LncRNA TUG1 attenuates ischaemia-reperfusion-induced apoptosis of renal tubular epithelial cells by sponging miR-144-3p via targeting Nrf2

Renal ischaemia/reperfusion (I/R) injury may induce kidney damage and dysfunction, in which oxidative stress and apoptosis play important roles. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are reported to be closely related to renal I/R, but the specific molecular mechanism is still unclear. The purpose of this research was to explore the regulatory effect of lncRNA TUG1 on oxidative stress and apoptosis in renal I/R injury. This research revealed that in renal I/R injury and hypoxia/reperfusion (H/R) injury in vitro, the expression level of lncRNA TUG1 was upregulated, and oxidative stress levels and apoptosis levels were negatively correlated with the expression level of lncRNA TUG1. Using bioinformatics databases such as TargetScan and microRNA.org, microRNA-144-3p (miR-144-3p) was predicted to be involved in the association between lncRNA TUG1 and Nrf2. This study confirmed that the level of miR-144-3p was significantly reduced following renal I/R injury and H/R injury in vitro, and miR-144-3p was determined to target Nrf2 and inhibit its expression. In addition, lncRNA TUG1 can reduce the inhibitory effect of miR-144-3p on Nrf2 by sponging miR-144-3p. In summary, our research shows that lncRNA TUG1 regulates oxidative stress and apoptosis during renal I/R injury through the miR-144-3p/Nrf2 axis, which may be a new treatment target for renal I/R injury.     

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress,mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1^S616, mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.     

RhoA modulates functional and physical interaction between ROCK1 and Erk1/2 in selenite-induced apoptosis of leukaemia cells

RhoA GTPase dysregulation is frequently reported in various tumours and haematologic malignancies. RhoA, regulating Rho-associated coiled-coil-forming kinase 1 (ROCK1), modulates multiple cell functions, including malignant transformation, metastasis and cell death. Therefore, RhoA/ROCK1 could be an ideal candidate target in cancer treatment. However, the roles of RhoA/ROCK1 axis in apoptosis of leukaemia cells remain elusive. In this study, we explored the effects of RhoA/ROCK1 cascade on selenite-induced apoptosis of leukaemia cells and the underlying mechanism. We found selenite deactivated RhoA/ROCK1 and decreased the association between RhoA and ROCK1 in leukaemia NB4 and Jurkat cells. The inhibited RhoA/ROCK1 signalling enhanced the phosphorylation of Erk1/2 in a Mek1/2-independent manner. Erk1/2 promoted apoptosis of leukaemia cells after it was activated. Intriguingly, it was shown that both RhoA and ROCK1 were present in the multimolecular complex containing Erk1/2. GST pull-down analysis showed ROCK1 had a direct interaction with GST-Erk2. In addition, selenite-induced apoptosis in an NB4 xenograft model was also found to be associated with the RhoA/ROCK1/Erk1/2 pathway. Our data demonstrate that the RhoA/ROCK1 signalling pathway has important roles in the determination of cell fates and the modulation of Erk1/2 activity at the Mek–Erk interplay level.     

Retracted: Deletion of long noncoding RNA EFNA3 aggravates hypoxia-induced injury in PC-12 cells by upregulation of miR-101a

Long noncoding RNAs (lncRNAs) have been reported to be involved in several neurological pathogenesis conditions including cerebral ischemia. In the current study, the functions of lncRNA EFNA3 on hypoxia-injured rat adrenal pheochromocytoma (PC-12) cells and the underlying molecular mechanism were studied. The expression of lncRNA EFNA3 was silenced by short hairpin RNA transfection, after which the cells were subjected with hypoxia. Cell viability, migration, invasion, and apoptosis were, respectively, determined by trypan blue staining, Transwell assay, annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining, and Western blot analysis. The cross regulation between lncRNA EFNA3 and miR-101a, as well as between miR-101a and Rho associated coiled-coil containing protein kinase 2 (ROCK2) were detected by performing quantitative real-time polymerase chain reaction, RNA pull-down, RNA immunoprecipitation, luciferase activity assay, and Western blot analysis. Studies showed that lncRNA EFNA3 was highly expressed in response to hypoxia. Deletion of lncRNA EFNA3 significantly aggravated hypoxia-induced injury in PC-12 cells, as the impairment of cell viability, migration, and invasion, and the inducement of apoptosis. LncRNA EFNA3 worked as a sponging molecule for miR-101a and miR-101a suppression-protected PC-12 cells against hypoxia-induced injury even when lncRNA EFNA3 was silenced. ROCK2 was a target gene of miR-101a. ROCK2 overexpression exhibited neuroprotective activities. Besides, ROCK2 overexpression activated Wnt/β-catenin pathway whereas it deactivated JAK/STAT pathway upon hypoxia. Our study suggests that deletion of lncRNA EFNA3 aggravates hypoxia-induced injury in PC-12 cells by upregulating miR-101a, which further targets ROCK2.     

Fluid shear stress regulates osteoblast proliferation and apoptosis via the lncRNA TUG1/miR-34a/FGFR1 axis

LncRNAs and microRNAs play critical roles in osteoblast differentiation and bone formation. However, their exact roles in osteoblasts under fluid shear stress (FSS) and the possible mechanisms remain unclear. The aim of this study was to explore whether and how miR-34a regulates osteoblast proliferation and apoptosis under FSS. In this study, FSS down-regulated miR-34a levels of MC3T3-E1 cells. MiR-34a up-regulation attenuated FSS-induced promotion of proliferation and suppression of apoptosis. Luciferase reporter assay revealed that miR-34a directly targeted FGFR1. Moreover, miR-34a regulated osteoblast proliferation and apoptosis via FGFR1. Further, we validated that lncRNA TUG1 acted as a competing endogenous RNA (ceRNA) to interact with miR-34a and up-regulate FGFR1 protein expression. Furthermore, lncRNA TUG1 could promote proliferation and inhibit apoptosis. Taken together, our study revealed the key role of the lncRNA TUG1/miR-34a/FGFR1 axis in FSS-regulated osteoblast proliferation and apoptosis and may provide potential therapeutic targets for osteoporosis.     

RhoA/ROCK/PTEN signaling is involved in AT-101-mediated apoptosis in human leukemia cells in vitro and in vivo

R-(−)-gossypol acetic acid (AT-101) is a natural cottonseed product that exhibits anticancer activity. However, the molecular mechanism behind the antileukemic activity of AT-101 has not been well characterized. In this study, we investigated how AT-101 induces apoptosis in human leukemia cells. Exposure to AT-101 significantly increased apoptosis in both human leukemia cell lines and primary human leukemia cells. This increase was accompanied by the activation of caspases, cytochrome c release, Bcl2-associated X protein (Bax) translocation, myeloid cell leukemia-1 (Mcl-1) downregulation, Bcl-2-associated death promoter (Bad) dephosphorylation, Akt inactivation, and RhoA/Rho-associated coiled-coil containing protein kinase 1/phosphatase and tensin homolog (RhoA/ROCK1/PTEN) activation. RhoA, rather than caspase-3 cleavage, mediated the cleavage/activation of ROCK1 that AT-101 induced. Inhibiting RhoA and ROCK1 activation by C3 exoenzyme (C3) and Y27632, respectively, attenuated the ROCK1 cleavage/activation, PTEN activity, Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and apoptosis mediated by AT-101. Knocking down ROCK1 expression using a ROCK1-specific siRNA also significantly abrogated AT-101-mediated apoptosis. Constitutively active Akt prevented the AT-101-induced Mcl-1 downregulation, Bad dephosphorylation, and apoptosis. Conversely, AT-101 lethality was potentiated by the phosphatidylinositol 3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. In vivo, the tumor growth inhibition caused by AT-101 was also associated with RhoA/ROCK1/PTEN activation and Akt inactivation in a mouse leukemia xenograft model. Collectively, these findings suggest that AT-101 may preferentially induce apoptosis in leukemia cells by interrupting the RhoA/ROCK1/PTEN pathway, leading to Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and Bax translocation, which culminate in mitochondrial injury and apoptosis.     

CircTIMELESS regulates the proliferation and invasion of lung squamous cell carcinoma cells via the miR-136-5p/ROCK1 axis

Numerous studies demonstrate that circular RNAs (circRNAs) are critical regulators of the occurrence and progression of tumors. However, research on the involvement of circRNAs in lung squamous cell carcinoma (LUSC) is limited. In our study, circTIMELESS (also named hsa_circ_0000408 in the Human circRNA Database) was upregulated in both LUSC tissues and LUSC cells, and circTIMELESS expression was positively associated with the TNM stage. Moreover, circTIMELESS silencing markedly suppressed invasion in vitro and disrupted proliferation in vitro as well as in vivo. Additional investigations have shown that circTIMELESS functions as a miR-136-5p “sponge” and regulates miR-136-5p expression. Furthermore, the impact of miR-136-5p upregulation was consistent with the results of circTIMELESS silencing, both of which inhibited the proliferation and invasion of LUSC cells. Additional results showed that Rho-associated coiled-coil containing protein kinase 1 (ROCK1) is targeted by miR-136-5p. The results of recovery experiments showed that ROCK1 overexpression partly rescued the impact of circTIMELESS silencing and miR-136-5p upregulation on proliferation and invasion. Consequently, our findings confirmed that circTIMELESS exists in LUSC and acts as a tumor promoter through the miR-136-5p/ROCK1 axis. Based on these findings, circTIMELESS may be potentially utilized as a therapeutic target for LUSC.     

Long noncoding RNA PTPRG-AS1 acts as a microRNA-194-3p sponge to regulate radiosensitivity and metastasis of nasopharyngeal carcinoma cells via PRC1

Protein regulator of cytokinesis 1 (PRC1) has been reported in correlation with various malignancies. Functionality of PRC1 in nasopharyngeal carcinoma (NPC) was investigated, in perspective of long noncoding RNA (lncRNA) regulatory circuitry. Aberrant expressed messenger RNA and lncRNA were screened out from the Gene Expression Omnibus microarray database. NPC cell line CNE-2 was adopted for in vitro study and transfected with mimic or short hairpin RNA of miR-194-3p and PTPRG-AS1. The radioactive sensitivity, cell viability, migration, invasion, and apoptosis were detected. PTPRG-AS1 and PRC1 were upregulated in NPC, whereas miR-194-3p was downregulated. PTPRG-AS1 was found to specifically bind to miR-194-3p as a competing endogenous RNA and miR-194-3p targets and negatively regulates PRC1. Overexpressed miR-194-3p or silenced PTPRG-AS1 resulted in enhanced sensitivity to radiotherapy and cell apoptosis along with suppressed cell migration, invasion and proliferation in NPC. Furthermore, impaired tumor formation was also caused by miR-194-3p overexpression or PTPRG-AS1 suppression through xenograft tumor in nude mice. In our study, PTPRG-AS1/miR-194-3p/PRC1 regulatory circuitry was revealed in NPC, the mechanism of which can be of clinical significance for treatment of NPC.     

Long non-coding RNA TUG1/microRNA-187-3p/TESC axis modulates progression of pituitary adenoma via regulating the NF-κB signaling pathway

The molecule mechanisms of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in human diseases have been broadly studied recently, therefore, our research aimed to assess the effect of lncRNA taurine upregulated gene 1 (TUG1)/miR-187-3p/tescalcin (TESC) axis in pituitary adenoma (PA) by regulating the nuclear factor-kappa B (NF-κB) signaling pathway. We observed that TUG1 was upregulated in PA tissues and was associated with invasion, knosp grade and tumor size. TUG1 particularly bound to miR-187-3p. TUG1 knockdown inhibited cell proliferation, invasion, migration, and epithelial–mesenchymal transition, promoted apoptosis, and regulated the expression of NF-κB p65 and inhibitor of κB (IκB)-α in PA cells lines in vitro, and also inhibited tumor growth in vivo, and these effects were reversed by miR-187-3p reduction. Similarly, miR-187-3p elevation inhibited PA cell malignant behaviors and modulated the expression of NF-κB p65 and IκB-α in PA cells, and reduced in vivo tumor growth as well. TUG1 inhibition downregulated TESC, which was targeted by miR-187-3p. In conclusion, this study suggests that TUG1 sponges miR-187-3p to affect PA development by elevating TESC and regulating the NF-κB signaling pathway.Subject terms: Cell biology, Diseases     

miR-145-5p attenuates inflammatory response and apoptosis in myocardial ischemia-reperfusion injury by inhibiting (NADPH) oxidase homolog 1

Myocardial ischemia-reperfusion (I/R) injury is a common complication following reperfusion therapy that involves a series of immune or apoptotic reactions. Studies have revealed the potential roles of miRNAs in I/R injury. Herein, we established a myocardial I/R model in rats and a hypoxia/reoxygenation (H/R) model in H9c2 cells and investigated the effect of miR-145-5p on myocardial I/R injury. After 3 h or 24 h of reperfusion, left ventricular end-systolic pressure (LVESP), ejection fraction (EF), and fractional shortening (FS) were obviously decreased, and left ventricular end-diastolic pressure (LVEDP) was increased. Meanwhile, I/R induced an increase in myocardial infarction area. Moreover, a decrease in miR-145-5p and increase in (NADPH) oxidase homolog 1 (NOH-1) were observed following I/R injury. With this in mind, we performed a luciferase reporter assay and demonstrated that miR-145-5p directly bound to NOH-1 3’ untranslated region (UTR). Furthermore, miR-145-5p mimics decreased the levels of tumor necrosis factor (TNF)-α, IL-1β, and IL-6 via oxygen and glucose deprivation/reperfusion (OGD/R) stimulation. Upregulation of miR-145-5p increased cell viability and reduced apoptosis accompanied by downregulation of Bax, cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP) and upregulation of Bcl2. In addition, miR-145-5p overexpression increased superoxide dismutase (SOD) activity and reduced reactive oxygen species (ROS) and malondialdehyde (MDA) content under OGD/R stress. Notably, NOH-1 could significantly abrogate the above effects, suggesting that it is involved in miR-145-5p-regulated I/R injury. In summary, our findings indicated that miR-145-5p/NOH-1 has a protective effect on myocardial I/R injury by inhibiting the inflammatory response and apoptosis.     

MicroRNA-340 suppresses osteosarcoma tumor growth and metastasis by directly targeting ROCK1

MicroRNAs (miRNAs) play key roles in cancer development and progression. In the present study, we investigated the role of miR-340 in the progression and metastasis of osteosarcoma (OS). Our results showed that miR-340 was frequently downregulated in OS tumors and cell lines. Overexpression of miR-340 in OS cell lines significantly inhibited cell proliferation, migration, and invasion in vitro, and tumor growth and metastasis in a xenograft mouse model. ROCK1 was identified as a target of miR-340, and ectopic expression of miR-340 downregulated ROCK1 by direct binding to its 3′ untranslated region. siRNA-mediated silencing of ROCK1 phenocopied the effects of miR-340 overexpression, whereas restoration of ROCK1 in miR-340-overexpressing OS cells reversed the suppressive effects of miR-340. Together, these findings indicate that miR-340 acts as a tumor suppressor and its downregulation in tumor tissues may contribute to the progression and metastasis of OS through a mechanism involving ROCK1, suggesting miR-340 as a potential new diagnostic and therapeutic target for the treatment of OS.     

Circ_0004712 promotes endometriotic epithelial cell proliferation,migration and invasion by regulating miR-488-3p/ROCK1 axis in vitro

Recent evidence indicates that circular RNAs (circRNAs) play crucial regulatory roles in the pathogenesis and development of endometriosis. Circ_0004712 was found to be differentially expressed in endometriosis. However, the detailed function and mechanism of circ_0004712 in endometriosis are still unclear. Quantitative real-time polymerase chain reaction and Western blot were used for the detection of circ_0004712, miR-488-3p and ROCK1 (Rho Associated Coiled-Coil Containing Protein Kinase 1) levels. In vitro experiments in endometrial endothelial cells were performed by cell counting kit-8, EdU, transwell, wound healing assays, and flow cytometry, respectively. The molecular mechanism of circ_0004712 function was investigated using bioinformatics target predication, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assays. The expression of circ_0004712 was higher in endometriotic endometrial tissues and epithelial cells. Knockdown of circ_0004712 suppressed cell proliferation, migration, invasion, EMT process and induced apoptosis in ectopic endometrial epithelial cells in vitro. Mechanistically, circ_0004712 acted as a ceRNA to sponge miR-488-3p, thus elevating the expression of ROCK1, which was confirmed to be a target of miR-488-3p. Rescue experiments suggested that miR-488-3p inhibition reversed the inhibitory effects of circ_0004712 silencing on cell growth and metastasis. Moreover, miR-488-3p restoration restrained the proliferation and metastasis in ectopic endometrial epithelial cells, which were attenuated by ROCK1 overexpression. Circ_0004712 knockdown suppressed the proliferation and metastasis of ectopic endometrial epithelial cells via miR-488-3p/ROCK1 axis in vitro, suggesting a new insight into the pathogenesis of endometriosis.     

LncRNA TUG1 inhibits neuronal apoptosis in status epilepticus rats via targeting the miR-421/mTOR axis

Status epilepticus (SE) induces apoptosis of hippocampal neurons. However, the underlying mechanism in SE is not fully understood. Recently, lncRNA TUG1 is reported as a significant mediator in neuronal development. In present study, we aimed to investigate whether lncRNA TUG1 induces apoptosis of hippocampal neurons in SE rat models. TUG1 expression in serum of normal volunteers and SE patients, SE rats and neurons with epileptiform discharge was detected. SE rat model was established and intervened with TUG1 to evaluate hippocampal neuronal apoptosis. The experiments in vitro were further performed in neurons with epileptiform discharge to verify the effects of TUG1 on neuronal apoptosis of SE rats. The downstream mechanism of TUG1 was predicted and verified. miR-421 was intervened to perform the rescue experiments. Levels of oxidative stress and inflammation-related factors and mTOR pathway-related proteins in SE rats and hippocampal neurons were detected. TUG1 was highly expressed in serum of SE patients, SE rats and neurons with epileptiform discharge. Inhibition of TUG1 relieved pathological injury, oxidative stress and inflammation and reduced neuronal apoptosis in SE rats, which were further verified in hippocampal neurons. TUG1 upregulated TIMP2 expression by targeting miR-421. Overexpressed miR-421 inhibited hippocampal neuronal apoptosis. TUG1 knockout inactivated the mTOR pathway via the miR-421/TIMP2 axis to relieve neuronal apoptosis, oxidative stress and inflammation in SE rats and hippocampal neurons. Taken together, these findings showed that downregulation of lncRNA TUG1 inhibited apoptosis of hippocampal neurons in SE rats, and attenuated oxidative stress and inflammation damage through regulating the miR-421/mTOR axis.     

Long noncoding RNA TUG1 regulates the development of oral squamous cell carcinoma through sponging miR-524-5p to mediate DLX1 expression as a competitive endogenous RNA

Long noncoding RNA (lncRNA) exerts a potential regulatory role in tumorigenesis. LncRNA TUG1 expression remains high in oral squamous cell carcinoma (OSCC) tissues. However, its biological mechanism in OSCC remains unknown. In this study, TUG1 expression in OSCC cells was detected by quantitative real-time polymerase chain reaction. Proliferative and migratory potentials of OSCC cells were determined by Cell Counting Kit 8, 5-Ethynyl-2′- deoxyuridine (EdU), and Transwell assay, respectively. We identified the potential target of TUG1 through bioinformatics and dual-luciferase reporter gene assay. Furthermore, their interaction and functions in regulating the development of OSCC were clarified by western blot and RNA immunoprecipitation assay. Our results demonstrated a high expression of TUG1 in OSCC cells. Overexpression of TUG1 markedly accelerated proliferative and migratory potentials of OSCC cells. Besides, TUG1 could positively regulate the expression of distal-less homeobox 1 (DLX1) by competing with miR-524-5p. These results indicated that TUG1 participated in the development of OSCC as a competing endogenous RNA to competitively bind to miR-524-5p and thus mediate DLX1 expression.