LncRNA GAS5/miR-137 Is a Hypoxia-Responsive Axis Involved in Cardiac Arrest and Cardiopulmonary Cerebral Resuscitation

BackgroundCardiac arrest/cardiopulmonary resuscitation (CA/CPR) represents one of the devastating medical emergencies and is associated with high mortality and neuro-disability. Post-cardiac arrest syndrome (PCAS) is mechanistically ascribed to acute systemic ischemia/reperfusion(I/R) injury. The lncRNA/microRNA/mRNA networks have been found to play crucial roles in the pathogenesis of the hypoxia-responsive diseases. Nonetheless, the precise molecular mechanisms by which lncRNA/miRNA/mRNA axes are involved in the astrocyte–microglia crosstalk in CA/CPR have not been fully elucidated.MethodsWe collected and purified the exosomes from the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. On the basis of microarray analysis, bioinformatic study, and luciferase activity determination, we speculated that lncRNA GAS5/miR-137 is implicated in the astrocyte–microglia crosstalk under the insult of systemic I/R injury. The regulation of lncRNA GAS5/miR-137 on INPP4B was examined by cellular transfection in OGD/R cell culture and by lateral ventricle injection with miR-137 agomir in CA/CPR mice model. Flow cytometry and immunofluorescence staining were performed to detect the microglial apoptosis, M1/M2 phenotype transformation, and neuroinflammation. Neurological scoring and behavior tests were conducted in CA/CPR group, with miR-137 agomir lateral-ventricle infusion and in their controls.ResultsIn all the micRNAs, miR-137 was among the top 10 micRNAs that experienced greatest changes, in both the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. Bioinformatic analysis revealed that miR-137 was sponged by lncRNA GAS5, targeting INPP4B, and the result was confirmed by Luciferase activity assay. qRT-PCR and Western blotting showed that lncRNA GAS5 and INPP4B were over-expressed whereas miR-137 was downregulated in the blood of CA/CPR patients, OGD/R-stimulated astrocytes, and brain tissue of CA/CPR mice. Silencing lncRNA GAS5 suppressed INPP4B expression, but over-expression of miR-137 negatively modulated its expression. Western blotting exhibited that PI3K and Akt phosphorylation was increased when lncRNA GAS5 was silenced or miR-137 was over-expressed. However, PI3K and Akt phosphorylation was notably suppressed in the absence of miR-137, almost reversing their phosphorylation in the silencing lncRNA GAS5 group. Then we found that GAS5 siRNA or miR-137 mimic significantly increased cell viability and alleviated apoptosis after OGD/R injury. Furthermore, over-expression of miR-137 attenuated microglial apoptosis and neuroinflammation in CA/CPR mice model, exhibiting significantly better behavioral tests after CA/CPR.ConclusionLncRNA GAS5/miR-137 may be involved in the astrocyte–microglia communication that inhibits PI3K/Akt signaling activation via regulation of INPP4B during CA/CPR.

lncRNA GAS5 Sensitizes Breast Cancer Cells to Ionizing Radiation by Inhibiting DNA Repair

Radioresistance of breast cancer is a major reason for therapeutic failure and limits further increases in the dose of radiation due to severe adverse effects. Recently, long noncoding RNAs (lncRNAs) have been shown to regulate cancer proliferation, chemoresistance, and radioresistance. Among these lncRNAs, lncRNA GAS5 expression was shown to be downregulated in breast cancer and related to trastuzumab resistance. However, its role in the radiation response is unclear. In this study, we demonstrated that lncRNA GAS5 expression was reduced in irradiated cells and that overexpression of GAS5 reduced cell viability and promoted cell apoptosis after irradiation. Moreover, overexpression of GAS5 resulted in increased G2/M arrest and unrepaired DNA damage, indicating a radiosensitizing role of GAS5 in breast cancer cells. Finally, we found that a GAS5-interacting miRNA, miR-21, reversed the radiosensitizing effects of GAS5 by inhibiting the apoptotic pathway. In conclusion, we found that lncRNA GAS5 sensitized breast cancer cells to ionizing radiation by inhibiting DNA repair and suppressing miR-21, identifying novel targets for breast cancer radiosensitization.

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.

Expression patterns of LncRNA-GAS5 and its target APOBEC3C gene through miR-103 in breast cancer patients

Early diagnosis of breast cancer can increase the survivability of the patients and the patient’s quality of life. There is growing evidence demonstrating the active role of LncRNA-GAS5 and miR-103 in cancer biology. APOBEC enzymes are important players in immunity and may contribute to carcinogenesis. Mutation and expression alteration in the APOBEC gene family was found to have a strong correlation with breast cancer risk. This study aimed to evaluate the expression level of lncRNA-GAS5 and its target APOBEC3C in women with breast cancer through expression evaluation of miR-103. Moreover, the interaction between lncRNA-GAS5 and miR-103 was studied. In the present study, forty paired tumor and normal samples classified based on breast cancer subtypes and clinical features of patients were analyzed using gene expression studies. Immunohistochemical analysis of the gene products was performed to classify tumors. The RNA samples were extracted from breast tissue. Real-time PCR was conducted for APOBEC3C and Lnc-RNA GAS5 expression. In addition, miR-103a miScript Primer Assay was utilized for the expression of miR-103-5p. It was revealed that the expression level of APOBEC3C and lncRNA-GAS5 were significantly down-regulated; however, the miRNA-103 expression level was significantly up-regulated. GAS5 expression was positively correlated with APOBEC3C expression and negatively correlated with miR-103 expression. In conclusion, we observed down-regulation of APOBEC3C and LncRNA-GAS5 and up-regulation of miRNA 103 in breast cancer patients. The expression of GAS5 may provide a new potential treatment target for breast cancer. To clarify the role of these molecules in the cellular signaling pathways, further studies are required.

LncRNA GAS5 participates in childhood pneumonia by inhibiting cell apoptosis and promoting SHIP-1 expression via downregulating miR-155

Background LncRNA GAS5 and miR-155 are reported to play opposite roles in lung inflammatory responses. Lung inflammation participates in childhood pneumonia, indicating the involvement of GAS5 and miR-155 in pneumonia. The study aimed to analyze the potential interaction between GAS5 and miR-155 in childhood pneumonia. Methods GAS5 and miR-155 levels in plasma samples from pneumonia patients and controls were detected using RT-qPCR. The role of GAS5 in miR-155 RNA gene methylation in human bronchial epithelial cells (HBEpCs) was analyzed by methylation analysis. Flow cytometry and RT-qPCR were applied to analyze cell apoptosis and SHIP-1 expression, respectively. Results GAS5 was downregulated in pneumonia, and miR-155 was upregulated in pneumonia. GAS5 and miR-155 were inversely correlated. GAS5 overexpression decreased miR-155 expression in HBEpCs, while miR-155 overexpression showed no significant effects on GAS5 expression. In addition, GAS5 suppressed LPS-induced HBEpC apoptosis, promoted SHIP-1 expression, and reduced the enhancing effect of miR-155 on cell apoptosis and SHIP-1 expression. Conclusions GAS5 may participate in childhood pneumonia by inhibiting cell apoptosis and promoting SHIP-1 expression via downregulating miR-155.

Long noncoding RNA GAS5 in age-related diseases

: Aging refers to a natural process and a universal phenomenon in all cells, tissues, organs and the whole organism. Long non-coding RNAs (lncRNAs) are non-coding RNAs with the length of 200 nucleotides. LncRNA growth arrest-specific 5 (lncRNA GAS5) is often down-regulated in cancer. The accumulation of lncRNA GAS5 has been found to be able to inhibit cancer growth, invasion and metastasis, while enhancing the sensitivity of cells to chemotherapy drugs. LncRNA GAS5 can be a signaling protein, which is specifically transcribed under different triggering conditions. Subsequently, it is involved in signal transmission in numerous pathways as a signal node. LncRNA GAS5, with a close relationship to multiple miRNAs, was suggested to be involved in the signaling pathway under three action modes (i.e., signal, bait and guidance). LncRNA GAS5 was found to be involved in different age-related diseases (e.g., rheumatoid arthritis, type 2 diabetes, atherosclerosis, osteoarthritis, osteoporosis, multiple sclerosis, cancer etc.). This study mainly summarized the regulatory effect exerted by lncRNA GAS5 on age-related diseases.