microRNA-21在食管癌细胞增殖、迁移和凋亡中的作用

本研究检测了40例食管癌组织和40例癌旁组织中的miR-21、PTEN、PI3K和AKT表达,并通过转染miR-21抑制剂来敲低人食管癌细胞系EC9706的miR-21表达,考察了miR-21对食管癌细胞生长的影响。研究发现,食管癌组织中PTEN蛋白的阳性染色评分低于癌旁组织(p<0.05),而PI3K和AKT蛋白的阳性染色评分高于癌旁组织(p<0.05)。miR-21在人食管癌组织中被上调(3.56 vs 1.21,p<0.05)。转染miR-21抑制剂导致PTEN蛋白表达升高,而PI3K和AKT蛋白表达降低(p<0.05)。转染miR-21抑制剂抑制了EC9706细胞的增殖和迁移,但促进了细胞凋亡(p<0.05)。miR-21的上调可通过激活PTEN/PI3K/AKT信号通路来促进食道癌细胞的增殖和迁移,并抑制细胞凋亡。     

miR-885 mediated cardioprotection against hypoxia/reoxygenation-induced apoptosis in human cardiomyocytes via inhibition of PTEN and BCL2L11 and modulation of AKT/mTOR signaling

Ischemia/reperfusion (I/R) injury could cause the enhanced cell apoptosis of cardiomyocytes, which is one of key contributors for the development of ischemic heart disease. Recent studies emphasized the role of microRNAs (miRNAs) in regulating cardiomyocyte apoptosis. The study planned to elucidate the molecular actions of miR-885 on mediating human cardiomyocytes (HCMs) apoptosis induced by hypoxia/reoxygenation (H/R) and to explore the potential molecular mechanisms. The present data revealed that H/R stimulation inhibited HCM viability and potentiated HCM apoptosis, and more importantly, the expression of miR-885 in HCMs was markedly repressed after H/R stimulation. Further experimental examinations demonstrated that overexpression of miR-885 attenuated H/R-induced increased in HCM apoptotic rates, while miR-885 knockdown impaired HCM viability and increased HCM apoptotic rates. Moreover, the mechanistic studies showed that miR-885 inversely regulated the expression of phosphatase and tensin homolog (PTEN) and BCL2 like 11 (BCL2L11) in HCMs, and enforced expression of PTEN and BCL2L11 partially antagonized the protective actions of miR-885 overexpression on H/R-induced HCM injury. Moreover, H/R suppressed AKT/mTOR signaling, which was attenuated by miR-885 overexpression in HCMs. In conclusion, the present study for the first time showed the downregulation of miR-885 induced by H/R in HCMs, and provided the evidence that miR-885 attenuated H/R-induced cell apoptosis via inhibiting PTEN and BLC2L11 and modulation of AKT/mTOR signaling in HCMs.     

过表达MicroRNA-21通过PTEN/PI3K/AKT信号通路调控人退变髓核细胞自噬的研究

目的:探讨过表达mi R-21通过PTEN/PI3K/AKT通路对人退变髓核细胞自噬的影响。方法:构建稳定过表达mi R-21 mimic人退变髓核细胞,转染无意义序列作为mi R-21 mimic control组,采用RT-qPCR检测转染效率;利用MDC荧光染色法观察细胞自噬泡;Western-Blot检测细胞自噬相关蛋白LC3和P62的表达以及PTEN/PI3K/AKT信号通路中关键蛋白PTEN、PI3K及AKT的表达水平。结果:RT-qPCR结果表明mi R-21 mimic转染成功且效率较高,与mi R-21 mimic control组及空白细胞对照组相比,差异显著(P0.05)。荧光显微镜观察MDC染色情况,mi R-21 mimic组的细胞中几乎没有发现自噬体,而mi R-21 mimic control组以及空白对照组细胞中自噬体均较多,与前者相比差异均明显,具有统计学意义(P0.05)。mi R-21 mimic组细胞中LC3-II/LC3-I表达量的比值均显著低于mi R-21 mimic control组及空白对照组细胞(P0.05);而P62在mi R-21 mimic组细胞中表达量显著高于mi R-21 mimic control组及空白细胞对照组,具有统计学意义(P0.05)。mi R-21 mimic组中PTEN蛋白的表达水平较低,与另外两组相比具有统计学意义(P0.05);磷酸化的PI3K(p-PI3K)和AKT(p-Akt)在mi R-21 mimic组中均明显高于mi R-21 mimic control组和空白细胞对照组,差异具有统计学意义(P0.05)。结论:mi R-21可以通过靶向沉默PTEN,促进PI3K和AKT发生磷酸化,进而使PTEN/PI3K/AKT信号通路被激活,最终抑制人椎间盘退变髓核细胞的自噬。     

Pyrroloquinoline quinone inhibits oxygen/glucose deprivation-induced apoptosis by activating the PI3K/AKT pathway in cardiomyocytes

The purposes of this study were to examine the protective effect of pyrroloquinoline quinone (PQQ) on oxygen/glucose deprivation (OGD)-induced injury to H9C2 rat cardiomyocytes and to investigate the mechanism. Using H9C2 cells cultured in vitro, we examined changes in cell viability with an MTT assay at 12, 24, and 48 h after injury induced by OGD. Various concentrations of PQQ (1, 10, and 100 μM) were added, and the effect of PQQ on cell viability after OGD was assessed using the MTT assay. Thus, the optimal concentration of PQQ for the protection of cardiomyocytes against oxygen and glucose deprivation injury was determined. We also used flow cytometry analysis to examine the effect of PQQ on H9C2 cells with OGD-induced injury. The molecular probe 2′,7′-dichlorofluorescin diacetate was used to label the H9C2 cells, and flow cytometry was used to detect the effect of PQQ on reactive oxygen species (ROS) content. After labeling the H9C2 cells using a mitochondrial green fluorescent probe (Mito-Tracker Green), we measured the change in the mitochondrial content of PQQ-treated H9C2 cells. Western blotting was used to examine the effect of PQQ on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the H9C2 cells. The results of the MTT assay showed that 48 h of OGD significantly injured the H9C2 cells (p < 0.01) and that treatment with 100 μM PQQ effectively decreased the level of OGD-induced injury (p < 0.01). The results of the flow cytometry analysis showed that PQQ significantly reduced apoptosis in H9C2 cells subjected to OGD (p < 0.05). In addition, OGD significantly increased the ROS level in H9C2 cells (p < 0.01), and PQQ significantly inhibited this increase (p < 0.05). The results of the Mito-Tracker Green staining suggested that PQQ effectively inhibited the decrease in mitochondrial content caused by OGD (p < 0.05). Western blot analysis showed that PQQ partially reversed the decrease in Akt phosphorylation that was caused by OGD (p < 0.05). PQQ treatment dose-dependently protects H9C2 cells from OGD-induced injury by reducing apoptosis, decreasing intracellular ROS levels, and rescuing the OGD-induced decrease in mitochondrial content. The protective effect of PQQ may be related to its effects on the PI3K/Akt pathway.     

miR-21 promotes proliferation and inhibits apoptosis of hepatic stellate cells through targeting PTEN/PI3K/AKT pathway

Abstract

To investigate the effect of microRNA 21 (miR-21) on hepatic stellate cells (HSCs) proliferation and apoptosis, and further to study its potential mechanisms. LX-2 cells were divided into miR-21 mimic group (Mimic), miR-21 mimic negative control group (NM), miR-21 inhibitor group (Inhibitor), miR-21 inhibitor negative control group (NC), and blank control group (Control). The cell proliferation was detected by CCK-8 assay and the cell migration and invasion were detected by scratch and transwell assay. Cell cycle and apoptosis were detected by flow cytometry. The levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β1 were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation, apoptosis, and phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway related genes and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. The cells proliferation, migration, and invasion were promoted in Mimic group. The levels of IL-6, TNF-α, and TGF-β1 were increased after miR-21 administration. The expression of α-smooth muscle actin (SMA) and collagen 1 (Colla1) were increased, while Bax/B-cell lymphoma (Bcl)-2 ratio and programed cell death 4 (PDCD4) were reduced after miR?21 treatment. Meanwhile, the mRNA and protein expression of PTEN were reduced and PI3K/AKT pathway been promoted. Our study demonstrated that miR-21 could promote proliferation and inhibit apoptosis of HSCs, and its mechanism may be related to PTEN/PI3K/AKT pathway.     

MiR-210 in exosomes derived from CAFs promotes non-small cell lung cancer migration and invasion through PTEN/PI3K/AKT pathway

ObjectiveCancer-associated fibroblasts (CAFs) function as a crucial factor in tumor progression by carrying exosomes to neighboring cells. This study was assigned to expound the underlying mechanism of CAFs-derived exosomal miR-210 in non-small cell lung cancer (NSCLC) progression.MethodCAFs and normal fibroblasts (NFs) were isolated and identified. Exosomes secreted from CAFs and NFs were isolated to analyze their effects on tumor volume and epithelial-mesenchymal transition (EMT). Exosomal miR-210 expression level was measured. The effects of exosomal miR-210 and UPF1 on cell viability, EMT, PTEN/PI3K/AKT signal pathway were determined. Dual-luciferase reporter gene assay was utilized to validate the binding of UPF1 to miR-210.ResultsCAFs-derived exosomes (CAFs-exo) were successfully extracted and proven to be uptake by lung cancer cells. Up-regulated expression level of miR-210 was found in CAFs-exo, which was then proved to enhance cell migration, proliferation, invasion abilities and EMT in NSCLC cells. Overexpression of miR-210 can also inhibit UPF1 and PTEN, but activate the PTEN/PI3K/AKT pathway. UPF1 was a target gene of miR-210. MiR-210 can up-regulate UPF1 expression level to activate PTEN/PI3K/AKT pathway.ConclusionMiR-210 secreted by CAFs-exo could promote EMT by targeting UPF1 and activating PTEN/PI3K/AKT pathway, thereby promoting NSCLC migration and invasion.     

Manipulation of Cardiac Phosphatidylinositol 3-Kinase (PI3K)/Akt Signaling by Apoptosis Regulator through Modulating IAP Expression (ARIA) Regulates Cardiomyocyte Death during Doxorubicin-induced Cardiomyopathy

PI3K/Akt signaling plays an important role in the regulation of cardiomyocyte death machinery, which can cause stress-induced cardiac dysfunction. Here, we report that apoptosis regulator through modulating IAP expression (ARIA), a recently identified transmembrane protein, regulates the cardiac PI3K/Akt signaling and thus modifies the progression of doxorubicin (DOX)-induced cardiomyopathy. ARIA is highly expressed in the mouse heart relative to other tissues, and it is also expressed in isolated rat cardiomyocytes. The stable expression of ARIA in H9c2 cardiac muscle cells increased the levels of membrane-associated PTEN and subsequently reduced the PI3K/Akt signaling and the downstream phosphorylation of Bad, a proapoptotic BH3-only protein. When challenged with DOX, ARIA-expressing H9c2 cells exhibited enhanced apoptosis, which was reversed by the siRNA-mediated silencing of Bad. ARIA-deficient mice exhibited normal heart morphology and function. However, DOX-induced cardiac dysfunction was significantly ameliorated in conjunction with reduced cardiomyocyte death and cardiac fibrosis in ARIA-deficient mice. Phosphorylation of Akt and Bad was substantially enhanced in the heart of ARIA-deficient mice even after treatment with DOX. Moreover, repressing the PI3K by cardiomyocyte-specific expression of dominant-negative PI3K (p110α) abolished the cardioprotective effects of ARIA deletion. Notably, targeted activation of ARIA in cardiomyocytes but not in endothelial cells reduced the cardiac PI3K/Akt signaling and exacerbated the DOX-induced cardiac dysfunction. These studies, therefore, revealed a previously undescribed mode of manipulating cardiac PI3K/Akt signaling by ARIA, thus identifying ARIA as an attractive new target for the prevention of stress-induced myocardial dysfunction.     

Reciprocal regulation of miR-23a and lysophosphatidic acid receptor signaling in cardiomyocyte hypertrophy

Earlier, our study demonstrated that lysophosphatidic acid (LPA) receptor mediated cardiomyocyte hypertrophy. However, the subtype-specific functions for LPA1 and LPA3 receptors in LPA-induced hypertrophy have not been distinguished. Growing evidence indicates that microRNAs (miRNAs) are involved in the pathogenesis of cardiac hypertrophy by down-regulating target molecules. The present work therefore aimed at elucidating the functions mediated by different subtypes of LPA receptors and investigating the modulatory role of miRNAs during LPA induced hypertrophy. Experiments were done with cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and we showed that knockdown of LPA1 by small interfering RNA (siRNA) enhanced LPA-induced cardiomyocyte hypertrophy, whereas LPA3 silencing repressed hypertrophy. miR-23a, a pro-hypertrophic miRNA, was up-regulated by LPA in cardiomyocytes and its down-regulation reduced LPA-induced cardiomyocyte hypertrophy. Importantly, luciferase reporter assay confirmed LPA1 to be a target of miR-23a, indicating that miR-23a is involved in mediating the LPA-induced cardiomyocyte hypertrophy by targeting LPA1. In addition, knockdown of LPA3, but not LPA1, eliminated miR-23a elevation induced by LPA. And PI3K inhibitor, LY294002, effectively prevented LPA-induced miR-23a expression in cardiomyocytes, suggesting that LPA might induce miR-23a elevation by activating LPA3 and PI3K/AKT pathway. These findings identified opposite subtype-specific functions for LPA1 and LPA3 in mediating cardiomyocyte hypertrophy and indicated LPA1 to be a target of miR-23a, which discloses a link between miR-23a and the LPA receptor signaling in cardiomyocyte hypertrophy.     

Retracted: 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.     

Pseudogene PTENP1 sponges miR-214 to regulate the expression of PTEN to modulate osteoclast differentiation and attenuate osteoporosis

Background aimsOsteoporosis (OP) is a common bone metabolic disease with a high incidence. Our study aimed to explore the pseudogene PTENP1/miR-214/PTEN axis to modulate the osteoclast differentiation in osteoporosis.MethodsPatients with osteoporosis were recruited in our study, and RANKL-induced osteoclast differentiation and ovariectomy-induced osteoporosis mouse model were established in vitro and in vivo, respectively.ResultsPseudogene PTENP1 and PTEN were significantly down-regulated and miR-214 was up-regulated in osteoporosis patients. In addition, overexpression of PTENP1 or silence of miR-214 inhibited the expression levels of osteoclast specific markers and osteoclast differentiation induced by RANKL. Overexpression of PTENP1 or silence of miR-214 also inhibited the levels of phosphorylation of PI3K and AKT, p65 nuclear translocation, IκBα degradation and the expression level of NFATc1. AlsoSilence of PTENP1 or overexpression of miR-214 induced the osteoclast differentiation under normal physiological condition. Pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN.ConclusionsIn an ovariectomy-induced osteoporosis mouse model, obvious pathological changes in bone tissues were found, and bone marrow mononuclear cells in this group were more likely to differentiate into osteoclasts. Therefore, pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN to inhibit osteoclast differentiation and attenuate osteoporosis by suppressing the PI3K/AKT/NF-κB signaling pathway.     

HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway

Kaposi''s sarcoma (KS) is an AIDS-defining cancer with aberrant neovascularization caused by KS-associated herpesvirus (KSHV). Although the interaction between HIV-1 and KSHV plays a pivotal role in promoting the aggressive manifestations of KS, the pathogenesis underlying AIDS-KS remains largely unknown. Here we examined HIV-1 Nef protein promotion of KSHV oncoprotein K1-induced angiogenesis. We showed that both internalized and ectopic expression of Nef in endothelial cells synergized with K1 to facilitate vascular tube formation and cell proliferation, and enhance angiogenesis in a chicken CAM model. In vivo experiments further indicated that Nef accelerated K1-induced angiogenesis and tumorigenesis in athymic nu/nu mice. Mechanistic studies revealed that Nef and K1 synergistically activated PI3K/AKT/mTOR signaling by downregulating PTEN. Furthermore, Nef and K1 induced cellular miR-718, which inhibited PTEN expression by directly targeting a seed sequence in the 3′ UTR of its mRNA. Inhibition of miR-718 expression increased PTEN synthesis and suppressed the synergistic effect of Nef- and K1-induced angiogenesis and tumorigenesis. These results indicate that, by targeting PTEN, miR-718 mediates Nef- and K1-induced angiogenesis via activation of AKT/mTOR signaling. Our results demonstrate an essential role of miR-718/AKT/mTOR axis in AIDS-KS and thus may represent an attractive therapeutic target.     

Retracted: Downregulation of microRNA-1469 promotes the development of breast cancer via targeting HOXA1 and activating PTEN/PI3K/AKT and Wnt/β-catenin pathways

Breast cancer (BC) is a common malignancy which is the most frequently diagnosed cancer in women all over the worldwide. This study aimed to investigate the roles of miR-1469 in the development of BC, as well as its regulatory mechanism. The expression levels of miR-1469 in BC tissues, serum, and cell lines were determined. Effects of overexpression of miR-1469 on MCF7 cell viability, colony-forming ability, apoptosis, migration, and invasion were then investigated. Furthermore, the potential target of miR-1469 in MCF7 cells was explored. Besides, the association between miR-1469, PTEN/PI3K/AKT, and Wnt/β-catenin pathways was elucidated. Notably, confirmatory experiments by downregulation of miR-1469 in SK-BR-3 cells were further performed. The miR-1469 expression was significantly downregulated in BC tissues, serum, and cell lines. The overexpression of miR-1469 significantly inhibited the proliferation, arrested cell-cycle at G2/M phase, increased apoptosis, suppressed migration, and invasion of MCF-7 cells. In addition, HOXA1 was verified as a direct target of miR-1469, and the effects of overexpression of miR-1469 on the malignant behaviors of MCF7 cells were significantly counteracted by overexpression of HOXA1 concurrently. Furthermore, the overexpression of miR-1469 suppressed the activation of PTEN/PI3K/AKT and Wnt/β-catenin pathways, which was reversed overexpression of HOXA1 concurrently. Besides, confirmatory experiments showed that the inhibition of miR-1469 promoted the malignant behaviors of SK-BR-3 cells, which was inversed after miR-1469 inhibition and HOXA1 knockdown at the same time. Our findings reveal that downregulation of miR-1469 may promote the development of BC by targeting HOXA1 and activating PTEN/PI3K/AKT and Wnt/β-catenin pathways. MiR-1469 may serve as a promising target for BC therapy.