were performed to detect the GAS5 expression. In combination with high-throughput sequencing technology, qRT-PCR, Western blotting, and immunofluorescence were performed to detect the effects of GAS5 on cytokines and proteins in the NF-κB pathway. GAS5 expression decreased in PDLSCs subjected to compressive force. GAS5 expression was downregulated in the gingiva tissues from patients with periodontitis. Consistent with the results of clinical samples, GAS5 expression decreased in the mouse ligature-induced periodontitis model. GAS5 expression was downregulated in PDLSCs under tumour necrosis factor (TNF)-α stimulation. Knockdown and overexpression of GAS5 increased and decreased the expression of cytokines induced by TNF-α in PDLSCs, respectively. The sequencing results showed that overexpressing GAS5 was related to genes in the NF-κB pathway. Overexpressing GAS5 alleviated p65 phosphorylation and inhibited the entry of p65 into the nucleus in the TNF-α activated NF-κB pathway, whereas GAS5 knockdown resulted in contrasting results. GAS5 alleviated the expression of cytokines in PDLSCs by inhibiting activation of the TNF-α-mediated NF-κB pathway. These findings provide new insight into the regulation of the PDLSCs inflammation response.

LncRNA GAS5 promotes spermidine‑induced autophagy through the miRNA‑31‑5p/NAT8L axis in pulmonary artery endothelial cells of patients with CTEPH

Chronic thromboembolic pulmonary hypertension (CTEPH) is a leading cause of pulmonary hypertension. The present study investigated the mechanisms of long non-coding RNA growth arrest-specific transcript 5 (GAS5) on spermidine (SP)-induced autophagy. Pulmonary artery endothelial cells (PAECs) were collected from patients with CTEPH and the rat model. Immunofluorescence, Western blots, reverse transcription-quantitative polymerase chain reaction, bioinformatics, rapid amplification of cDNA ends assays, luciferase reporter assays, RNA-binding protein immunoprecipitation assays, GFP-LC3 adenoviruses, tfLC3 assays and transmission electron microscopy were performed. The results revealed that SP-induced autophagy increased GAS5 in PAECs. The upregulation of GAS5 enhanced and the downregulation of GAS5 reversed the roles of SP in PAECs. Furthermore, GAS5 promoted SP-induced autophagy in PAECs by targeting miRNA-31-5p. The miRNA-31-5p mimic suppressed and the inhibitor promoted SP-induced autophagy. Furthermore, N-Acetyltransferase 8 Like (NAT8L) was a target gene of miRNA-31-5p and knockdown of NAT8L inhibited the autophagic levels of PAECs. In vivo, SP treatment decreased miRNA-31-5p and increased NAT8L levels, which was reversed by the knockdown of GAS5. The downregulation of GAS5 abolished the stimulatory role of SP in PAECs of CTEPH rats. In conclusion, GAS5 promoted SP-induced autophagy through miRNA-31-5p/NAT8L signaling pathways in vitro and in vivo and GAS5 may be a promising molecular marker for therapies of CTEPH.

Research Progress of Long Non-Coding RNA GAS5 in Malignant Tumors.

With completing the whole genome sequencing project, awareness of lncRNA further deepened. The growth arrest-specific transcript 5 (GAS5) was initially identified in growth-inhibiting cells. GAS5 is a lncRNA (long non-coding RNA), and it plays a crucial role in various human cancers. There are small ORFs (open reading frames) in the exons of the GAS5 gene sequence, but they do not encode functional proteins. In addition, GAS5 is also the host gene of several small nucleolar RNAs (snoRNA). These snoRNAs are believed to play a suppressive role during tumor progression by methylating ribosomal RNA (rRNA). As a result, GAS5 expression levels in tumor tissues are significantly reduced, leading to increased malignancy, poor prognosis, and drug resistance. Recent studies have demonstrated that GAS5 can interact with miRNAs by base-pairing and other functional proteins to inhibit their biological functions, impacting signaling pathways and changing the level of intracellular autophagy, oxidative stress, and immune cell function in vivo. In addition, GAS5 participates in regulating proliferation, invasion, and apoptosis through the above molecular mechanisms. This article reviews the recent discoveries on GAS5, including its expression levels in different tumors, its biological behavior, and its molecular regulation mechanism in human cancers. The value of GAS5 as a molecular marker in the prevention and treatment of cancers is also discussed.

Protective role of Achyranthes bidentata polysaccharides against chondrocyte extracellular matrix degeneration through lncRNA GAS5 in osteoarthritis.

Achyranthes bidentata polysaccharides (ABPS) is an active ingredient of the flowering plant Achyranthes bidentata that has been previously reported to be effective for the treatment of osteoarthritis (OA). However, the underlying molecular mechanism remain to be fully clarified. Emerging studies have shown that the long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) is involved in the pathogenesis of OA. Therefore, the present study aimed to investigate the potential mechanism of ABPS by focusing on its effects on the regulation of chondrocyte extracellular matrix (ECM) homeostasis, with particular emphasis on lncRNA GAS5. In the present study, the modified Hulth method was used to construct OA rats, which were gavaged with 400 mg/kg ABPS for 8 weeks. Histopathological changes in cartilage and subchondral bone were evaluated by hematoxylin-eosin staining and Safranin O-fast green staining. In in vitro experiments, IL-1β-treated chondrocytes were infected with Lenti-lncRNA GAS5. Fluorescence in situ hybridization assay was performed to measure the expression of the lncRNA GAS5 in chondrocytes. Moreover, the relative expression level of lncRNA GAS5 in cartilage tissue and chondrocytes was detected using reverse transcription-quantitative PCR. Western blot analysis was used to detect protein expression levels of MMP-9, MMP-13, TIMP-1, TIMP-3 and type II collagen in cartilage tissue and chondrocytes. The results indicated that ABPS delayed the degradation of the ECM by chondrocytes in addition to reducing lncRNA GAS5 expression both in vivo and in vitro. Furthermore, silencing of lncRNA GAS5 expression in IL-1β-treated chondrocytes downregulated the protein expression of MMP-9 and MMP-13 whilst upregulating the expression of tissue inhibitor matrix metalloproteinase (TIMP)-1, TIMP-3 and type II collagen. To conclude, the present study provides evidence that ABPS can inhibit the expression of lncRNA GAS5 in chondrocytes to regulate the homeostasis of ECM, which in turn may delay the occurrence of cartilage degeneration during OA.

Growth arrest-specific transcript 5 represses endometrial cancer development by promoting antitumor function of tumor-associated macrophages.

The tumor‐suppressor role of long noncoding RNA (lncRNA) growth arrest‐specific transcript 5 (GAS5) has been proven in various types of cancer. However, the specific function of GAS5 in tumor‐associated macrophages (TAMs) of endometrial cancer (EC) is elusive. Quantitative PCR results showed that GAS5 expression decreased in EC tissues and primary TAMs from EC tumors. Tumor‐associated macrophage infiltration was significantly positively associated with the developmental stage of EC. Direct coculture of GAS5‐overexpressing TAMs and EC cells showed that GAS5 enhanced phagocytosis, antigen presentation, and activation of cytotoxic T cells, and repressed “Don’t eat me” signals between TAMs and EC cells. Tumor formation in immunodeficient mice showed that GAS5‐overexpressing macrophages could repress EC formation in vivo. GAS5 promoted M1 polarization by activating the microRNA‐21– phosphatase and tensin homolog (PTEN)–AKT signaling pathway and directly repressing the nuclear accumulation and phosphorylation of oncogenic yes‐associated protein 1 (YAP1) in TAMs. GAS5 inhibited the development of EC from both innate and adaptive immunity by transforming TAMs from a protumor to an antitumor phenotype. These antitumor effects of GAS5 on TAMs were mediated by the activation of the miR‐21‐PTEN‐AKT pathway and inhibition of YAP1.

Effect of berberine on global modulation of lncRNAs and mRNAs expression profiles in patients with stable coronary heart disease

BackgroundBerberine (BBR) is an isoquinoline alkaloid found in the Berberis species. It was found to have protected effects in cardiovascular diseases. Here, we investigated the effect the regulatory function of long noncoding RNAs (lncRNAs) during the treatment of stable coronary heart disease (CHD) using BBR. We performed microarray analyses to identify differentially expressed (DE) lncRNAs and mRNAs between whole blood samples from 5 patients with stable CHD taking BBR and 5 no BBR volunteers. DE lncRNAs and mRNAs were validated by quantitative real-time PCR.ResultsA total of 1703 DE lncRNAs and 912 DE mRNAs were identified. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated DE mRNAs might be associated with mammalian target of rapamycin and mitogen-activated protein kinase pathway. These pathways may be involved in the healing process after CHD. To study the relationship between mRNAs encoding transcription factors (DNA damage inducible transcript 3, sal-like protein 4 and estrogen receptor alpha gene) and CHD related de mRNAs, we performed protein and protein interaction analysis on their corresponding proteins. AKT and apoptosis pathway were significant enriched in protein and protein interaction network. BBR may affect downstream apoptosis pathways through DNA damage inducible transcript 3, sal-like protein 4 and estrogen receptor alpha gene. Growth arrest-specific transcript 5 might regulate CHD-related mRNAs through competing endogenous RNA mechanism and may be the downstream target gene regulated by BBR. Verified by the quantitative real-time PCR, we identified 8 DE lncRNAs that may relate to CHD. We performed coding and non-coding co-expression and competing endogenous RNA mechanism analysis of these 8 DE lncRNAs and CHD-related DE mRNA, and predicted their subcellular localization and N6-methyladenosine modification sites.ConclusionOur research found that BBR may affect mammalian target of rapamycin, mitogen-activated protein kinase, apoptosis pathway and growth arrest-specific transcript 5 in the process of CHD. These pathways may be involved in the healing process after CHD. Our research might provide novel insights for functional research of BBR.

5‑Aza‑dC suppresses melanoma progression by inhibiting GAS5 hypermethylation.

The in-depth study of melanoma pathogenesis has revealed that epigenetic modifications, particularly DNA methylation, is a universal inherent feature of the development and progression of melanoma. In the present study, the analysis of the tumor suppressor gene growth arrest-specific transcript 5 (GAS5) demonstrated that its expression was downregulated in melanoma, and its expression level had a certain negative association with its methylation modification level. The promoter of GAS5 presented with detectable CpG islands, and methylation-specific polymerase chain reaction analysis demonstrated that GAS5 was actually modified by methylation in melanoma tissues and cells; however, no methylation modification of GAS5 was detected in normal tissues. Following the treatment of melanoma cells with 5-Aza-2′-deoxycytidine (5-Aza-dC), GAS5 methylation was significantly reversed. The analysis of melanoma cell proliferation revealed that 5-Aza-dC inhibited A375 and SK-MEL-110 cell proliferation in a time-dependent manner. Further analysis of apoptosis demonstrated that 5-Aza-dC significantly increased the apoptosis level of the two cell lines. Moreover, migration analysis of melanoma cells revealed that 5-Aza-dC significantly reduced cell migration. Furthermore, 5-Aza-dC significantly decreased the invasive ability of the two cell lines. However, when the expression of GAS5 was silenced, the effects of 5-Aza-dC on cell proliferation, apoptosis, invasion and migration were not significant. Furthermore, the subcutaneous injection of A375 cells in nude mice successfully resulted in xenograft tumor formation. However, following an intraperitoneal injection of 5-Aza-dC, the volume and weight of xenograft tumors and Ki-67 expression were significantly reduced, and caspase-3 activity and GAS5 expression were enhanced; following the silencing of GAS5, the antitumor effect of 5-Aza-dC was significantly blocked. On the whole, the present study demonstrates that 5-Aza-dC inhibits the growth of melanoma, and its function may be related to the methylation modification of GAS5.

Level of LncRNA GAS5 and Hippocampal Volume are Associated with the Progression of Alzheimer's Disease.

PurposeWe evaluated the diagnostic value of long non-coding RNA growth arrest-specific transcript 5 (GAS5) and its relationship with hippocampal volume in Alzheimer’s disease (AD).Patients and MethodsOne hundred and eight patients with AD and 83 healthy controls were included, and demographic data, biochemical parameters, GAS5 levels, and hippocampal volume were recorded. Chi-squared tests or independent sample t-tests were used to compare the baseline characteristics, relative expression of GAS5, and hippocampal volume. Correlations between variables were determined using Spearman’s rank correlation test. Receiver operating characteristic (ROC) curves were generated to compare the diagnostic value of GAS5 and total hippocampal volume in AD.ResultsThe levels of GAS5 were significantly upregulated in patients with AD compared with those in controls and were negatively correlated with MMSE score. There were differences in left hippocampal volume, right hippocampal volume, and total hippocampal volume between the two groups. Total hippocampal volume was positively correlated with MMSE score and negatively correlated with GAS5 expression in patients with AD. The area under the curve (AUC) of for GAS5 expression was 0.831, the sensitivity was 61.1%, and the specificity was 95.2%. The AUC of the combined total hippocampal volume was 0.891, the sensitivity was 74.1%, and the specificity was 92.8%.ConclusionThe results suggested that GAS5 may be an excellent indicator of AD progression alone or in combination with hippocampal volume.

LncRNA GAS5 regulates migration and epithelial-to-mesenchymal transition in lens epithelial cells via the miR-204-3p/TGFBR1 axis

AbstractDiabetic cataract (DC) is a major ocular complication secondary to diabetes mellitus. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is an important event in DC progression. Long non-coding RNAs (lncRNAs) and microRNAs are involved in various biological processes and disorders. The aim of this study was to investigate the roles of lncRNA growth arrest-specific transcript 5 (GAS5) and microRNA-204-3p (miR-204-3p) deregulation in the pathogenic mechanism of high glucose (HG)-stimulated LECs. The results show that GAS5 was up-regulated, whereas miR-204-3p was down-regulated in anterior lens capsule tissues of DC patients and in HG-treated LECs compared to their controls, respectively. Functional experiments suggest that the lentivirus-mediated depletion of GAS5, as well as overexpression of miR-204-3p, suppressed migration and EMT in HG-treated LECs. Further mechanistic studies revealed that lncRNA GAS5/miR-204-3p/type 1 receptor of transforming growth factor-beta (TGFBR1) has a regulatory role in the process. Collectively, we demonstrated that dysregulation of GAS5 affects lens epithelial cell migration and EMT under HG conditions via the miR-204-3p/TGFBR1 axis. The current findings may provide new insights into the molecular mechanisms of DC development.Under high-glucose conditions, LECs obtain characteristics of mesenchymal cells such as high migratory capacity and invasiveness, which is the foundational basis for DC progression. The authors demonstrated that lncRNA GAS5 facilitates high glucose-induced lens epithelial cell migration and epithelial-to-mesenchymal transition by regulating the miR-204-3p/TGFBR1 axis. This study, therefore, provides novel insights into the pathogenesis of DCs.

LncRNA growth arrest specific transcript 5 inhibits the growth of pituitary neuroendocrine tumors via miR-27a-5p/cylindromatosis axis

ABSTRACT The long noncoding RNA growth arrest-specific transcript 5 (GAS5) has been reported to function as a suppressor in many cancers. However, the role and mechanism of lncRNA GAS5 in pituitary neuroendocrine tumors (PitNETs) remain unclear. Here, we found that lncRNA GAS5 and cylindromatosis (CYLD) expression was downregulated in invasive PitNET tissues and was negatively correlated with miR-27a-5p expression. LncRNA GAS5 overexpression inhibited proliferation of PitNETs cell line MMQ and GH3 cells and induced cell apoptosis, simultaneously, inhibited miR-27a-5p expression and increased CYLD expression. Moreover, miR-27a-5p mimic significantly decreased the luciferase activities of lncRNA GAS5 and CYLD luciferase reporter vector and downregulated CYLD expression, while miR-27a-5p inhibitor increased the expression of CYLD in MMQ and GH3 cells. Furthermore, RNA-immunoprecipitation assay revealed the direct binding between lncRNA GAS5 and miR-27a-5p. Additionally, miR-27a-5p mimic or silenced CYLD attenuated the effect of lncRNA GAS5 on MMQ and GH3 cell proliferation. In vivo lncRNA GAS5 overexpression inhibited GH3 cell tumor growth, while miR-27a-5p mimic or silenced CYLD attenuated the effect of lncRNA GAS5 on GH3 cell tumor growth. These results suggest that lncRNA GAS5 acts as an endogenous sponge by binding miR-27a-5p to increase the expression of its target gene CYLD, thereby inhibits PitNETs cell proliferation and tumor growth.

Long Noncoding RNA GAS5 Inhibits Osteogenic Differentiation through MicroRNA 382-3p/TAF1 Signaling.

Long noncoding RNAs (lncRNAs) have been confirmed as important regulators during osteogenic differentiation. Previous research has disclosed that growth arrest-specific transcript 5 (GAS5) can promote osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), but the underlying regulatory mechanism of GAS5 during the osteogenic differentiation of hBMSCs is unclear. Osteogenic differentiation was induced in hBMSCs by using osteogenic medium (OM). Gene expression was assessed by quantitative real-time PCR (RT-qPCR) or Western blot assays as needed. Alkaline phosphatase (ALP) activity, ALP staining, and alizarin red S (ARS) staining assays were performed to evaluate the impact of GAS5, microRNA 382-3p (miR-382-3p), and TATA box binding protein-associated factor 1 (TAF1) on osteogenic differentiation in vitro. The interaction among GAS5, miR-382-3p, and TAF1 was determined by RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and luciferase reporter assays. Expression of GAS5 (transcript variant 2) was downregulated during the osteogenic differentiation of hBMSCs, and its overexpression retarded the osteogenic differentiation of hBMSCs. GAS5 inhibited miR-382-3p by targeting RNA-directed microRNA degradation (TDMD). miR-382-3p downregulation partially offset the promoted osteogenic differentiation of hBMSCs upon GAS5 silencing. TAF1 negatively modulated osteogenic differentiation, and it activated GAS5 transcription so as to form a positive GAS5-miR-382-3p-TAF1 feedback loop in hBMSCs. This research was the first to reveal that the GAS5-miR-382-3p-TAF1 feedback loop inhibited the osteogenic differentiation of hBMSCs, which provided new clues for exploring the mechanism of osteogenic differentiation and disclosed the potential of GAS5 as a promising target during osteogenic differentiation.

Highly Expressed lncRNA GAS5 in the Serum of Children with Mycoplasma pneumoniae Pneumonia and Its Effect on LAMPs-Induced Apoptosis and Inflammation.

The aim of the study was to explore the serum expression of long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in Mycoplasma pneumoniae pneumonia (MPP) and its effect on lipid-associated membrane proteins (LAMPs)-induced apoptosis and inflammation. Totally, 56 children with MPP (MPP group) and 56 healthy children (NC group) were enrolled. lncRNA GAS5 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Serum levels of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) were detected using ELISA, and the high mobility family protein B1 (HMGBl) was detected by qRT-PCR. The methylated binding protein 2 (MECP2) was inhibited by gene silencing, and the expression of MECP2, TNF-α, IL-6, HMGBl, p-p65, and p-IκBα was measured. lncRNA GAS5 and TNF-α, IL-6, and HMGBl in the peripheral blood of the MPP group were positively correlated (P < 0.05). The expression of TNF-α, IL-6, HMGBl, and lncRNA GAS5 showed a positive correlation with that of LAMPs. The GAS5-siRNA group showed an increased cell survival rate compared with the scrambled-RNAi group (P < 0.05) while showing decreased apoptosis and cell death rates (P < 0.05). In addition, the expression of IL-6, TNF-α, HMGBl, p-p65, and p-IκBα was significantly reduced (P < 0.05). lncRNA GAS5 is highly expressed in the serum of children with MPP and inhibits LAMPs-induced apoptosis and alveolar macrophage inflammation.

LncRNA FGD5-AS1 acts as a ceRNA to regulate lipopolysaccharide-induced injury via the miR-223-3p-3p/GAS5 axis in cardiomyocytes.

Long noncoding RNAs (lncRNAs) are abnormally expressed in numerous diseases, and they are closely associated with cardiac diseases. However, the role of lncRNAs in lipopolysaccharide (LPS)-induced cardiotoxicity as well as the potential mechanism remain largely unclear. In the present study, IncRNA microarray assays were performed to analyze differential lncRNA expression in LPS-treated cardiomyocytes, and lncRNA FGD5-AS1 was one of the downregulated lncRNAs. H9C2 cells were treated with LPS, and the expression of lncRNA FGD5-AS1 was markedly downregulated. LncRNA FGD5 overexpression decreased the LPS-induced cardiomyocyte apoptosis and inflammation. Bioinformatics analysis and a luciferase reporter assay indicated that lncRNA FGD5-AS1 directly binds to miR-223-3p. A miR-222-3p mimic partially reversed the inhibitory effect of lncRNA FGD5-AS1 on the LPS-induced H9C2 cell apoptosis and inflammatory response. Moreover, miR-223-3p directly targeted growth arrest-specific transcript 5 (GAS5). LncRNA FGD5-AS1 regulated LPS-induced H9C2 cell inflammation and apoptosis via the miR-223-3p/GAS5 axis.

Expression Profiles of Long Non-Coding RNA GAS5 and MicroRNA-222 in Younger AML Patients.

Acute myeloid leukemia (AML) is a heterogeneous malignant disease both on clinical and genetic levels. AML has poor prognosis and, therefore, there is a constant need to find new prognostic markers, as well as markers that can be used as targets for innovative therapeutics. Recently, the search for new biomarkers has turned researchers’ attention towards non-coding RNAs, especially long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs). We investigated the expression level of growth arrest-specific transcript 5 (GAS5) lncRNA in 94 younger AML patients, and also the expression level of miR-222 in a cohort of 39 AML patients with normal karyotype (AML-NK), in order to examine their prognostic potential. Our results showed that GAS5 expression level in AML patients was lower compared to healthy controls. Lower GAS5 expression on diagnosis was related to an adverse prognosis. In the AML-NK group patients had higher expression of miR-222 compared to healthy controls. A synergistic effect of GAS5low/miR-222high status on disease prognosis was not established. This is the first study focused on examining the GAS5 and miR-222 expression pattern in AML patients. Its initial findings indicate the need for further investigation of these two non-coding RNAs, their potential roles in leukemogenesis, and the prognosis of AML patients.

LncRNA GAS5 positively regulates IL-10 expression in patients with generalized myasthenia gravis.

IntroductionLncRNA growth arrest‐specific transcript 5 (GAS5) has been proven to be involved in autoimmune diseases. Rheumatoid arthritis is a type of autoimmune disease that may affect myasthenia gravis (MG) patients. However, its direct role in MG is unknown.MethodsOur study included 62 generalized MG patients. GAS5 expression was analyzed with real‐time quantitative RT‐PCR (qRT‐PCR). The interaction between GAS5 and interleukin 10 (IL‐10) was explored in overexpressed cells using real time quantitative polymerase chain reactions (RT‐qPCRs) and western blot. The correlation of GAS5 and IL‐10 was analyzed using Pearson's correlation analysis. The diagnostic value of GAS5 for MG was analyzed using receiver operating characteristic (ROC) curve analysis.ResultsGAS5 and IL‐10 mRNA levels in peripheral blood mononuclear cells (PBMCs) were significantly lower in MG patients than healthy controls. Downregulated GAS5 effectively distinguished MG patients from healthy controls. GAS5 expression was positively correlated with IL‐10 expression in both MG patients and healthy controls. GAS5 overexpression significantly upregulated IL‐10 expression in PBMCs derived from both MG patients and healthy controls.ConclusionLncRNA GAS5 may improve generalized MG by positively regulating IL‐10 expression.

Oxytocin Protects Against Isoproterenol-Induced Cardiac Hypertrophy by Inhibiting PI3K/AKT Pathway via a lncRNA GAS5/miR-375-3p/KLF4-Dependent Mechanism.

Cardiac hypertrophy is caused by cardiac volume or pressure overload conditions and ultimately leads to contractile dysfunction and heart failure. Oxytocin (OT), an endocrine nonapeptide, has been identified as a cardiovascular homeostatic hormone with anti-hypertrophic effects. However, the underlying mechanism remains elusive. In this study, we aimed to investigate the role and mechanism of OT in cardiac hypertrophy. The rats with cardiac hypertrophy induced by isoproterenol (ISO) were treated with or without oxytocin. Cardiac functional parameters were analyzed by echocardiography. The changes in cell surface area were observed using wheat germ agglutinin (WGA) or immunofluorescence staining. The expressions of cardiac hypertrophy markers (B-Natriuretic Peptide, BNP and β-myosin heavy chain, β-MHC), long non-coding RNA Growth (LcRNA) Arrest-Specific transcript 5 (lncRNA GAS5), miR-375-3p, and Kruppel-like factor 4 (Klf4) were detected by qRT-PCR. KLF4 protein and PI3K/AKT pathway related proteins were detected by Western blot. The interactions among lncRNA GAS5, miR-375-3p, and Klf4 were verified by dual-luciferase reporter assays. The findings showed that OT significantly attenuated cardiac hypertrophy, increased expressions of lncRNA GAS5 and KLF4, and decreased miR-375-3p expression. In vitro studies demonstrated that either knock-down of lncRNA GAS5 or Klf4, or over-expression of miR-375-3p blunted the anti-hypertrophic effects of OT. Moreover, down-regulation of lncRNA GAS5 promoted the expression of miR-375-3p and inhibited KLF4 expression. Similarly, over-expression of miR-375-3p decreased the expression of KLF4. Dual-luciferase reporter assays validated that lncRNA GAS5 could sponge miR-375-3p and Klf4 was a direct target gene of miR-375-3p. In addition, OT could inactivate PI3K/AKT pathway. The functional rescue experiments further identified OT regulated PI3K/AKT pathway through lncRNA GAS5/miR-375-3p/KLF4 axis. In summary, our study demonstrates that OT ameliorates cardiac hypertrophy by inhibiting PI3K/AKT pathway via lncRNA GAS5/miR-375-3p/KLF4 axis.

LncRNA GAS5 suppresses ovarian cancer progression by targeting the miR-96-5p/PTEN axis.

BackgroundLong non-coding RNAs (lncRNAs) play critical roles in the occurrence and progression of various tumors, including ovarian cancer (OC). The lncRNA growth arrest-specific transcript 5 (GAS5) has been shown to be an important modulator in the growth and metastasis of OC cells. Our previous studies confirmed that GAS5 was down-regulated in OC; however, the potential underlying molecular mechanism underlying has not yet been elucidated.MethodsWe screened the Gene Expression Profiling Interactive Analysis (GEPIA) database for the expression of the lncRNA GAS5 in OC. Cell Counting Kit-8 (CCK-8), transwell assay, colony formation assay, flow cytometry analysis, and western blotting were applied to determine the various functions of GAS5 in OC progression. The competing endogenous RNA (ceRNA) mechanism was verified through bioinformatics analysis, dual-spectral luciferase reporter gene assay, and RNA immunoprecipitation assay (RIPA). Finally, the expression interactions between microRNA-96-5p, phosphatase and tensin homolog deleted on chromosome ten (PTEN), and GAS5 were measured.ResultsOur results demonstrated decreased expression levels of GAS5 and PTEN in OC samples and cell lines, while miR-96-5p was up-regulated when compared with the controls. GAS5 overexpression could significantly reduce OC cell proliferation and invasion ability via suppression of miR-96-5p expression. Moreover, GAS5 could influence the PTEN/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway.ConclusionsOur study identified GAS5 as a ceRNA that can regulate the PTEN/AKT/mTOR axis by sponging miR-96-5p in OC.

Knockdown of long noncoding RNA GAS5 reduces vascular smooth muscle cell apoptosis by inactivating EZH2-mediated RIG-I signaling pathway in abdominal aortic aneurysm

BackgroundAbdominal aortic aneurysm (AAA), an irreversible cardiovascular disease prevalent in the artery, causes the increase of the aneurysm diameter over time, and is a fatal phenomenon inducing sidewall rupture. Long noncoding RNAs (lncRNAs) serve as promising biomarkers for AAA. In the present study, we sought to define the role of lncRNA growth-arrest-specific transcript 5 (GAS5) in growth of smooth muscle cells (SMC) and progression of AAA.MethodsInitially, we established angiotensin II (Ang II)-induced AAA mouse models and Ang II-treated vascular SMC model. RT-qPCR and Western blot analysis were adopted to determine expression of GAS5 and zeste homolog 2 (EZH2). After ectopic expression and depletion experiments in Ang II-treated mice and vascular SMCs, cell apoptosis was detected in SMCs using flow cytometry and in mice using TUNEL staining. The binding of GAS5 and EZH2 was evaluated using RNA binding protein immunoprecipitation (RIP) and Co-IP assays.ResultsIncreased GAS5 and RIG-I but decreased EZH2 were found in aortic tissues of AAA mice. EZH2 overexpression inhibited AAA formation and suppressed SMC apoptosis. Functionally, EZH2 blocked the RIG-I signaling pathway and consequently inhibited SMC apoptosis. GAS5 regulated EZH2 transcription in a negative manner in SMCs. Knockdown of GAS5 attenuated SMC apoptosis, which was reversed by EZH2 inhibition or RIG-I overexpression.ConclusionsThe current study demonstrated that GAS5 induced SMC apoptosis and subsequent AAA onset by activating EZH2-mediated RIG-I signaling pathway, highlighting GAS5 as a novel biomarker for AAA.

Long noncoding RNA Gas5 induces cell apoptosis and inhibits tumor growth via activating the CHOP-dependent endoplasmic reticulum stress pathway in human hepatoblastoma HepG2 cells.

In recent years, long noncoding RNAs (lncRNAs) have been demonstrated to be important tumor-associated regulatory factors. LncRNA growth arrest-specific transcript 5 (Gas5) acts as an anti-oncogene in most cancers. Whether Gas5 acts as an oncogene or anti-oncogene in hepatocellular carcinoma (HCC) remains unclear. In the present study, the expression and role of Gas5 in HCC were investigated in vitro and in vivo. Lower expression levels of Gas5 were determined in HCC tissues and cells by quantitative reverse transcription-polymerase chain reaction. Overexpressed Gas 5 lentiviral vectors were constructed to analyze their influence on cell viability, migration, invasion, and apoptosis. Fluorescence in situ hybridization was used to identify the subcellular localization of Gas5. Protein complexes that bound to Gas5 were isolated from HepG2 cells through pull-down experiments and analyzed by mass spectrometry. A series of novel Gas5-interacting proteins were identified and bioinformatics analysis was carried out. These included ribosomal proteins, proteins involved in protein folding, sorting, and transportation in the ER, some nucleases and protein enzymes involved in gene transcription, translation, and other proteins with various functions.78 kDa glucose-regulated protein (GRP78) was identified as a direct target of Gas5 by Rip-qPCR and Western blot analysisassay. Gas5 inhibited HepG2 cell growth and induced cell apoptosis via upregulating CHOP to activate the ER stress signaling pathway. Further studies indicated that the knockdown of CHOP by shRNA partially reversed Gas5-mediated apoptosis in HepG2 cells. Magnetic resonance imagingshowed that the ectopic expression of Gas5 inhibited the growth of HCC in nude mice. These findings suggest that Gas5 functions as a tumor suppressor and induces apoptosis through activation of ER stress by targeting the CHOP signal pathway in HCC.

Long‑chain non‑coding RNA GAS5 promotes cell autophagy bymodulating the miR‑181c‑5p/ATG5 and miR‑1192/ATG12 axes.

The main aim of the present study was to explore the role of long-chain non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in macrophage autophagy. Firstly, the expression of lncRNA GAS5 during cell starvation or following treatment with 3-methyladenine was determined using reverse transcription-quantitative PCR (RT-qPCR). Additionally, fluorescent in situ hybridization (FISH) assay was utilized to determine the localization of the expression of lncRNA GAS5 in RAW264.7 cells. In vitro cell models were established through the transfection of LV5-lncRNA GAS5 (LV5-GAS5) or LV3-shRNA-lnc GAS5 (sh-GAS5), in order to overexpress or knockdown lncRNA GAS5 expression in RAW264.7 cells. The potential target microRNAs (miRNAs/miRs) of lncRNA GAS5 were analyzed using bioinformatics. The formation of autophagic bodies was detected with the use of laser confocal and transmission electron microscopy. Dual-luciferase reporter assay was performed to determine the target specificities of miR-181c-5p or miR-1192 to lncRNA GAS5 and autophagy-related gene (ATG) or ATG12. The mRNA levels of miR181c-5p, miR-1192, as well as ATG5 and ATG12 were detected using RT-qPCR. The protein levels of microtubule-associated proteins 1A/1B light chain 3B (LC3), p62, ATG5 and ATG12 were measured using western blot analysis. It was revealed that lncRNA GAS5 expression in RAW264.7 macrophages increased significantly during starvation-induced autophagy, and that lncRNA GAS5 overexpression was able to markedly promote the formation of autophagic bodies. Bioinformatics analysis demonstrated that miR-181c-5p and miR-1192 were potential targets of lncRNA GAS5, which was further confirmed by RT-qPCR, western blot analysis and the dual-luciferase reporter assay. Finally, it was confirmed that lncRNA GAS5 promoted autophagy by sponging miR-181c-5p and miR-1192, and upregulating the expression levels of the key autophagic regulators, ATG5 and ATG12. On the whole, the present study demonstrates that total, lncRNA GAS5 promotes macrophage autophagy by targeting the miR-181c-5p/ATG5 and miR-1192/ATG12 axes.