Rutin treats myocardial damage caused by pirarubicin via regulating miR‐22‐5p‐regulated RAP1/ERK signaling pathway

Our experiments have previously demonstrated that rutin (RUT) can improve myocardial damage caused by pirarubicin (THP). However, the underlying molecular mechanisms remain uncertain. In this study, we developed an microRNA (miRNA) chip by replicating the rat model of THP‐induced myocardial injury and identified miR‐22‐5p and the RAP1‐member of RAS oncogene family/extracellular regulated protein kinases (RAP1/ERK) signaling pathway as an object of study. Also, in vivo experiments demonstrated that THP caused abnormal changes in the electrocardiogram, cardiac function, and histomorphology in rats (P < .01). THP also reduces the expression of miR‐22‐5p (P < .01) and increases the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). RUT significantly improved THP‐induced myocardial damage (P < .01), increased the expression of miR‐22‐5p (P < .01), and decreased the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). In vitro studies confirmed that Rap1a is one of the target genes of miR‐22‐5p. miR‐22‐5p overexpression in cardiomyocytes can affect the RAP1/ERK pathway and reduce reactive oxygen species production and cardiomyocyte apoptosis caused by THP (P < .01), which is consistent with the effect of RUT. Our results indicate that RUT treats THP‐induced myocardial damage, which may be achieved by upregulating miR‐22‐5p, causing changes in its target gene Rap1a and the RAP1/ERK pathway.     

MicroRNA‐21 protects against cardiac hypoxia/reoxygenation injury by inhibiting excessive autophagy in H9c2 cells via the Akt/mTOR pathway

MicroRNAs and autophagy play critical roles in cardiac hypoxia/reoxygenation (H/R)‐induced injury. Here, we investigated the function of miR‐21 in regulating autophagy and identified the potential molecular mechanisms involved. To determine the role of miR‐21 in regulating autophagy, H9c2 cells were divided into the following six groups: control group, H/R group, (miR‐21+ H/R) group, (miR‐21‐negative control + H/R) group, (BEZ235+ H/R) group and (miR‐21+ BEZ235+ H/R) group. The cells underwent hypoxia for 1 hr and reoxygenation for 3 hrs. Cell count kit‐8 was used to evaluate cell function and apoptosis was analysed by Western blotting. Western blotting and transmission electron microscopy were used to investigate autophagy. We found that miR‐21 expression was down‐regulated, and autophagy was remarkably increased in H9c2 cells during H/R injury. Overexpression of miR‐21 with a miR‐21 precursor significantly inhibited autophagic activity and decreased apoptosis, accompanied by the activation of the AKT/mTOR pathway. In addition, treatment with BEZ235, a novel dual Akt/mTOR inhibitor, resulted in a significant increase in autophagy and apoptosis. However, we found that miR‐21‐mediated inhibition of apoptosis and autophagy was partly independent of Akt/mTOR activation, as demonstrated in cells treated with both miR‐21 and BEZ235. We showed that miR‐21 could inhibit H/R‐induced autophagy and apoptosis, which may be at least partially mediated by the Akt/mTOR signalling pathway.     

miR‐1‐3p and miR‐206 sensitizes HGF‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT

Hepatocyte growth factor (HGF) overexpression is an important mechanism in acquired epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib resistance in lung cancers with EGFR activating mutations. MiR‐1‐3p and miR‐206 act as suppressors in lung cancer proliferation and metastasis. However, whether miR‐1‐3p and miR‐206 can overcome HGF‐induced gefitinib resistance in EGFR mutant lung cancer is not clear. In this study, we showed that miR‐1‐3p and miR‐206 restored the sensitivities of lung cancer cells PC‐9 and HCC‐827 to gefitinib in present of HGF. For the mechanisms, we demonstrated that both miR‐1‐3p and miR‐206 directly target HGF receptor c‐Met in lung cancer. Knockdown of c‐Met mimicked the effects of miR‐1‐3p and miR‐206 transfections Meanwhile, c‐Met overexpression attenuated the effects of miR‐1‐3p and miR‐206 in HGF‐induced gefitinib resistance of lung cancers. Furthermore, we showed that miR‐1‐3p and miR‐206 inhibited c‐Met downstream Akt and Erk pathway and blocked HGF‐induced epithelial‐mesenchymal transition (EMT). Finally, we demonstrated that miR‐1‐3p and miR‐206 can increase gefitinib sensitivity in xenograft mouse models in vivo. Our study for the first time indicated the new function of miR‐1‐3p and miR‐206 in overcoming HGF‐induced gefitinib resistance in EGFR mutant lung cancer cell.     

Neuroprotective effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on ischaemia/reperfusion‐induced neuronal injury by activating the Nrf2/HO‐1 pathway

1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a lipophilic phenolic agent, has an antioxidant activity and reactive oxygen species (ROS) scavenging property. However, the role of HTHQ on cerebral ischaemic/reperfusion (I/R) injury and the underlying mechanisms remain poorly understood. In the present study, we demonstrated that HTHQ treatment ameliorated cerebral I/R injury in vivo, as demonstrated by the decreased infarct volume ration, neurological deficits, oxidative stress and neuronal apoptosis. HTHQ treatment increased the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream antioxidant protein, haeme oxygenase‐1 (HO‐1). In addition, HTHQ treatment decreases oxidative stress and neuronal apoptosis of PC12 cells following hypoxia and reperfusion (H/R) in vitro. Moreover, we provided evidence that PC12 cells were more vulnerable to H/R‐induced oxidative stress after si‐Nrf2 transfection, and the HTHQ‐mediated protection was lost in PC12 cells transfected with siNrf2. In conclusion, these results suggested that HTHQ possesses neuroprotective effects against oxidative stress and apoptosis after cerebral I/R injury via activation of the Nrf2/HO‐1 pathway.     

miR‐150‐5p suppresses the stem cell‐like characteristics of glioma cells by targeting the Wnt/β‐catenin signaling pathway

Glioma is the most common brain tumor malignancy with high mortality and poor prognosis. Emerging evidence suggests that cancer stem cells are the key culprit in the development of cancer. MicroRNAs have been reported to be dysregulated in many cancers, while the mechanism underlying miR‐150‐5p in glioma progression and proportion of stem cells is unclear. The expression levels of miR‐150‐5p and catenin beta 1 (CTNNB1, which encodes β‐catenin) were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot. The expression levels of downstream genes of the Wnt/β‐catenin pathway and stem cell markers were detected by qRT‐PCR. Tumorigenesis was investigated by cell viability, colony formation, and tumor growth in vitro and in vivo. The interaction between miR‐150‐5p and β‐catenin was explored via bioinformatics analysis and luciferase activity assay. We found that miR‐150‐5p was downregulated in glioma and its overexpression inhibited cell proliferation, colony formation, and tumor growth. Moreover, miR‐150‐5p directly suppressed CTNNB1 and negatively regulated the abundances of downstream genes of the Wnt/β‐catenin pathway and stem cell markers. Furthermore, miR‐150‐5p expression was decreased and β‐catenin level was enhanced in CD133+ glioma stem cells. Knockdown of miR‐150‐5p contributed to CD133? cells with stem cell‐like phenotype, whereas overexpression of miR‐150‐5p suppressed CD133+ glioma stem cell‐like characteristics. In conclusion, miR‐150‐5p inhibited the progression of glioma by controlling stem cell‐like characteristics via regulating the Wnt/β‐catenin pathway, providing a novel target for glioma treatment.     

Curcumin reinforces MSC‐derived exosomes in attenuating osteoarthritis via modulating the miR‐124/NF‐kB and miR‐143/ROCK1/TLR9 signalling pathways

Curcumin treatment was reported to delay the progression of OA, but its underlying mechanism remains unclear. In this study, we aimed to investigate the molecular mechanism underlying the role of curcumin in OA treatment. Accordingly, by conducting MTT and flow cytometry assays, we found that the exosomes derived from curcumin‐treated MSCs helped to maintain the viability while inhibiting the apoptosis of model OA cells. Additionally, quantitative real‐time PCR and Western blot assays showed that the exosomes derived from curcumin‐treated MSCs significantly restored the down‐regulated miR‐143 and miR‐124 expression as well as up‐regulated NF‐kB and ROCK1 expression in OA cells. Mechanistically, curcumin treatment decreased the DNA methylation of miR‐143 and miR‐124 promoters. In addition, the 3’ UTRs of NF‐kB and ROCK1 were proven to contain the binding sites for miR‐143 and miR‐124, respectively. Therefore, the up‐regulation of miR‐143 and miR‐124 in cellular and mouse OA models treated with exosomes remarkably restored the normal expression of NF‐kB and ROCK1. Consequently, the progression of OA was attenuated by the exosomes. Our results clarified the molecular mechanism underlying the therapeutic role of MSC‐derived exosomes in OA treatment.     

The mechanism of long non‐coding RNA MEG3 for hepatic ischemia‐reperfusion: Mediated by miR‐34a/Nrf2 signaling pathway

To investigate the function of MEG3 in hepatic ischemia‐reperfusion (HIR) progress, involving its association with the level of miR‐34a during hypoxia‐induced hypoxia re‐oxygenation (H/R) in vitro. HIR mice model in vivo was established. MEG3, miR‐34a expression, along with Nrf2 mRNA and protein level were detected in tissues and cells. Serum biochemical parameters (ALT and AST) were assessed in vivo. A potential binding region between MEG3 and miR34a was confirmed by luciferase assays. Hepatic cells HL7702 were subjected to hypoxia treatment in vitro for functional studies, including TUNEL‐positive cells detection and ROS analysis. MEG3, Nrf2 expression was significantly down‐regulated in infarction lesion from HIR mice, as opposed to increased miR‐34a production, while similar results were also observed in H/R HL7702 cells, while the above effects were reversed by MEG3 over‐expression. By using bioinformatics study and RNA pull down combined with luciferase assays, we demonstrated that MEG3 functioned as a competing endogenous RNA (ceRNA) for miR‐34a, and there was reciprocal repression between MEG3 and miR‐34a in an Argonaute 2‐dependent manner. Functional studies demonstrated that MEG3 showed positive regulation on TUNEL‐positive cells and ROS level. Further in vivo study confirmed that MEG3 over‐expression could improve hepatic function of HIR mice, and markedly decreased the expression of serum ALT and AST. MEG3 protected hepatocytes from HIR injury through down‐regulating miR‐34a expression, which could add our understanding of the molecular mechanisms in HIR injury.     

ACE2‐EPC‐EXs protect ageing ECs against hypoxia/reoxygenation‐induced injury through the miR‐18a/Nox2/ROS pathway

Oxidative stress is one of the mechanisms of ageing‐associated vascular dysfunction. Angiotensin‐converting enzyme 2 (ACE2) and microRNA (miR)‐18a have shown to be down‐regulated in ageing cells. Our previous study has shown that ACE2‐primed endothelial progenitor cells (ACE2‐EPCs) have protective effects on endothelial cells (ECs), which might be due to their released exosomes (EXs). Here, we aimed to investigate whether ACE2‐EPC‐EXs could attenuate hypoxia/reoxygenation (H/R)‐induced injury in ageing ECs through their carried miR‐18a. Young and angiotensin II‐induced ageing ECs were subjected to H/R and co‐cultured with vehicle (medium), EPC‐EXs, ACE2‐EPCs‐EXs, ACE2‐EPCs‐EXs + DX600 or ACE2‐EPCs‐EXs with miR‐18a deficiency (ACE2‐EPCs‐EXs^{anti‐miR‐18a}). Results showed (1) ageing ECs displayed increased senescence, apoptosis and ROS production, but decreased ACE2 and miR‐18a expressions and tube formation ability; (2) under H/R condition, ageing ECs showed higher rate of apoptosis, ROS overproduction and nitric oxide reduction, up‐regulation of Nox2, down‐regulation of ACE2, miR‐18a and eNOS, and compromised tube formation ability; (3) compared with EPC‐EXs, ACE2‐EPC‐EXs had better efficiencies on protecting ECs from H/R‐induced changes; (4) The protective effects were less seen in ACE2‐EPCs‐EXs + DX600 and ACE2‐EPCs‐EXs^{anti‐miR‐18a} groups. These data suggest that ACE‐EPCs‐EXs have better protective effects on H/R injury in ageing ECs which could be through their carried miR‐18a and subsequently down‐regulating the Nox2/ROS pathway.     

Tau overexpression impairs neuronal endocytosis by decreasing the GTPase dynamin 1 through the miR‐132/MeCP2 pathway

Tauopathies are a class of neurodegenerative diseases that are characterized by pathological aggregation of tau protein, which is accompanied by synaptic disorders. However, the role of tau in endocytosis, a fundamental process in synaptic transmission, remains elusive. Here, we report that forced expression of human tau (hTau) in mouse cortical neurons impairs endocytosis by decreasing the level of the GTPase dynamin 1 via disruption of the miR‐132‐MeCP2 pathway; this process can also be detected in the brains of Alzheimer's patients and hTau mice. Our results provide evidence for a novel role of tau in the regulation of presynaptic function.     

MicroRNA‐132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras‐MAPK pathway

Arteriogenesis is a complicated process induced by increased local shear‐and radial wall‐stress, leading to an increase in arterial diameter. This process is enhanced by growth factors secreted by both inflammatory and endothelial cells in response to physical stress. Although therapeutic promotion of arteriogenesis is of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades via microRNAs. We observed that miR‐132/212 expression was significantly upregulated after occlusion of the femoral artery. miR‐132/212 knockout (KO) mice display a slower perfusion recovery after hind‐limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although Ex vivo aortic ring assays score similar number of branches in miR‐132/212 KO mice compared to WT, it can be stimulated with exogenous miR‐132, a dominant member of the miR‐132/212 family. Moreover, in in vitro pericyte‐endothelial co‐culture cell assays, overexpression of miR‐132 and mir‐212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR‐132/212 may exert their effects by enhancing the Ras‐Mitogen‐activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR‐132/212 cluster promotes arteriogenesis by modulating Ras‐MAPK signalling via direct targeting of its inhibitors Rasa1 and Spred1.