Growth Arrest-specific 5 Overexpression Research Articles

Long non‑coding RNA growth arrest‑specific 5 (GAS5) acts as a tumor suppressor by promoting autophagy in breast cancer.

Growth arrest-specific 5 (GAS5) is a known tumor suppressor which negatively regulates cell survival and malignancy in several cancer cell types. The present study aimed to establish the correlation between GAS5 and unc-51 like autophagy activating kinase (ULK)1/2, two key regulators of autophagy initiation in breast cancer (BC). To address this, expression levels of these genes were quantitively analyzed in BC clinical samples by performing reverse transcription-quantitative PCR. GAS5 was downregulated in BC clinical samples compared with adjacent samples and was positively correlated with ULK1/2. Detection methods including cell cycle analysis, annexin V-FITC/PI double staining and flow cytometry analysis, Transwell cell invasion assay, transfection and western blotting were used for BC cells. In MCF-7 cells, it was also observed that overexpression of GAS5 upregulated ULK1/2 protein levels without disturbing other autophagy initiation-associated proteins and inhibited cell proliferation, invasion and tumor formation. These effects were reversed by blocking autophagy with 3-methyladenine (3-MA). These results demonstrated that the suppressive effects of overexpressed GAS5 were mediated via autophagy induction, at least in part. Overexpression of GAS5 induced chemoresistance to cisplatin, which was not reversed by 3-MA-mediated inhibition of autophagy, indicating that GAS5 promotes chemosensitivity in an autophagy-independent manner. Collectively, these results indicated that GAS5 contributes to the pathogenesis of BC potentially by promoting autophagy. However, the mechanism by which GAS5 functions as a tumor suppressor in an autophagy-independent manner remains unknown.

LncRNA GAS5 participates in the regulation of dexamethasone on androgen receptor -negative and -positive prostate cancer cell proliferation

BackgroundAndrogen receptor (AR) and long non-coding RNAs (lncRNA) play important roles in the initiation and progression of prostate cancer (PCa). The present study was designed to investigate whether lncRNA growth arrest-specific 5 (GAS5) is involved in the regulation of dexamethasone on the proliferation of AR+ PCa and AR− PCa cell lines. MethodsCell proliferation and cell cycle distribution were assessed using MTT assay and flow cytometry, respectively. GAS5 expression was examined by quantitative real-time PCR. AR protein level was examined by Western blot. RNA immunoprecipitation and RNA pull-down were performed to analyze the binding of GAS5 to AR. ResultsIn AR− PCa cell line PC3, dexamethasone upregulated GAS5 expression, induced cell cycle arrest in the G0/G1 phase and inhibited cell proliferation, which were enhanced by GAS5 overexpression and attenuated by GAS5 silencing. However, in AR+ PCa cell line 22Rv1, dexamethasone had no obvious effects on GAS5 expression, cell cycle distribution and cell proliferation. AR was localized in the cytoplasm and bound to GAS5, counteracting the proliferation-inhibitory effect of GAS5. ConclusionTaken together, GAS5 participates in the regulation of dexamethasone on the proliferation of AR+ PCa and AR− PCa cell lines.

LncRNA GAS5 promotes apoptosis as a competing endogenous RNA for miR-21 via thrombospondin 1 in ischemic AKI

Mounting evidence has indicated that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) played important roles in renal ischemia/reperfusion (I/R) injury. However, the involvement of lncRNA growth arrest specific 5 (GAS5) in acute kidney injury (AKI) remained largely unexplored. This study aimed to determine possible mechanisms of GAS5 in the renal I/R process. We found that GAS5, noticeably upregulated by renal I/R injury, was further suppressed by delayed IPC while knockdown of miR-21 in vivo before IPC could significantly increased the GAS5 levels. Concurrently, TSP-1 was negatively regulated by miR-21 in vivo and vitro. Additionally, Reciprocal repression of GAS5 and miR-21 was identified. Knockdown of miR-21 in H6R0.5 treated HK-2 cells promoted apoptosis. Co-transfection of miR-21 mimic and pcDNA-GAS5 or pcDNA-Vector were performed, results of which showed that inhibition of miR-21 on TSP-1 could be rescued by overexpression of GAS5. This study suggested that GAS5 facilitated apoptosis by competitively sponging miR-21, which negatively regulated TSP-1 in renal I/R injury. This novel regulatory axis could act as a therapeutic target for AKI in the future.

The lncRNA-GAS5/miR-221-3p/DKK2 Axis Modulates ABCB1-Mediated Adriamycin Resistance of Breast Cancer via the Wnt/β-Catenin Signaling Pathway

Drug resistance, including adriamycin (ADR)-based therapeutic resistance, is a crucial cause of chemotherapy failure in breast cancer treatment. Acquired chemoresistance has been identified to be closely associated with the overexpression of P-glycoprotein (P-gp/ABCB1). Long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) can be involved in carcinogenesis; however, its roles in ABCB1-mediated ADR resistance are poorly understood. In this study, we identified a panel of differentially expressed lncRNAs, mRNAs, and microRNAs (miRNAs) in MCF-7 and MCF-7/ADR cell lines through RNA sequencing (RNA-seq) technologies. GAS5 level was downregulated whereas ABCB1 level was upregulated in the resistant breast cancer tissues and cells. Overexpression of GAS5 significantly enhanced the ADR sensitivity and apoptosis, and it inhibited the efflux function and expression of ABCB1 in vitro, while knockdown of GAS5 had the opposite effects. Further mechanism-related investigations indicated that GAS5 acted as an endogenous “sponge” by competing for miR-221-3p binding to regulate its target dickkopf 2 (DKK2), and then it inhibited the activation of the Wnt/β-catenin pathway. Functionally, GAS5 enhanced the anti-tumor effect of ADR in vivo. Collectively, our findings reveal that GAS5 exerted regulatory function in ADR resistance possibly through the miR-221-3p/DKK2 axis, providing a novel approach to develop promising therapeutic strategy for overcoming chemoresistance in breast cancer patients.

Retracted paper: The Long Noncoding RNA, Growth Arrest-Specific 5, Suppresses Gastric Cancer by Downregulating miR-21 Expression

Background: Gastric cancer has become the second major cause of cancer death. The aim of the present study was to explore the relationship between miR-21 and the long noncoding RNA growth arrest-specific 5 (GAS5) in gastric cancer and the effect on gastric cancer cells. Methods: The expression of miR-21 and GAS5 mRNA was analyzed by quantitative real-time-PCR. Overexpression of GAS5 was used to investigate the biological functions of GAS5 in cells. The cell proliferation was detected by cell counting kit-8 assay and the cell migration and invasion were detected by Transwell. Cell apoptosis was evaluated by Annexin V-FITC/PI staining and apoptosis-related proteins were detected by western blot. The mechanism of GAS5 in vivo was evaluated by the tumorigenesis of nude mice, and dual luciferase reporter was used to determine if miR-21 is a GAS5 target. The inhibition of miR-21 and the simultaneous overexpression of GAS5 and miR-21 were further performed, and the above indicators were detected again. Results: GAS5 was low expression and miR-21 was high expression in gastric cancer tissues and cells. GAS5 overexpression reduced the proliferation, migration, and invasion of gastric cancer cells and increased the apoptosis of gastric cancer cells. The growth rate of GAS5 group slowed down and the volume of tumor decreased. miR-21 is a GAS5 target and GAS5 inhibits the proliferation of gastric cancer cells by targeting miR-21. Conclusion: Our research shown that overexpression of GAS5 can significantly inhibit the proliferation, migration, invasion and tumor formation of gastric cancer cells, and promote the apoptosis of gastric cancer cells, which may be related to the targeting inhibition of miR-21 expression by GAS5.

LncRNA GAS5 suppresses inflammatory responses and apoptosis of alveolar epithelial cells by targeting miR-429/DUSP1

Acute lung injury (ALI) is a life-threatening syndrome characterized by excessive inflammation and apoptosis of alveolar epithelial cells. This study firstly investigated the role and mechanism of long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) in regulating lipopolysaccharide (LPS)-induced inflammatory response and apoptosis of murine alveolar epithelial cell line MLE-12. The expression of GAS5, miR-429, and dual-specificity phosphatase 1 (DUSP1) were examined using quantitative Real-Time PCR (qRT-PCR) and western blot. The inflammatory responses were evaluated by detecting the levels of pro-inflammatory cytokines using ELISA. Cell apoptosis was assessed by TUNEL assay. The interactions among GAS5, miR-429, and DUSP1 were examined using luciferase reporter assay. The results showed that GAS5 and DUSP1 expression were decreased, whereas miR-429 was increased in lung tissues from LPS-induced ALI mice and LPS-treated MLE-12 cells. Furthermore, GAS5 overexpression decreased cell inflammatory responses and apoptosis in LPS-treated MLE-12 cells, which was reversed by miR-429 mimic and DUSP1 knockdown. Mechanistically, GAS5 acted as a competitive endogenous RNA by sponging miR-429 to facilitate DUSP1 expression. Our findings suggest that GAS5 suppresses inflammatory responses and apoptosis of alveolar epithelial cell MLE-12 by targeting miR-429/DUSP1 axis.

GAS5/miR-21 Axis as a Potential Target to Rescue ZCL-082-Induced Autophagy of Female Germline Stem Cells In Vitro

Several studies have recently revealed the regulatory mechanisms underlying female germline stem cell (FGSC) differentiation, proliferation, and apoptosis, but other biological processes such as autophagy and its mechanism in FGSCs are largely unclear. The use of small chemical compounds may be a good approach to further investigate the process and mechanism of autophagy in FGSC development. In this study, we used ZCL-082, a derivative of benzoxaboroles, to treat FGSCs. Using a cell counting kit-8 (CCK8) and 5-ethynyl-2′-deoxyuridine (EdU) assays, we found that ZCL-082 could significantly reduce the viability, proliferation, and number of FGSCs in vitro. Moreover, western blotting revealed that the expression of light chain 3 beta 2 (LC3B-II) in FGSCs was significantly increased after treatment with ZCL-082 for 3 and 6 h. Meanwhile, the expression of sequestosome-1 (SQSTM1) was significantly decreased. These results suggested that ZCL-082 can induce autophagy of FGSCs in vitro. Regarding the molecular mechanism, ZCL-082 could significantly reduce the expression of growth arrest-specific 5 (GAS5) long non-coding RNA, which could directly bind to microRNA-21a (miR-21a) and negatively regulate each other in FGSCs. Knockdown of GAS5 induced the autophagy of FGSCs, while GAS5 overexpression inhibited the autophagy of FGSCs in vitro and rescued FGSC autophagy induced by ZCL-082. Additionally, overexpression of miR-21a significantly enhanced LC3B-II protein expression while significantly reducing the expression of programmed cell death protein 4 (PDCD4) and SQSTM1 protein in FGSCs compared with control cells. The inhibition of miR-21a significantly reduced the basal or ZCL-082-induced upregulated expression of LC3B-II, and it significantly enhanced the expression of PDCD4 while downregulating the basal or ZCL-082-induced expression of SQSTM1 in FGSCs. Furthermore, the overexpression of GAS5 enhanced the protein expression of PDCD4, but knockdown of GAS5 reduced the protein expression of PDCD4. Taken together, these results suggested that ZCL-082 induced autophagy through GAS5 functioning as a competing endogenous RNA (ceRNA) sponge for miR-21a in FGSCs. It also suggested that the GAS5/miR-21a axis may be a potential therapeutic target for premature ovarian failure in the clinic.

RETRACTED: lncRNA GAS5 Inhibits Cell Migration and Invasion and Promotes Autophagy by Targeting miR-222-3p via the GAS5/PTEN-Signaling Pathway in CRC

Colorectal cancer (CRC) is a frequently occurring lethal disorder with heterogeneous outcomes and drug responses. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) play a critical role in carcinogenesis. Hence, the aim of this study was to investigate the role of lncRNA growth arrest-specific 5 (GAS5) in CRC cells via mediation of the microRNA-222-3p (miR-222-3p)/GAS5/phosphatase and tensin homolog (PTEN)-signaling pathway. HCT116 and SW480 cells were collected and treated with small interfering (si)-lncRNA GAS5, overexpressing (oe)-lncRNA GAS5, miR-222-3p mimic, miR-222-3p inhibitor, or si-lncRNA GAS5 + miR-222-3p mimic. The miR-222-3p level and mRNA and protein levels of GAS5, Beclin1, light-chain 3B (LC3B), PTEN, and Akt were detected. Besides, cell migration, invasion, and apoptosis as well as acidic vesicular organelles (AVOs) were examined respectively. Xenografts in nude mice were also performed to detect tumorigenesis in vivo. Results suggested that the downregulation of lncRNA GAS5 decreased the expressions of Beclin1, LC3B, and PTEN. When treated with oe-lncRNA GAS5 or miR-222-3p inhibitor, HCT116 and SW480 cells exhibited suppressed invasion and migration abilities and increased apoptotic cells and autophagosome and AVO activities. Moreover, overexpression of GAS5 inhibited the tumorigenesis of CRC cells in vivo. Taken together, lncRNA GAS5 upregulated the expression of PTEN by functioning as a competing endogenous RNA (ceRNA) of miR-222-3p, thus inhibiting CRC cell migration and invasion and promoting cell autophagy.

MicroRNA-145 inhibits proliferation and induces apoptosis in human prostate carcinoma by upregulating long non-coding RNA GAS5.

MicroRNA-145 (miR-145) and long non-coding RNA (lncRNA) growth arrest specific 5 (GAS5) function as tumor suppressors in prostate carcinoma. The aim of the present study was to investigate the role of miR-145 and lncRNA GAS5 in prostate carcinoma. In the present study, miR-145 and lncRNA GAS5 expression levels were demonstrated to be downregulated in tumor tissues compared with adjacent healthy tissues of patients with prostate carcinoma. miR-145 and lncRNA GAS5 expression levels were found to be positively and significantly correlated in tumor tissues, but not in adjacent healthy tissues. A follow-up study revealed that low miR-145 and lncRNA GAS5 expression levels were associated with poor survival. Overexpression of miR-145 resulted in upregulated lncRNA GAS5, whereas lncRNA GAS5 overexpression or silencing did not affect miR-145 expression. Overexpression of miR-145 and lncRNA GAS5 promoted apoptosis and inhibited cell proliferation in prostate carcinoma cell lines, whereas lncRNA GAS5 knockdown had an opposite effect. In addition, lncRNA GAS5 knockdown partially attenuated the effect of miR-145 overexpression of cancer cell proliferation and apoptosis. Therefore, miR-145 may inhibit cell proliferation and induce apoptosis in human prostate carcinoma by upregulating lncRNA GAS5.

Low Long Noncoding RNA Growth Arrest-Specific Transcript 5 Expression in the Exosomes of Lung Cancer Cells Promotes Tumor Angiogenesis.

Angiogenesis plays a key role in the development and progression of lung cancer. Recent studies have found that tumor cells can stimulate angiogenesis by secreting exosomes, which contain many long noncoding RNAs (lncRNAs), some of which are important for the development of lung cancer. However, the roles and mechanisms of exosomal lncRNAs in lung cancer angiogenesis have not yet been reported. In this study, lung cancer in mice was induced by urethane; we found that growth arrest specific 5 (GAS5) was lowly expressed in the serum exosomes and lung cancer tissues of mice with lung cancer. And there was a significant positive correlation between GAS5 expression in serum exosomes and lung cancer tissues. Furthermore, GAS5 was lowly expressed in human lung cancer tissues, lung cancer cells, and cells culture supernatant exosomes. The exosomes of lung cancer cells promoted human umbilical vein endothelial cells (HUVECs) proliferation and tube formation and inhibited their apoptosis. GAS5 overexpression in lung cancer cells increased GAS5 level in cell culture supernatant exosomes. And the exosomes of lung cancer cells containing high GAS5 level inhibited HUVECs proliferation and tube formation and increased their apoptosis. In addition, we found that GAS5 competitively bound miRNA-29-3p with phosphatase and tensin homolog (PTEN), upregulating PTEN mRNA and protein expression, and inhibited level of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3K) and serine/threonine kinase 1 (AKT) phosphorylation in HUVECs. Overall, our results suggest that exosomal GAS5 could be a new therapeutic target for lung cancer which inhibits angiogenesis.

Overexpression of lncRNA GAS5 attenuates cardiac fibrosis through regulating PTEN/MMP-2 signal pathway in mice.

The present study aimed at investigating the effect and mechanism of lncRNA Growth Arrest-Specific 5 (GAS5) in cardiac fibrosis induced by isoproterenol (ISO) in vivo. The C57BL/6 mice were injected subcutaneously with ISO to induce cardiac fibrosis and injected intracoronary with lentivirus pcDNA-GAS5. After 3 weeks, cardiac function was detected by echocardiography. The interstitial collagen volume was stained by Masson trichrome. The expression of GAS5 was measured by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The expressions of phosphatase and tensin homologue (PTEN), matrix metalloprotease-2 (MMP-2), α-smooth muscle actin (α-SMA), and collagen I protein were measured by Western blot. Our results indicated that the expression of GAS5 was significantly down-regulated in the fibrotic myocardium. Overexpression of GAS5 after injection with pcDNA-GAS5 could attenuate cardiac fibrosis and improve cardiac function through increasing the expression of PTEN and decreasing the expression of MMP-2, α-SMA, and collagen I. Overexpression of GAS5 could attenuate cardiac fibrosis induced by ISO. The molecular mechanism was associated with the regulation of PTEN/MMP-2 signaling pathway.

Effect of long non-coding RNA growth arrest-specific 5 on apoptosis in renal ischaemia/reperfusion injury.

Long non-coding RNA (lncRNAs) have been shown to play a critical role in a variety of pathophysiological processes, such as cell proliferation, apoptosis and migration. However, there were few studies addressing the function of lncRNAs in renal ischaemia/reperfusion (I/R) injury. Apoptosis is an important pathogenesis during I/R injury. Here, we identified the effect of hypoxia-responsive lncRNA growth arrest-specific 5 (GAS5) on apoptosis in renal I/R injury. Ischaemia/reperfusion injury in mice or hypoxia/re-oxygenation (H/R) in human proximal renal tubular epithelial cells (HK-2) was practiced to induce apoptosis. The kidneys and blood were collected at 24 h after reperfusion. The GAS5 messenger RNA (mRNA) expression and apoptosis-related gene mRNA and protein levels, including p53, cellular inhibitor of apoptosis protein 2 (cIAP2) and thrombospondin-1 (TSP-1), were analysed. GAS5 small-interfering RNA was transfected with H/R induced cells. Over-expression of GAS5 was performed by plasmid transfection. Apoptotic cells significantly increased in I/R-injured kidneys. GAS5 could be up-regulated in kidneys at 24 h after reperfusion and 3 h after re-oxygenation, combined with increased expression of its downstream apoptosis-related proteins p53 and cIAP2. GAS5 small-interfering RNA treatment down-regulated the mRNA and protein levels of p53 and TSP-1, and attenuated apoptosis induced by H/R in HK-2 cells. Conversely, over-expression of GAS5 up-regulated the mRNA and protein levels of p53 and TSP-1, and promoted apoptosis in HK-2 cells. Long non-coding RNA GAS5 induced by I/R injury could promote apoptosis in kidney. TSP-1 might be one of the downstream effectors of GAS5, which will be explored in the future.

Evidence for long noncoding RNA GAS5 up-regulationin patients with Klinefelter syndrome

BackgroundKlinefelter syndrome (KS) is characterized by the presence of at least one supernumerary X chromosome. KS typical symptoms include tall stature, gynecomastia, hypogonadism and azoospermia. KS patients show a higher risk of developing metabolic and cardiovascular diseases, inflammatory and autoimmune disorders, osteoporosis and cancer. Long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) has been shown to be involved in several biologic processes, including inflammatory and autoimmune diseases, vascular endothelial cells apoptosis and atherosclerosis, as well as cellular growth and proliferation, cellular development and cell-to-cell signaling and interaction. The lncRNA GAS5 expression profile in KS patients has never been evaluated so far.MethodsTo accomplish this, GAS5 mRNA levels were evaluated by Next Generation Sequencing (NGS) technology and qRT-PCR assay in 10 patients with KS and 10 age-matched controls.ResultsNGS results showed a significantly lncRNAGAS5up-regulation by 5.171-fold in patients with KS. Theresults of qRT-PCR confirmed the NGS data.ConclusionsThese findings showed the occurrence of lncRNA GAS5 over-expression in KS patients. Whether this lncRNA is involved in the pathogenesis of inflammation and autoimmune diseases, atherogenesis or germ cell depletion in KS patients is not known. Further studies are needed.

The CtBP1-HDAC1/2-IRF1 transcriptional complex represses the expression of the long noncoding RNA GAS5 in human osteosarcoma cells.

Growth Arrest Specific 5 (GAS5), a long noncoding RNA (lncRNA), functions as a tumor suppressor in multiple cancers. However, its function, downstream targets and upstream regulatory mechanism are still obscure in osteosarcoma cells. Here, we discovered that GAS5 was downregulated in cancerous osteosarcoma tissues and cells. Using a microarray analysis, we identified that GAS5 can regulate the expression of TP53, Bax, Bim, DDB2, TGFB and ROS1 in osteosarcoma cells. Specifically, GAS5 overexpression in the U2OS osteosarcoma cell line induced TP53, Bax and Bim levels but inhibited DDB2, TGFB and ROS1 expression, resulting in the inhibition of cell proliferation, invasion, colony formation and in vivo tumor formation. By analyzing the GAS5 promoter region (-2000), we identified several potential transcription factor-binding sites including NF-ĸB, IK-1, AP-1, SP1 and IRF1. By individually knocking down these transcription factors, we found that only knockdown of IRF1 affected GAS5 expression. Using immunoprecipitation (IP), mass spectrometry assays, and co-IP assays, we identified that IRF1 formed a transcriptional complex with Histone Deacetylase 1 and 2 (HDAC1/2) and C-terminal binding protein 1 (CtBP1). Functional analyses indicated that the CtBP1-HDAC1/2-IRF1 complex specifically bound to the GAS5 promoter and regulated its expression and downstream events. Knockdown of CtBP1 or overexpression of IRF1 in osteosarcoma cells can significantly reverse their oncogenic phenotypes. Altogether, our results indicated that the CtBP1-HDAC1/2-IRF1 transcriptional complex inhibited GAS5-mediated signaling in osteosarcoma cells, and it might be a potential therapeutic target for osteosarcoma treatment.

Long Non-Coding RNA (lncRNA) Growth Arrest Specific 5 (GAS5) Suppresses Esophageal Squamous Cell Carcinoma Cell Proliferation and Migration by Inactivating Phosphatidylinositol 3-kinase (PI3K)/AKT/Mammalian Target of Rapamycin (mTOR) Signaling Pathway.

BackgroundlncRNA GAS5 acts as a tumor-suppressor gene in various types of malignancies, but its involvement in esophageal cancer has not been well studied.Material/MethodsA total of 112 patients with esophageal cancer and 55 volunteers with normal physiological conditions were included in this study. Tumor tissues and adjacent healthy tissues were collected from esophageal cancer patients and blood was extracted from patients and controls. Expression of GAS5 in those tissues was detected by qRT-PCR. All patients were followed up for 5 years and diagnostic and prognostic values of serum GAS5 for esophageal cancer were investigated by ROC curve analysis and survival curve analysis, respectively. Effects of GAS5 expression on cell proliferation and migration were investigated by CCK-8 assay and Transwell cell migration assay, respectively. Effects of GAS5 overexpression on expression of PI3K/AKT/mTOR-related proteins were explored by Western blot analysis.ResultsGAS5 expression level was lower in tumor tissues than in adjacent healthy tissues. Serum level of GAS5 was lower in cancer patients than in healthy controls, and serum level of GAS5 was decreased with increase in stage of primary tumor (T stage). GAS5 overexpression inhibited tumor cell proliferation and migration, while treatment with PI3K activator reduced the inhibitory effects. GAS5 overexpression decreased the expression level of PI3K and phosphorylation levels of Akt and mTOR in esophageal cancer cells, while PI3K activator treatment showed no significant effects on GAS5 expression.ConclusionsGAS5 was downregulated in esophageal cancer patients compared to healthy controls, and GAS5 overexpression suppressed proliferation and migration of esophageal cancer cells by inactivating the PI3K/AKT/mTOR pathway.

Growth arrest-specific 5 attenuates cisplatin-induced apoptosis in cervical cancer by regulating STAT3 signaling via miR-21.

Cervical cancer is the most common cause of female cancer-related mortality worldwide. Decreased expression of long noncoding RNA growth arrest-specific 5 (GAS5) is found in human cervical cancer tissues and associated with poor prognosis. However, the studies on associations between GAS5 level and malignant phenotypes, as well as sensitivity to chemotherapeutic drug in cervical cancer cells are limited. In this study, overexpression of GAS5 in cervical cancer cells resulted in prohibited cell proliferation and colony formation, which were promoted by siGAS5. Enhanced GAS5 increased cell percentage in the G0/G1 phase and decreased cells percentage in the S phase, whereas reduced expression did not. The malignant behaviors of cervical cancer cells, manifested by cell migration and invasion, could be weakened by the GAS5 overexpression and enhanced by siGAS5. Furthermore, in cisplatin-induced cell, overexpression of GAS5 reduced cells viability and enhanced apoptosis, whereas in cells transfected with siGAS5, apoptosis eliminated. We have reported the upregulation of microRNA-21 (miR-21) and its oncogenetic roles in cervical cancer previously. In this study, we found the negative relationship between the GAS5 and miR-21. Moreover, the decrease of miR-21 associated proteins phosphorylated STAT3 and E2F3 was seen in GAS5 overexpressed cells, both of which could be increased by siGAS5. The GAS5 deficiency also reduced miR-21 target proteins TIMP3 and PDCD4 expressions. Taken together, the GAS5 expression level is inversely associated with malignancy, but positively associated with sensitivity to cisplatin-induced apoptosis, suggesting that GAS5 could be a biomarker of cisplatin-resistance in clinical therapy of human cervical cancer.

Long noncoding RNA growth arrest-specific 5 facilitates glioma cell sensitivity to cisplatin by suppressing excessive autophagy in an mTOR-dependent manner.

Malignant glioma is a severe type of brain tumor with a grim prognosis. The occurrence of resistance compromises the efficacy of chemotherapy for glioma. Long noncoding RNA growth arrest-specific 5 (GAS5) has recently become an attractive target for cancer therapy by regulating cell growth, invasion, and migration. Nevertheless, its role in glioma chemoresistance remains elusive. In the current study, the expression of GAS5 was decreased in glioma cell lines, and lower levels of GAS5 were observed in U138 and LN18 glioma cells that had low sensitivity to cisplatin. Functional assay confirmed that knockdown of GAS5 enhanced cell resistance to cisplatin in U87 cells, which had a relatively high expression of GAS5. Conversely, elevation of GAS5 increased cell sensitivity to cisplatin in U138 cells that had a relatively low expression of GAS5. Mechanistically, cisplatin exposure evoked excessive autophagy concomitant with an increase in autophagy-related LC3II expression and a decrease in autophagy substrate p62 expression, which was reversely muted after GAS5 overexpression. In addition, GAS5 restored cisplatin-inhibited mammalian target of rapamycin (mTOR) activation. Preconditioning with mTOR antagonist rapamycin engendered not only mTOR inhibition but also abrogated GAS5-mediated depression in cisplatin-evoked autophagy. Notably, blocking the mTOR pathway also attenuated GAS5-increased sensitivity to cisplatin in U138 cells. Cumulatively, these findings indicate that GAS5 may blunt the resistance of glioma cells to cisplatin by suppressing excessive autophagy through the activation of mTOR signaling, implying a promising therapeutic strategy against chemoresistance in glioma.

LncRNA GAS5 Reverses EMT and Tumor Stem Cell-Mediated Gemcitabine Resistance and Metastasis by Targeting miR-221/SOCS3 in Pancreatic Cancer

Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) mediating chemotherapeutic drug effects and metastasis in pancreatic cancer (PC) are key reasons for the poor prognosis of this disease. lncRNA growth arrest-specific 5 (GAS5) is reported to be a tumor suppressor in multiple cancers. However, the functions of GAS5 and its related miRNAs in PC are poorly understood. This study explored the potential functions and mechanisms of GAS5 in PC gemcitabine resistance and metastasis. The results show that overexpression of GAS5 suppressed the proliferation, migration, gemcitabine resistance, stem cell-like properties, and epithelial-mesenchymal transition (EMT) of PC cells by directly binding to and suppressing miR-221 expression and enhancing suppressor of cytokine signaling 3 (SOCS3) expression. The effects of miR-221 overexpression on proliferation, migration, gemcitabine resistance, stem cell-like properties, and EMT inhibition were reversed by SOCS3 overexpression in PC cells. Additionally, GAS5 promoted gemcitabine-induced tumor growth and metastasis inhibition, as determined by Ki-67 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), bioluminescence imaging, and the detection of cell-like properties and EMT in vivo. Thus, lncRNA GAS5 functioned as a competing endogenous RNA for miR-221, and it suppressed cell growth, metastasis, and gemcitabine resistance in PC by regulating the miR-221/SOCS3 pathway mediating EMT and tumor stem cell self-renewal.

Long noncoding RNA GAS5 regulates the proliferation, migration, and invasion of glioma cells by negatively regulating miR-18a-5p.

Long noncoding RNAs, transcribed from a recently discovered class of noncoding genes, may play a critical role in regulating cellular processes, such as cell proliferation and apoptosis, as well as in cancer progression and metastasis. We previously detected the induction of growth arrest-specific 5 (GAS5) during glioma cell death. However, the function and underlying mechanism of GAS5 in human gliomas remain to be elucidated. Cell proliferation was detected using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) and tumorigenicity assay in nude mice. Wound-healing assay and transwell assay were utilized to examine the effects of GAS5 expression on glioma cells migration and invasion. In situ hybridization (ISH) was performed to evaluate GAS5 and microRNA (miR)-18a-5p levels in tissue microarrays. The relationship between GAS5 and miR-18a-5p was evaluated by quantitative reverse-transcription polymerase chain reaction and RNA precipitation. In this study, we demonstrated that overexpression of GAS5 inhibits malignant phenotypes in glioma cells, including proliferation, migration, and invasion, whereas GAS5 knockdown enhances these phenotypes. We further observed Argonaute 2-dependent reciprocal repression between GAS5 and miR-18a-5p in glioma cells. Downregulation of GAS5 and upregulation of miR-18a-5p were observed in glioma tissue microarrays relative to normal brain tissue by ISH. By deletion analysis, we identified one miR-18a-5p-binding site within exon 2 of GAS5 that is partially responsible for the tumor-suppressor functions of GAS5. Taken together, our findings suggest that GAS5 is a tumor suppressor in human gliomas that acts in part by repressing miR-18a-5p.

High expression of GAS5 promotes neuronal death after cerebral infarction by regulating miR-365a-3p.

To investigate whether growth arrest specific5 (GAS5) could regulate the expression of DCDC2 through competitive binding to miR-365a-3p, thus leading to increased neuronal death in cerebral cortex. The expression levels of GAS5 and DCDC2 in cerebral cortical neurons of mice with mouse middle cerebral artery occlusion (MCAO) cerebral infarction and in control mice were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Meanwhile, the expression levels of GAS5 and DCDC2 in primary neurons cultured in vitro were also detected by qRT-PCR. The effects of GAS5 and DCDC2 on neuronal death were evaluated by calculating the cerebral infarct area by Tunel assay and TTC staining. Dual luciferase reporter assays were performed to detect the binding of miR-365a-3p to GAS5 and DCDC2. Western blot was applied to detect the protein expression of DCDC2, Bcl-2 and Bax after overexpression and knockdown of GAS5. The expression of GAS5 and DCDC2 were significantly higher in cerebral cortical neurons and primary cultured neurons in vitro than those in control mice, respectively. Inhibition of GAS5 and DCDC2 in primary neurons decreased the neuronal cells death rate, while overexpression of GAS5 and DCDC2 increased the cell death rate. The dual luciferase reporter gene results showed that GAS5 regulated the expression of DCDC2 through competitive binding of miR-365a-3p thus forming a GAS5/miR-365a-3p/DCDC2 regulatory network. In addition, GAS5 inhibited Bcl-2 while promoting the expression of Bax. High expression of GAS5 could promote neuronal death after cerebral infarction in mice, possibly through competitive binding to miR-365a-3p and promoting the expression of DCDC2.