The long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) level was demonstrated as involved in pediatric inflammatory bowel disease (IBD) pathogenesis. Since its antisense transcript GAS5-AS1 has never been investigated in IBD, this study aims to detect whether GAS5-AS1 and GAS5 levels are related to IBD clinical parameters and investigate their correlation in vitro. Twenty-six IBD pediatric patients were enrolled; paired inflamed and non-inflamed intestinal biopsies were collected. We evaluated GAS5 and GAS5-AS1 levels by real-time PCR. The role of GAS5 and GAS5-AS1 was assessed in vitro by transient silencing in THP1-derived macrophages. GAS5-AS1 and GAS5 levels were associated with patients\' clinical parameters; GAS5-AS1 expression was downregulated in inflamed tissues and inversely correlated with disease activity. A positive correlation between GAS5-AS1 and GAS5 levels was observed in non-inflamed biopsies. On THP1-derived macrophages, a reduced amount of both GAS5-AS1 and GAS5 was observed; accordingly, matrix metalloproteinase (MMP) 9 was increased. After GAS5-AS1 silencing, a downregulation of GAS5 was found, whereas no effect was detected on GAS5-AS1 after GAS5 silencing.    Conclusion: This study provided for the first time new insights into the potential role of GAS5-AS1 in IBD. GAS5-AS1 modulates GAS5 levels in vitro and may serve as a potential IBD diagnostic biomarker. What is Known: • GAS5 is involved in regulating intestinal MMP-2 and MMP-9 in pediatric patients with IBD; • GAS5-AS1 has never been investigated in the context of IBD; • GAS5-AS1 regulates the expression of GAS5, increasing its stability in tissues and in vitro cell models of cancer. What is New: • GAS5-AS1 correlated with GAS5 and IBD clinical parameters; • GAS5-AS1 can modulate GAS5 levels in macrophages; • GAS5-AS1 may serve as potential IBD diagnostic biomarker.

Bone marrow (BM)-derived CD45 (BM45) cells were demonstrated to exhibit an improved antifibrotic effect on the treatment of CCL4-induced liver fibrosis by significantly increasing the level of matrix metalloproteinase 9 (MMP-9). In this study, we aimed to validate the therapeutic effect of BM45 on the treatment of liver cirrhosis and to further investigate the molecular mechanism underlying the effect of growth arrest-specific transcript 5 (GAS5) on BM45. Accordingly, GAS5 significantly suppressed miR-222 and miR-21 expression but enhanced p27 and MMP-9 expression in HepG2 and LX2 cells. Additionally, GAS5 obstructed transforming growth factor (TGF)-β-induced dysregulation of miR-222, p27, and α-smooth muscle actin (α-SMA) in mice. GAS5 showed a considerable potential to enhance the capability of BM45 in restoring the normal expression of CCL4, miR-222, miR-21, MMP-9, p27, and α-SMA that was dysregulated by alanine aminotransferase (ALT), albumin, and fibrosis. In summary, our study validated the regulatory relationship between miR-21 and MMP-9, as well as between miR-222 and p27. The overexpression of GAS5 upregulated the expression of MMP-9 and p27 via respectively reducing the miR-222 and miR-21 expression, resulting in higher BM45-induced activation of hepatic stellate cells (HSCs). Accordingly, same results were obtained in an animal model, indicating that GAS5 may exert a positive effect on the treatment of BM45 of liver cirrhosis.

The aim of this study was to investigate the mechanism by which growth arrest-specific transcript 5 (GAS5) regulates bladder cancer cells. Bladder cancer samples were collected and tested for experiment. Dual-luciferase reporter assay was used to verify the downstream target genes for GAS5 and miR-21. The expression level of GAS5 was decreased and that of miR-21 was increased, indicating a negative correlation between the two. Patients with high GAS5 level and low miR-21 level had relatively longer survival rates. GAS5 inhibited bladder cancer cells proliferation and promoted apoptosis, and miR-21 had the opposite effects. MiR-21 was a direct target for GAS5, whereas phosphatase and tensin homolog (PTEN) was a direct target gene of miR-21. Low expression of miR-21 could reverse the proliferative and antiapoptotic effects caused by GAS5 silencing. High levels of GAS5 and low levels of miR-21 might be associated with a higher survival rate in bladder cancer patients. GAS5 could exert antiproliferative and proapoptotic effects on bladder cancer cells through miR-21 and PTEN.

Growth arrest-specific transcript 5 (GAS5), along non-coding RNA (LncRNA), is highly expressed in hypoxia/reoxygenation (H/R)-cardiomyocytes and promotes H/R-induced apoptosis. In this study, we determined whether down-regulation of GAS5 ameliorates myocardial ischaemia/reperfusion (I/R) injury and further explored its mechanism. GAS5 expression in cardiomyocytes and rats was knockdown by transfected or injected with GAS5-specific small interfering RNA or adeno-associated virus delivering small hairpin RNAs, respectively. The effects of GAS5 knockdown on myocardial I/R injury were detected by CCK-8, myocardial enzyme test, flow cytometry, TTC and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. qRT-PCR and luciferase reporter assay were carried out to analyse the relationship between GAS5 and miR-335. The regulation of GAS5 on Rho-associated protein kinase 1 (ROCK1) expression, the activation of PI3K/AKT/GSK-3β pathway and mitochondrial permeability transition pore (mPTP) opening was further evaluated. The results indicated that GAS5 knockdown enhanced the viability, decreased apoptosis and reduced the levels of lactate dehydrogenase and creatine kinase-MB in H/R-treatment cardiomyocytes. Meanwhile, down-regulation of GAS5 limited myocardial infarct size and reduced apoptosis in I/R-heart. GAS5 was found to bind to miR-335 and displayed a reciprocal inhibition between them. Furthermore, GAS5 knockdown repressed ROCK1 expression, activated PI3K/AKT, thereby leading to inhibition of GSK-3β and mPTP opening. These suppressions were abrogated by miR-335 inhibitor treatment. Taken together, our results demonstrated that down-regulation of GAS5 ameliorates myocardial I/R injury via the miR-335/ROCK1/AKT/GSK-3β axis. Our findings suggested that GAS5 may be a new therapeutic target for the prevention of myocardial I/R injury.

Retinal ganglion cells (RGCs) play a key role in the pathogenesis and development of glaucoma. The present study aims to investigate the underlying mechanism of long noncoding RNA growth arrest-specific transcript 5 (GAS5) in glaucoma development through regulating the apoptosis of RGCs. Rat models of chronic glaucoma were successfully established by translimbal laser photocoagulation. Retinal tissues were collected to determine the density of RGCs through Toluidine blue staining. The overexpression vector or short hairpin RNA for GAS5 or enhancer of zeste homolog 2 (EZH2) was transfected into RGCs after in vitro pressurization culture to examine the function of GAS5 in RGC apoptosis. The involvement of EZH2 and ATP-binding cassette transporter A1 (ABCA1) was further identified. Cell apoptosis after laser treatment and transfection was assessed by flow cytometry. We found abundant GAS5 expression and a reduction in RGC density in the retinal tissues of glaucoma rats. Silencing of GAS5 led to increased EZH2 expression and decreased ABCA1 expression in RGCs. In addition, upregulation of EZH2 promoted trimethylation of lysine 27 on histone H3, thereby suppressing ABCA1 expression and eventually leading to the inhibition of RGC apoptosis. These findings provide further understanding of the function of GAS5 in RGC apoptosis. We conclude that downregulation of GAS5 could help relieve glaucoma symptoms. GAS5 is therefore a promising target for developing novel therapeutic approaches for treating patients with glaucoma.

Hepatitis B virus (HBV) infection remains a severe health burden worldwide. Emerging long noncoding RNAs (lncRNAs) are hijacked to enhance virus replication or employed by the host to stimulate immune responses to clear the virus. LncRNA growth arrest-specific transcript 5 (GAS5) can regulate RNA virus by suppressing the replication of both hepatitis C virus and human immunodeficiency virus. In this study, we explored the changes of HBV replication by overexpressing or knocking down GAS5 in HepAD38 cell and HepG2 cell transfected with pHBV1.2. We found HBV can induce the expression of GAS5. However, GAS5 had no effect on extracellular HBsAg and HBeAg, nor intracellular HBV RNA and HBV DNA. In addition, GAS5 possessed similar expression levels between stable HBV-producing cell lines and hepatoma cell lines. Furthermore, GAS5 showed no difference between healthy subjects and patients with chronic HBV in multiple GEO microarray data sets by GEO2R analysis. Taken together these results, GAS5 does not modulate the replication of HBV but it inhibits cell proliferation in HepAD38. This provides insights into the possible roles of GAS5 in HBV infection.

Objective: To investigate whether long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5)/microRNA-200c-3p/angiotensin converting enzyme 2(ACE2) involved in the regulation of the apoptosis of human lung epithelial cell A549 in acute respiratory distress syndrome (ARDS). Methods: ARDS rat models were established and were divided into control, ARDS, ARDS+ pcDNA and ARDS+ pcDNA-GAS5 groups. Six hours after the establishment of ARDS rat model, arterial blood and lung tissues of the rats from the four groups were collected. The changes of partial pressure of oxygen (PO(2)) and partial pressure of CO(2) (PCO(2)) were analyzed and the expression of GAS5 in lung tissue was observed in these groups. Then, A549 cells were divided into control, lipopolysaccharide (LPS), LPS+ pcDNA, LPS+ pcDNA-GAS5, LPS+ pcDNA-GAS5+ pre-NC, LPS+ pcDNA-GAS5+ miR-200c-3p mimic groups. Quantitative real-time PCR (qRT-PCR) was conducted to measure lncRNA GAS5, ACE2 and miR-200c-3p levels. RNA immunoprecipitation and RNA pull-down assay were used to detect the combination between GAS5 and miR-200c-3p. Western blotting was used to detect the protein level of ACE2. Flow cytometry was used to observe the apoptosis of A549 cells in those groups. The data between groups were compared by t test. Results: In ARDS rat model, PO(2) value was significantly increased in ARDS+ pcDNA-GAS5 group than that in ARDS+ pcDNA group[(81.5±3.3) vs (57.5±5.1) mmHg, t=4.850, P<0.05], and PCO(2) value was significantly decreased in ARDS+ pcDNA-GAS5 group than that in ARDS+ pcDNA group[(50.6±1.9) vs (64.0±1.9) mmHg, t=5.940, P<0.05]. LncRNA GAS5 level in A549 cells of LPS group decreased significantly than that in control group (0.43±0.01 vs 1.01±0.01, t=0.242, P<0.05). Compared with LPS+ pcDNA group, ACE2 expression increased significantly in LPS+ pcDNA-GAS5 group (0.85±0.04 vs 0.34±0.02, t=1.800, P<0.05). Compared with LPS+ pcDNA-GAS5+ pre-NC group, ACE2 expression decreased significantly in LPS+ pcDNA-GAS5+ miR-200c-3p mimic group (0.62±0.01 vs 0.84±0.02, t=9.440, P<0.05). Compared with control group, the percentage of A549 cell apoptosis promoted significantly in LPS group (25.90±0.61 vs 7.90±0.22, t=0.257, P<0.05). Compared with LPS+ pcDNA group, the percentage of A549 cell apoptosis suppressed significantly in LPS+ pcDNA-GAS5 group (10.50±0.37 vs 26.37±0.45, t=1.760, P<0.05). Compared with LPS+ pcDNA-GAS5+ pre-NC group, the percentage of A549 apoptosis promoted significantly in LPS+ pcDNA-GAS5+ miR-200c-3p mimic group (19.07±0.56 vs 10.87±0.26, t=0.643, P<0.05). Conclusion: In ARDS, down-regulation of lncRNA GAS5 decreases ACE2 expression through increasing miR-200c-3p to promote the apoptosis of A549 cells, thus to promote the progression of ARDS.
目的： 探究长链非编码RNA生长阻滞特异转录物5/微小RNA 200c－3p/血管紧张素转换酶2(lncRNA GAS5/miR－200c－3p/ACE2)信号轴是否参与呼吸窘迫综合征(ARDS)肺上皮细胞的凋亡。 方法： 构建ARDS大鼠模型，实验分为对照组(Control组)、LPS处理组(ARDS组)、LPS＋pcDNA处理组(ARDS＋pcDNA组)和LPS＋pcDNA－GAS5处理组(ARDS＋pcDNA－GAS5组)。ARDS大鼠构建6 h后收集4组大鼠动脉血及肺组织，血气分析仪进行测定氧分压(PaO(2))和二氧化碳分压(PaCO(2))，并观察GAS5的表达变化。随后，用LPS处理人肺泡Ⅱ型上皮细胞A549，分为未处理、LPS、LPS＋pcDNA、LPS＋pcDNA－GAS5、LPS＋pcDNA－GAS5＋pre－NC和LPS＋pcDNA－GAS5＋miR－200c－3p mimic组。实时荧光定量聚合酶链反应(qRT－PCR)检测lncRNA GAS5、ACE2和miR－200c－3p的表达水平；RNA免疫沉淀和RNA下拉(pull－down)实验用于检测GAS5与miR－200c－3p的结合与调控；Western blotting法检测ACE2的蛋白表达；流式细胞术用于检测A549细胞的凋亡。组间数据比较采用t检验。 结果： ARDS大鼠实验表明与ARDS＋pcDNA组相比，ARDS＋pcDNA－GAS5组PO(2)值显著升高[(81.5±3.3)比(57.5±5.1)mmHg，t＝4.850，P<0.05]，PCO(2)值显著降低[(50.6±1.9)比(64.0±1.9)mmHg，t＝5.940，P<0.05]。细胞实验中，与Control组相比，LPS组A549细胞中lncRNA GAS5的表达显著下降(0.43±0.01比1.01±0.01，t＝0.242，P<0.05)。与LPS＋pcDNA组相比，LPS＋pcDNA－GAS5组ACE2的表达水平显著升高(0.85±0.04比0.34±0.02，t＝1.800，P<0.05)；与LPS＋pcDNA－GAS5＋pre－NC组相比，LPS＋pcDNA－GAS5＋miR－200c－3p mimic组ACE2的表达水平显著降低(0.62±0.01比0.84±0.02，t＝9.440，P<0.05)。与未处理组相比，LPS组A549细胞凋亡百分比明显升高(25.90±0.61比7.90±0.22，t＝0.257，P<0.05)；与LPS＋pcDNA组相比，LPS＋pcDNA－GAS5组凋亡百分比明显降低(10.50±0.37比26.37±0.45，t＝1.760，P<0.05)；与LPS＋pcDNA－GAS5＋pre－NC组相比，LPS＋pcDNA－GAS5＋miR－200c－3p mimic组细胞凋亡百分比显著升高(19.07±0.56比10.87±0.26，t＝0.643，P<0.05)。 结论： ARDS模型中，lncRNA GAS5下调促进miR－200c－3p表达，使ACE2表达下降，从而使肺上皮细胞凋亡增加，促进ARDS进展。.

The recognition of the biological relevance of long non-coding RNA (lncRNA) molecules has only recently been recognized as one of the most significant advances in contemporary molecular biology. A growing body of evidence indicates that lncRNAs act not only as the intermediary between DNA and protein but also as significant protagonists of cellular functions. The dysregulation of lncRNAs has increasingly been linked to numerous human diseases, particularly cancers. Recent studies have demonstrated that the lncRNA growth arrest-specific transcript 5 (GAS5) was pervasively downexpressed in most human cancers compared with non-cancerous adjacent tissues including gastric, breast, lung and prostate cancer. In addition, patients with decreased GAS5 expression have a significantly poorer prognosis than those with higher expression. Furthermore, GAS5 is involved in the control of cell apoptosis, proliferation, metastasis, angiogenesis, DNA repair and tumor cell metabolism. This review provides an overview of the current knowledge concerning the role of GAS5 in tumor expression and biology function.

Effective control of hepatic stellate cell (HSC) activation and proliferation is critical to the treatment of liver fibrosis. Long non-coding RNAs have been shown to play a pivotal role in the regulation of cellular processes. It has been reported that growth arrest-specific transcript 5 (GAS5) acts as a crucial mediator in the control of cell proliferation and growth. However, little is known about the role and underlying mechanism of GAS5 in liver fibrosis. In this study, our results indicated that GAS5 expression was reduced in mouse, rat, and human fibrotic liver samples and in activated HSCs. Overexpression of GAS5 suppressed the activation of primary HSCs in vitro and alleviated the accumulation of collagen in fibrotic liver tissues in vivo. We identified GAS5 as a target of microRNA-222 (miR-222) and showed that miR-222 could inhibit the expression of GAS5. Interestingly, GAS5 could also repress miR-222 expression. A pulldown assay further validated that GAS5 could directly bind to miR-222. As a competing endogenous RNAs, GAS5 had no effect on primary miR-222 expression. In addition, GAS5 was mainly localized in the cytoplasm. Quantitative RT-PCR further demonstrated that the copy numbers of GAS5 per cell are higher than those of miR-222. GAS5 increased the level of p27 protein by functioning as a competing endogenous RNA for miR-222, thereby inhibiting the activation and proliferation of HSCs. Taken together, a new regulatory circuitry in liver fibrosis has been identified in which RNAs cross-talk by competing for shared microRNAs. Our findings may provide a new therapeutic strategy for liver fibrosis.