MicroRNA-132 protects H9c2 cells against oxygen and glucose deprivation-evoked injury by targeting FOXO3A.

Myocardial ischemia/reperfusion injury (MIRI) is a clinically familiar disease, which possesses a great negative impact on human health. But, the effective treatment is still absent. MicroRNAs (miRNAs) have been testified to play a momentous role in MIRI. The purpose of the study aimed to probe the functions of miR-132 in oxygen and glucose deprivation (OGD)-evoked injury in H9c2 cells. miR-132 expression in H9c2 cells accompanied by OGD disposition was evaluated via real-time quantitative polymerase chain reaction. After miR-132 mimic and inhibitor transfections, the impacts of miR-132 on OGD-affected H9c2 cell viability, apoptosis, cell cycle, and the interrelated factors were appraised by exploiting cell counting kit-8, flow cytometry, and western blot analysis. FOXO3A expression was estimated in above-transfected cells, meanwhile, the correlation between miR-132 and FOXO3A was probed by dual-luciferase report assay. Ultimately, above mentioned cell processes were reassessed in H9c2 cells after preprocessing OGD administration and transfection with si-FOXO3A and si-NC plasmids. We got that OGD disposition obviously enhanced miR-132 expression in H9c2 cells. Overexpressed miR-132 evidently reversed OGD-evoked cell viability repression and apoptosis induction in H9c2 cells. In addition, overexpressed miR-132 mitigated OGD-evoked G0/G1 cell arrest by mediating p21, p27, and cyclin D1 expression. Repression of FOXO3A was observed in miR-132 mimic-transfected cells, which was also predicated as a direct gene of miR-132. We discovered that silenced FOXO3A alleviated OGD-evoked cell injury in H9c2 cells via facilitating cell viability, hindering apoptosis and restraining cell arrest at G0/G1 phase. In conclusion, these investigations corroborated that miR-132 exhibited the protective impacts on H9c2 cells against OGD-evoked injury via targeting FOXO3A.

RETRACTED: LncRNA MALAT1 knockdown alleviates oxygen-glucose deprivation and reperfusion induced cardiomyocyte apoptotic death by regulating miR-122.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. The journal was initially contacted by the corresponding author to request the retraction of the article. Given the comments of Dr Elisabeth Bik regarding this article "… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Quercetin supports cell viability and inhibits apoptosis in cardiocytes by down-regulating miR-199a.

Hypoxia-caused cardiocytes insults are closely correlated with ectopic expression of genes, which might be modulated by microRNAs (miRs). Quercetin exhibits a profound protective function against hypoxic damages in cardiomyocytes. Here, we aimed to investigate a possible underpinning. H9c2 cells were pre-administrated using quercetin before hypoxia treatment. The damages were assessed using viability, apoptosis and alteration of proteins associated with apoptosis and adenosine monophosphate-activated protein (AMPK) pathway. Transfection was conducted to enforce overexpression of miR-199a or silence of sirtuin 1 (sirt1) which were confirmed by qRT-PCR. Sirt1 protein was quantified by immunoblotting. A luciferase reporter was exploited to confirm the target relationship between miR-199a and sirt1 3'-untranslated region (3'-UTR). We found quercetin mitigated hypoxia-caused viability reduction and apoptosis with restoring apoptosis-associated protein and rescuing phosphorylation of AMPK. Quercetin flattened hypoxia-evoked overexpression of miR-199a. miR-199a abrogated the protective effects of quercetin against hypoxia-elicited damages. Quercetin elevated sirt1 which was repressed by hypoxia, while this effect was slight in miR-199a-overexpressed cells. miR-199a negatively mediated sirt1 expression through directly binding its 3'-UTR. Further, quercetin facilitated the phosphorylation of AMPK by up-regulating sirt1. Collectively, quercetin participated in repressing miR-199a which negatively modulated sirt1. Mechanically, through activating AMPK, quercetin protected cardiomyocytes cells against hypoxia-caused insults. Highlights Quercetin ameliorates hypoxia-evoked apoptosis and blockage of AMPK phosphorylation; The elevated miR-199a level is eased by quercetin, which might be a protective mechanism; Quercetin restores sirt1 level by repressing miR-199a expression; By mediating miR-199a and sirt1, AMPK phosphorylation is fortified by quercetin.

Oridonin relieves hypoxia-evoked apoptosis and autophagy via modulating microRNA-214 in H9c2 cells.

Oridonin (Orid) has been diffusely applied to remedy dissimilar cancers. Howbeit, the influence of Orid in ischemic heart disease (IHD) remains imprecise. The current study uncovered the functions of Orid in hypoxia-caused apoptosis and autophagy in H9c2 cells. H9c2 cells received hypoxia and Orid manipulation, cell viability, apoptosis, apoptosis-interrelated factors and autophagy-correlative factors were appraised. After the extraordinary vectors transfections, the impacts of miR-214 inhibition on hypoxia-triggered apoptosis and autophagy were investigated. Further, dual luciferase reporter assay was enforced for ascertaining the pertinence between miR-214 and PTEN. PI3K/AKT/mTOR pathway was finally determined using western blot. We found that, Orid significantly alleviated hypoxia-induced apoptosis and autophagy through regulation their associated proteins in H9c2 cells. Up-regulation of miR-214 was found in hypoxia and Orid co-managed cells, meanwhile, repression of miR-214 obviously annulled the modulatory functions of Orid in hypoxia-evoked apoptosis and autophagy. Additionally, PTEN was forecasted to be a firsthand target of miR-214. Besides, we observed that Orid evoked PI3K/AKT/mTOR activation through elevation of miR-214 in hypoxia-managed H9c2 cells. In conclusion, the amusing results corroborated that Orid relieved hypoxia-caused apoptosis and autophagy via adjusting PI3K/AKT/mTOR pathway through enhancement of miR-214 in H9c2 cells.

Novel missense mutation in PTPN22 in a Chinese pedigree with Hashimoto's thyroiditis.

BACKGROUND: Hashimoto's thyroiditis is a complex autoimmune thyroid disease, the onset of which is associated with environmental exposures and specific susceptibility genes. Its incidence in females is higher than its incidence in males. Thus far, although some susceptibility loci have been elaborated, including PTPN22, FOXP3, and CD25, the aetiology and pathogenesis of Hashimoto's thyroiditis remains unclear. METHODS: Four affected members from a Chinese family with Hashimoto's thyroiditis were selected for whole-exome sequencing. Missense, nonsense, frameshift, or splicing-site variants shared by all affected members were identified after frequency filtering against public and internal exome databases. Segregation analysis was performed by Sanger sequencing among all members with available DNA. RESULTS: We identified a missense mutation in PTPN22 (NM_015967.5; c. 77A > G; p.Asn26Ser) using whole-exome sequencing. PTPN22 is a known susceptibility gene associated with increased risks of multiple autoimmune diseases. Cosegregation analysis confirmed that all patients in this family, all of whom were female, carried the mutation. All public and private databases showed that the missense mutation was extremely rare. CONCLUSIONS: We found a missense mutation in PTPN22 in a Chinese HT pedigree using whole-exome sequencing. Our study, for the first time, linked a rare variant of PTPN22 to Hashimoto's thyroiditis, providing further evidence of the disease-causing or susceptibility role of PTPN22 in autoimmune thyroid disease. Functional studies regarding the effects of this variant on thyroid autoimmunity and thyroid function are warranted.

Astragaloside IV Protects Rat Cardiomyocytes from Hypoxia-Induced Injury by Down-Regulation of miR-23a and miR-92a.

BACKGROUND/AIMS: Astragaloside IV (AS-IV), a traditional Chinese medicine isolated from Astragalus membranaceus, has been shown to exert cardioprotective effect previously. This study aimed to reveal the effects of AS-IV on hypoxia-injured cardiomyocyte. METHODS: H9c2 cells were treated with various doses of AS-IV for 24 h upon hypoxia. CCK-8 assay, flow cytometry/Western blot, and qRT-PCR were respectively conducted to measure the changes in cell viability, apoptosis, and the expression of miR-23a and miR-92a. Sprague-Dawley rats were received coronary ligation, and were administrated by various doses of AS-IV for 14 days. The infarct volume and outcome of rats followed by ligation were tested by ultrasound, arteriopuncture and nitrotetrazolium blue chloride (NBT) staining. RESULTS: We found that 10 µg/ml of AS-IV exerted myocardioprotective effects against hypoxia-induced cell damage, as AS-IV significantly increased H9c2 cells viability and decreased apoptosis. Interestingly, the myocardioprotective effects of AS-IV were alleviated by miR-23a and/or miR-92a overexpression. Knockdown of miR-23a and miR-92a activated PI3K/AKT and MAPK/ ERK signaling pathways. Bcl-2 was a target gene for miR-23a, and BCL2L2 was a target gene for miR-92a. In the animal model of myocardial infarction (MI), AS-IV significantly reduced the infarct volume, ejection fraction (EF), shortening fraction (FS) and LV systolic pressure (LVSP), and significantly increased left ventricular end-diastolic internal diameter (LVEDd). And also, the elevated expression of miR-23a and miR-92a in MI rat was reduced by AS-IV. CONCLUSION: AS-IV protected cardiomyocytes against hypoxia-induced injury possibly via down-regulation of miR-23a and miR-92a, and via activation of PI3K/AKT and MAPK/ERK signaling pathways.

Knockdown of long non-coding RNA MEG3 protects H9c2 cells from hypoxia-induced injury by targeting microRNA-183.

Acute myocardial infarction (AMI) occurs when blood supply to the heart is diminished (ischemia) for long time, and ischemia is primarily caused due to hypoxia. This study evaluated the effects of long non-coding RNA maternally expressed gene 3 (MEG3) on hypoxic rat cardiomyocyte-drived H9c2 cells. Hypoxic injury was confirmed by alterations of cell viability, migration, invasion, apoptosis, and hypoxia-inducible factor 1α (HIF-1α) expression. MEG3 level in hypoxic cells and effects of its knockdown on hypoxic cells were assessed. The interactions between MEG3 and miR-183 as well as miR-183 and p27 were investigated. In addition, the effects of aberrantly expressed MEG3, miR-183, and p27 on hypoxic cells along with the activation of PI3K/AKT/FOXO3a signaling pathway were all assessed. Results showed that hypoxia induced decreases of cell viability, migration and invasion, and increases of apoptosis and expressions of HIF-1α and MEG3. Knockdown of MEG3 decreased hypoxia-induced injury in H9c2 cells. Knockdown of MEG3 also increased miR-183 expression, which was identified as a target of MEG3. The effects of MEG3 knockdown on the hypoxic cells were reversed by miR-183 silence. p27 was identified as a target gene of miR-183, and its expression negatively regulated by miR-183. The mechanistic studies revealed that knockdown of p27 decreased hypoxia-induced H9c2 cell injury by activating PI3K/AKT/FOXO3a signal pathways. These findings suggest that knockdown of MEG3 alleviates hypoxia-induced H9c2 cell injury by miR-183-mediated suppression of p27 through activation of PI3K/AKT/FOXO3a signaling pathway.

Long Non-Coding RNA H19 Protects H9c2 Cells against Hypoxia-Induced Injury by Targeting MicroRNA-139.

BACKGROUND/AIMS: Acute myocardial infarction (AMI) occurs when blood supply to the heart is diminished (ischemia) for long time; ischemia is primarily caused due to hypoxia. The present study evaluated the effects of long non-coding RNA H19 on hypoxic rat H9c2 cells and mouse HL-1 cells. METHODS: Hypoxic injury was confirmed by measuring cell viability, migration and invasion, and apoptosis using MTT, Transwell and flow cytometry assays, respectively. H19 expression after hypoxia was estimated by qRT-PCR. We then measured the effects of non-physiologically expressed H19, knockdown of miR-139 with or without H19 silence, and abnormally expressed Sox8 on hypoxia-induced H9c2 cells. Moreover, the interacted miRNA for H19 and downstream target gene were virtually screened and verified. The involved signaling pathways and the effects of abnormally expressed H19 on contractility of HL-1 cells were explored via Western blot analysis. RESULTS: Hypoxia induced decreases of cell viability, migration and invasion, increase of cell apoptosis and up-regulation of H19. Knockdown of H19 increased hypoxia-induced injury in H9c2 cells. H19 acted as a sponge for miR-139 and H19 knockdown aggravated hypoxia-induced injury by up-regulating miR-139. Sox8 was identified as a target of miR-139, and its expression was negatively regulated by miR-139. The mechanistic studies revealed that overexpression of Sox8 might decrease hypoxia-induced cell injury by activating the PI3K/AKT/mTOR pathway and MAPK. Besides, H19 promoted contractility of HL-1 cells. CONCLUSION: These findings suggest that H19 alleviates hypoxia-induced myocardial cell injury by miR-139-mediated up-regulation of Sox8, along with activation of the PI3K/AKT/mTOR pathway and MAPK.

Polyalanine repeat expansion mutation of the HOXD13 gene in a Chinese family with unusual clinical manifestations of synpolydactyly.

Synpolydactyly (SPD) is an autosomal dominant limb malformation caused by mutations in the gene HOXD13. We investigated a Chinese family in which three individuals across three generations were affected with distinctive limb malformations. We extracted genomic DNA from the affected and three unaffected individuals from this family as well as 100 unrelated controls, for mutation detection by DNA sequencing. The family was characterized by camptodactyly and symphalangism of fingers two to five, transverse phalanx and osseous fusion of the third metacarpal with the proximal phalanx, as well as the coexistence of mild and more severe bilateral phenotypes. We identified a duplication mutation, c. 186-212dup, in exon 1 of the HOXD13 gene in the affected individuals from this family; it was not present in the unaffected individuals or the 100 unrelated individuals. And we also did not find polymorphism among the controls. This study has expanded the phenotypic spectrum of known HOXD13 polyalanine repeat mutations and provided more information about the polymorphic nature of the polyalanine repeat. In addition, new clinical manifestations have been added to the spectrum of possible synpolydactyly phenotypes.