Long noncoding RNA PVT1 facilitates high glucose-induced cardiomyocyte death through the miR-23a-3p/CASP10 axis

Dilated cardiomyopathy (DCM) is the leading cause of morbidity and mortality in diabetic patients. Long noncoding RNA plasmacytoma variant translocation 1 (PVT1) has been shown to be related to the pathogenesis of DCM. However, the mechanism by which PVT1 regulates DCM pathogenesis is unclear. High glucose level was employed to construct a DCM cell model in vitro. Cell viability was determined via cell counting kit-8 assay. The level of lactate dehydrogenase (LDH) was measured with the corresponding kit. Expression levels of PVT1, miR-23a-3p, and caspase-10 (CASP10) messenger RNA were evaluated with a quantitative real-time polymerase chain reaction. Cell apoptosis was assessed by flow cytometry assay. Protein levels of B-cell lymphoma 2-associated X (Bax), cleaved-caspase-3 (cleaved-casp-3), and CASP10 were examined via western blot analysis. The relationship between PVT1 or CASP10 and miR-23a-3p was verified with dual-luciferase reporter assay. We observed that PVT1 and CASP10 were upregulated while miR-23a-3p was downregulated in high glucose-induced cardiomyocytes. High glucose levels repressed cardiomyocyte activity and induced cardiomyocyte apoptosis, but this influence was antagonized by PVT1 knockdown or miR-23a-3p overexpression. Furthermore, PVT1 acted as a sponge for miR-23a-3p, and miR-23a-3p inhibition counterbalanced the influence of PVT1 silencing on viability and apoptosis of cardiomyocytes under high glucose level treatment. PVT1 could increase CASP10 expression via sponging miR-23a-3p. In conclusion, PVT1 acted as a deleterious lncRNA in DCM. PVT1 facilitated cardiomyocyte death by regulating the miR-23a-3p/CASP10, which offered a new mechanism to comprehend the pathogenesis of DCM.     

Knockdown of lncRNA AK139328 alleviates myocardial ischaemia/reperfusion injury in diabetic mice via modulating miR‐204‐3p and inhibiting autophagy

This study was aimed at investigating the effects of lncRNA AK139328 on myocardial ischaemia/reperfusion injury (MIRI) in diabetic mice. Ischaemia/reperfusion (I/R) model was constructed in normal mice (NM) and diabetic mice (DM). Microarray analysis was utilized to identify lncRNA AK139328 overexpressed in DM after myocardial ischaemia/reperfusion (MI/R). RT‐qPCR assay was utilized to investigate the expressions of lncRNA AK139328 and miR‐204‐3p in cardiomyocyte and tissues. Left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF) and fractioning shortening (FS) were obtained by transthoracic echocardiography. Haematoxylin‐eosin (HE) staining and Masson staining were utilized to detect the damage of myocardial tissues degradation of myocardial fibres and integrity of myocardial collagen fibres. Evans Blue/TTC staining was used to determine the myocardial infarct size. TUNEL staining was utilized to investigate cardiomyocyte apoptosis. The targeted relationship between lncRNA AK139328 and miR‐204‐3p was confirmed by dual‐luciferase reporter gene assay. MTT assay was used for analysis of cardiomyocyte proliferation. Western blot was utilized to investigate the expression of alpha smooth muscle actin (α‐SMA), Atg7, Atg5, LC3‐II/LC3‐I and p62 marking autophagy. Knockdown of lncRNA AK139328 relieved myocardial ischaemia/reperfusion injury in DM and inhibited cardiomyocyte autophagy as well as apoptosis of DM. LncRNA AK139328 modulated miR‐204‐3p directly. MiR‐204‐3p and knockdown of lncRNA AK139328 relieved hypoxia/reoxygenation injury via inhibiting cardiomyocyte autophagy. Silencing lncRNA AK139328 significantly increased miR‐204‐3p expression and inhibited cardiomyocyte autophagy, thereby attenuating MIRI in DM.     

Long noncoding RNA ATB participates in the development of renal cell carcinoma by downregulating p53 via binding to DNMT1

Long noncoding RNA (lncRNA) exerts an essential role in the pathological processes of many diseases. Our previous study found that lncRNA ATB was highly expressed in renal cell carcinoma (RCC). Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), and migration-related assays were conducted to access the regulatory effects of lncRNA ATB on proliferative and migratory capacities of RCC cells. Flow cytometry was carried out to determine cell cycle and apoptosis influenced by lncRNA ATB. The interaction among lncRNA ATB, DNMT1, and p53 was evaluated through RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and western blot analyses. The results showed that lncRNA ATB knockdown in RCC cell line ACHN inhibited proliferative and migratory capacities and promoted apoptosis. Meanwhile, overexpression of lncRNA ATB in RCC cell line A-498 promoted proliferative and migratory capacities but inhibited apoptosis. RIP and ChIP assays confirmed that lncRNA ATB can bind to DNMT1 and stabilize its expression; meanwhile, it can promote the binding of DNMT1 to p53. Overexpression of p53 partially reversed the proliferative and migratory changes caused by lncRNA ATB. To sum up, our study revealed that high expression of lncRNA ATB could accelerate the proliferative and migratory rates of RCC cells and inhibit cell apoptosis through downregulating p53 via binding to DNMT1.     

Retracted: Long noncoding RNA MEG3 is a tumor suppressor in choriocarcinoma by upregulation of microRNA-211

Tumor suppressor long noncoding RNA maternally expressed gene 3 (lncRNA MEG3) exists in various cancers. Nonetheless, the functions of lncRNA MEG3 in choriocarcinoma (CC) are still not well studied. We explored the effects of lncRNA MEG3 on human CC JEG-3 and BeWo cells. lncRNA MEG3 was overexpressed, and the effects of lncRNA MEG3 on cell viability, proliferation, apoptosis, migration, and invasion were assessed by the cell counting kit-8 assay, western blot analysis, flow cytometry (plus western blot analysis), and transwell assay (plus western blot analysis), respectively. Then, the expression level of miR-211 was detected by real-time quantitative polymerase chain reaction. After that, the effects of dysregulated microRNA-211 (miR-211) with overexpressing lncRNA MEG3 on JEG-3 cells and BeWo cells were testified. Western blot analysis was used to study the involvements of the signaling pathways in the lncRNA MEG3-associated modulation. We found that lncRNA MEG3 upregulation reduced cell viability, inhibited proliferation, migration and invasion, and promoted apoptosis. Expression of miR-211 was upregulated after lncRNA MEG3 overexpression. Effects of lncRNA MEG3 overexpression were augmented by miR-211 overexpression, while they were declined by miR-211 silencing. Phosphorylated levels of PI3K, AKT, and AMP-activated protein kinase (AMPK) were decreased by lncRNA MEG3 overexpression via regulation of miR-211. To sum up, lncRNA MEG3 could repress proliferation, migration and invasion, and promote apoptosis of JEG-3 and BeWo cells through upregulating miR-211. The PI3K/AKT and AMPK pathways were inhibited by lncRNA MEG3 overexpression via regulation of miR-211.     

A novel long noncoding RNA FAF inhibits apoptosis via upregulating FGF9 through PI3K/AKT signaling pathway in ischemia–hypoxia cardiomyocytes

Long noncoding RNAs (lncRNAs) have been increasingly considered to play an important role in the pathological process of various cardiovascular diseases, which often bind to the proximal promoters of the protein-coding gene to regulate the protein expression. However, the functions and mechanisms of lncRNAs in cardiomyocytes have not been fully elucidated. High-throughput RNA sequencing was performed to identify the differently expressed lncRNAs and messenger RNAs (mRNAs) between acute myocardial infarction (AMI) rats and healthy controls. One novel lncRNA FGF9-associated factor (termed FAF) and mRNAs in AMI rats were verified by bioinformatics, real-time polymerase chain reaction or western blot. Moreover, RNA fluorescence in situ hybridization was performed to determine the location of lncRNA. Subsequently, a series of in vitro assays were used to observe the functions of lncRNA FAF in cardiomyocytes. The expression of lncRNA FAF and FGF9 were remarkably decreased in ischemia–hypoxia cardiomyocytes and heart tissues of AMI rats. Overexpression of FAF could significantly inhibit cardiomyocytes apoptosis induced by ischemia and hypoxia. Conversely, knockdown of lncRNA FAF could promote apoptosis in ischemia–hypoxia cardiomyocytes. Moreover, overexpression of lncRNA FAF could also increase the expression of FGF9. Knockdown of the FGF9 expression could promote apoptosis in cardiomyocytes with the insult of ischemia and hypoxia, which was consistent with the effect of lncRNA FAF overexpression on cardiomyocyte apoptosis. Mechanistically, FGF9 inhibited cardiomyocytes apoptosis through activating signaling tyrosine kinase FGFR2 via phosphoinositide 3-kinase/protein kinase B signaling pathway. Thus, lncRNA FAF plays a protective role in ischemia–hypoxia cardiomyocytes and may serve as a treatment target for AMI.     

Long noncoding RNA FTX regulates cardiomyocyte apoptosis by targeting miR-29b-1-5p and Bcl2l2

Cardiomyocyte apoptosis correlates with the pathogenesis of heart disease. Long noncoding RNA (LncRNA) emerges as a class of noncoding RNAs that regulate gene expression and participate in various cellular processes. However, the role of lncRNAs in cardiomyocyte apoptosis remains to be elucidated. In our study, we found that lncRNA FTX is significantly down-regulated upon ischemia/reperfusion injury and hydrogen peroxide treatment. Enhanced expression of FTX inhibits cardiomyocyte apoptosis induced by hydrogen peroxide. miR-29b-1-5p was found to interact with FTX and regulate the expression of Bcl2l2. Inhibition of miR-29b-1-5p attenuated cardiomyocyte apoptosis upon hydrogen peroxide treatment. We then found that FTX functions as endogenous sponge for miR-29b-1-5p and regulates the activity of miR-29b-1-5p. The results demonstrate that FTX regulates cardiomyocyte apoptosis through modulating the expression of Bcl2l2 which is mediated by miR-29b-1-5p. Our findings reveal a novel regulatory model which is composed of FTX, miR-29b-1-5p and Bcl2l2. Manipulating of their levels may become a new approach to tackling cardiomyocyte apoptosis related heart diseases.     

LncRNA FAF attenuates hypoxia/ischaemia‐induced pyroptosis via the miR‐185‐5p/PAK2 axis in cardiomyocytes

Pyroptosis is associated with various cardiovascular diseases. Increasing evidence suggests that long noncoding RNAs (lncRNAs) have been implicated in gene regulation, but how lncRNAs participate in the regulation of pyroptosis in the heart remains largely unknown. In this study, we aimed to explore the antipyroptotic effects of lncRNA FGF9‐associated factor (FAF) in acute myocardial infarction (AMI). The expression patterns of lncRNA FAF, miR‐185‐5p and P21 activated kinase 2 (PAK2) were detected in hypoxia/ischaemia‐induced cardiomyocytes. Hoechst 33342/PI staining, lactate dehydrogenase (LDH) release assay, immunofluorescence and Western blotting were conducted to assay cell pyroptosis. The interaction between lncRNA FAF, miR‐185‐5p and PAK2 was verified by bioinformatics analysis, small RNA sequencing luciferase reporter assay and qRT‐PCR. The expression of LncRNA FAF was downregulated in hypoxic cardiomyocytes and myocardial tissues. Overexpression of lncRNA FAF could attenuate cardiomyocyte pyroptosis, improve cell viability and reduce infarct size during the procession of AMI. Moreover, lncRNA FAF was confirmed as a sponge of miR‐185‐5p and promoted PAK2 expression in cardiomyocytes. Collectively, our findings reveal a novel lncRNA FAF/miR‐185‐5p/PAK2 axis as a crucial regulator in cardiomyocyte pyroptosis, which might be a potential therapeutic target of AMI.     

MiR-320 regulates cardiomyocyte apoptosis induced by ischemia–reperfusion injury by targeting AKIP1

Background

MicroRNAs play important roles in regulation of the cardiovascular system. The purpose of this study was to investigate microRNA-320 (miR-320) expression in myocardial ischemia-reperfusion (I/R) injury and the roles of miR-320 in cardiomyocyte apoptosis by targeting AKIP1 (A kinase interacting protein 1).

Methods

The level of miR-320 was detected using quantitative real-time polymerase chain reaction (qRT-PCR), and cardiomyocyte apoptosis was detected via terminal dUTP nick end-labeling assay. Cardiomyocyte apoptosis and the mitochondrial membrane potential were evaluated via flow cytometry. Bioinformatics tools were used to identify the target gene of miR-320. The expression levels of AKIP1 mRNA and protein were detected via qRT-PCR and Western blot, respectively.

Results

Both the level of miR-320 and the rate of cardiomyocyte apoptosis were substantially higher in the I/R group and H9c2 cells subjected to H/R than in the corresponding controls. Overexpression of miR-320 significantly promoted cardiomyocyte apoptosis and increased the loss of the mitochondrial membrane potential, whereas downregulation of miR-320 had an opposite effect. Luciferase reporter assay showed that miR-320 directly targets AKIP1. Moreover, knock down and overexpression of AKIP1 had similar effects on the H9c2 cells subjected to H/R.

Conclusions

miR-320 plays an important role in regulating cardiomyocyte apoptosis induced by I/R injury by targeting AKIP1 and inducing the mitochondrial apoptotic pathway.

     

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.     

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.     

Retracted: Downregulation of long noncoding RNA H19 rescues hippocampal neurons from apoptosis and oxidative stress by inhibiting IGF2 methylation in mice with streptozotocin-induced diabetes mellitus

The diabetes mellitus (DM)-induced reduction of neurogenesis in the hippocampus is consequently accompanied by cognitive decline. The present study set out to define the critical role played by long noncoding RNA H19 (lncRNA H19) in the apoptosis of hippocampal neurons, as well as oxidative stress (OS) in streptozotocin (STZ)-induced DM mice through regulation of insulin-like growth factor 2 (IGF2) methylation. The expression of lncRNA H19 in the hippocampal neurons and surviving neurons were detected. Hippocampal neurons were cultured and transfected with oe-H19, sh-H19, oe-IGF2, or sh-IGF2, followed by detection of the expressions of IGF2 and apoptosis-related genes. Determination of the lipid peroxide and glutathione levels was conducted, while antioxidant enzyme activity was identified. The IGF2 methylation, the binding of lncRNA H19 to DNA methyltransferase, and the binding of lncRNA H19 to IGF2 promoter region were detected. DM mice exhibited high expressions of H19, as well as a decreased hippocampal neurons survival rate. Higher lncRNA H19 expression was found in DM. Upregulated lncRNA H19 significantly increased the expression of Bax and caspase-3 but decreased that of Bcl-2, thus promoting the apoptosis of hippocampal neuron. Besides, upregulation of lncRNA H19 induced OS. LncRNA H19 was observed to bind specifically to the IGF2 gene promoter region and promote IGF2 methylation by enriching DNA methyltransferase, thereby silencing IGF2 expression. Taken together, downregulated lncRNA H19 reduces IGF2 methylation and enhances its expression, thereby suppressing hippocampal neuron apoptosis and OS in STZ-induced (DM) mice.     

LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR‐9‐5p/QKI‐5 axis

This study was designed to detecting the influences of lncRNA MEG3 in prostate cancer. Aberrant lncRNAs expression profiles of prostate cancer were screened by microarray analysis. The qRT‐PCR and Western blot were employed to investigating the expression levels of lncRNA MEG3, miR‐9‐5p and QKI‐5. The luciferase reporter assay was utilized to testifying the interactions relationship among these molecules. Applying CCK‐8 assay, wound healing assay, transwell assay and flow cytometry in turn, the cell proliferation, migration and invasion abilities as well as apoptosis were measured respectively. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer tissues and cells and could inhibit the expression of miR‐9‐5p, whereas miR‐9‐5p down‐regulated QKI‐5 expression. Overexpressed MEG3 and QKI‐5 could decrease the abilities of proliferation, migration and invasion in prostate cancer cells effectively and increased the apoptosis rate. On the contrary, miR‐9‐5p mimics presented an opposite tendency in prostate cancer cells. Furthermore, MEG3 inhibited tumour growth and up‐regulated expression of QKI‐5 in vivo. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer and impacted the abilities of cell proliferation, migration and invasion, and cell apoptosis rate, this regulation relied on regulating miR‐9‐5p and its targeting gene QKI‐5.