microRNA-186 inhibition of PI3K–AKT pathway via SPP1 inhibits chondrocyte apoptosis in mice with osteoarthritis

Chondrocyte apoptosis has been implicated as a major pathological osteoarthritis (OA) change in humans and experimental animals. We evaluate the ability of miR-186 on chondrocyte apoptosis and proliferation in OA and elucidate the underlying mechanism concerning the regulation of miR-186 in OA. Gene expression microarray analysis was performed to screen differentially expressed messenger RNAs (mRNAs) in OA. To validate the effect of miR-186 on chondrocyte apoptosis, we upregulated or downregulated endogenous miR-186 using mimics or inhibitors. Next, to better understand the regulatory mechanism for miR-186 governing SPP1, we suppressed the endogenous expression of SPP1 by small interfering RNA (siRNA) against SPP1 in chondrocytes. We identified SPP1 is highly expressed in OA according to an mRNA microarray data set GSE82107. After intra-articular injection of papain into mice, the miR-186 is downregulated while the SPP1 is reciprocal, with dysregulated PI3K–AKT pathway in OA cartilages. Intriguingly, miR-186 was shown to increase chondrocyte survival, facilitate cell cycle entry in OA chondrocytes, and inhibit chondrocyte apoptosis in vitro by modulation of pro- and antiapoptotic factors. The determination of luciferase activity suggested that miR-186 negatively targets SPP1. Furthermore, we found that the effect of miR-186 suppression on OA chondrocytes was lost when SPP1 was suppressed by siRNA, suggesting that miR-186 affected chondrocytes by targeting and depleting SPP1, a regulator of PI3K–AKT pathway. Our findings reveal a novel mechanism by which miR-186 inhibits chondrocyte apoptosis in OA by interacting with SPP1 and regulating PI3K–AKT pathway. Restoring miR-186 might be a future therapeutic strategy for OA.     

Berberine reduces ischemia/reperfusion-induced myocardial apoptosis via activating AMPK and PI3K–Akt signaling in diabetic rats

Diabetes increases the risk of cardiovascular diseases. Berberine (BBR), an isoquinoline alkaloid used in Chinese medicine, exerts anti-diabetic effect by lowering blood glucose and regulating lipid metabolism. It has been reported that BBR decreases mortality in patients with chronic congestive heart failure. However, the molecular mechanisms of these beneficial effects are incompletely understood. In the present study, we sought to determine whether BBR exerts cardioprotective effect against ischemia/reperfusion (I/R) injury in diabetic rats and the underlying mechanisms. Male Sprague-Dawley rats were injected with low dose streptozotocin and fed with a high-fat diet for 12 weeks to induce diabetes. The diabetic rats were intragastrically administered with saline or BBR (100, 200 and 400 mg/kg/d) starting from week 9 to 12. At the end of week 12, all rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion. BBR significantly improved the recovery of cardiac systolic/diastolic function and reduced myocardial apoptosis in diabetic rats subjected to myocardial I/R. Furthermore, in cultured neonatal rat cardiomyocytes, BBR (50 μmol/L) reduced hypoxia/reoxygenation-induced myocardial apoptosis, increased Bcl-2/Bax ratio and decreased caspase-3 expression, together with enhanced activation of PI3K–Akt and increased adenosine monophosphate-activated protein kinase (AMPK) and eNOS phosphorylation. Pretreatment with either PI3K/Akt inhibitor wortmannin or AMPK inhibitor Compound C blunted the anti-apoptotic effect of BBR. Our findings demonstrate that BBR exerts anti-apoptotic effect and improves cardiac functional recovery following myocardial I/R via activating AMPK and PI3K–Akt–eNOS signaling in diabetic rats.     

αB-crystallin regulates oxidative stress-induced apoptosis in cardiac H9c2 cells via the PI3K/AKT pathway

The present study was carried out to observe the protective effects of αB-crystallin protein on hydrogen peroxide (H₂O₂)-induced injury in rat myocardial cells (H9c2) and to investigate the mechanisms of these protective effects at the cellular level, which could provide the experimental basis for future applications of αB-crystallin in the treatment of cardiovascular disease. Western blotting was used to measure the expression of αB-crystallin in cultured H9c2 cells in vitro. A αB-crystallin recombinant expression vector, pcDNA3.1-Cryab, was constructed to transfect H9c2 cells for the establishment of cells that stably expressed αB-crystallin. A tetrazolium-based colorimetric assay (MTT test) was used to measure changes in the viability of the H9c2 cells at 1, 2, 3 and 4 h after induced by 150 μM H₂O₂ to establish a model of H₂O₂ injury to cells. H₂O₂ was applied to H9c2 cells that were stably transfected with αB-crystallin, and the effect of αB-crystallin overexpression on the viability of myocardial cells subjected to H₂O₂-induced injury was measured by the MTT assay. The effect of αB-crystallin overexpression on the H₂O₂-induced injury of H9c2 cells was also analyzed by flow cytometry. The mitochondrial components and cytoplasmic components of H9c2 cells were separated, and western blotting was used to measure the effect of αB-crystallin overexpression on the release of cytochrome c from the mitochondria. Western blotting was also used to measure the effect of αB-crystallin overexpression on the expression of the anti-apoptosis protein Bcl-2 and components of the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathway. The αB-crystallin recombinant expression vector pcDNA3.1-Cryab successfully transfected H9c2 cells, and H9c2 cells that were stably transfected with αB-crystallin were established after G418 selection. The measurements carried out by western blotting showed that αB-crystallin proteins are expressed in normal H9c2 cells, but the proteins’ expression was much higher in pcDNA3.1-Cryab transfected cells (P < 0.01). The MTT assays showed that 4 h of H₂O₂ treatment induced significant injury in H9c2 cells (P < 0.01), but αB-crystallin overexpression can effectively antagonize the H₂O₂-induced injury to H9c2 cells (P < 0.05). The results of flow cytometry analysis showed that αB-crystallin overexpression can significantly reduce apoptosis in H₂O₂-injured H9c2 cells (P < 0.05). The results of western blotting showed that αB-crystallin overexpression in myocardial cells can reduce the H₂O₂-induced release of cytochrome c from the mitochondria (P < 0.05), antagonize the H₂O₂-induced downregulation of Bcl-2 (P < 0.05) and magnify the decrease in phosphorylated AKT levels induced by H₂O₂ injury (P < 0.05). The overexpression of αB-crystallin has a protective effect on H₂O₂-injured H9c2 cells, and αB-crystallin can play a protective role by reducing apoptosis, reducing the release of cytochrome c from the mitochondria and antagonizing the downregulation of Bcl-2 expression. The protective effects of αB-crystallin may be related to the PI3K/AKT pathway.     

SDF-1α inhibits hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells through PI3K/Akt and ERK1/2 signaling pathways

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be a promising cell sources for cardiac regeneration. Poor survival rate of transplanted BMSCs in infarcted myocardium attenuated its clinical application. It’s reported that stromal-derived factor-1 (SDF-1) could protect progenitor cells including endothelial progenitor cells and embryonic stem cells from apoptosis. But little is known whether SDF-1α protein has the same protective effects on BMSCs under conditions of hypoxia and serum deprivation (hypoxia/SD). In present study, we verified that SDF-1α (0.50–2.0 μg/ml) inhibited hypoxia/SD induced apoptosis of BMSCs through mitochondrial pathway. After administration of SDF-1α, the loss of mitochondrial membrane potential and cytochrome c released from mitochondria to cytosol were significantly inhibited, and caspase 3 activity also declined. Furthermore, the effect of SDF-1α on mitochondrial pathway was neutralized by using PI3K inhibitor (Wortmannin) and ERK1/2 inhibitor (U0126). Our observations suggested that SDF-1α inhibits hypoxia/SD induced BMSCs apoptosis through PI3K/Akt and ERK1/2 signaling pathways. These data also imply that the anti-apoptotic effect mediated by SDF-1α may enhance cell survival after cell transplantation.     

USP49 inhibits ischemia–reperfusion-induced cell viability suppression and apoptosis in human AC16 cardiomyocytes through DUSP1–JNK1/2 signaling

Dual-specificity protein phosphatases (DUSP) also known as mitogen-activated protein kinase (MAPK) phosphatases (MKPs) can dephosphorylate MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38. DUSP1-mediated JNK dephosphorylation has been found to play an antiapoptotic role against cardiac ischemia–reperfusion (I/R) injury. However, the regulation of DUSP1–JNK pathway remains unclear. In the current study, ubiquitin-specific peptidase 49 (USP49) expression in human AC16 cardiomyocytes following I/R injury was measured by real-time polymerase chain reaction and western blot analysis. Cell viability, apoptosis, the Bax, Bcl-2, and DUSP1 expression, and the activity of MAPKs in AC16 cardiomyocytes following indicated treatment was measured by CCK-8, flow cytometry, and western blot analysis. The direct interaction between USP49 and DUSP1 was measured by coimmunoprecipitation and ubiquitination analysis. The effect of USP49 on apoptosis and JNK activity in rat cardiomyocytes following I/R injury was also measured by TUNEL and western blot analysis. Here, we found that USP49 expression was time-dependently increased in AC16 cardiomyocytes following I/R. I/R-induced cell apoptosis and JNK1/2 activation both in in vivo and in vitro reversed by USP49 overexpression in AC16 cardiomyocytes. Inhibiting JNK1/2 activation significantly inhibited USP49 knockdown-induced the cell viability inhibition, apoptosis and the JNK1/2 activation in AC16 cardiomyocytes. Moreover, USP49 positively regulated DUSP1 expression through deubiquitinating DUSP1. Overall, our findings establish USP49 as a novel regulator of DUSP1–JNK1/2 signaling pathway with a protective role in cardiac I/R injury.     

PSMB4 inhibits cardiomyocyte apoptosis via activating NF-κB signaling pathway during myocardial ischemia/reperfusion injury

Journal of Molecular Histology - Myocardial ischemia/reperfusion (I/R) injury induces cardiomyocyte apoptosis to deteriorate heart function. Thus, how to inhibit cardiomyocyte apoptosis is the...     

Long noncoding RNA MALAT-1 inhibits apoptosis and matrix metabolism disorder in interleukin-1β-induced inflammation in articular chondrocytes via the JNK signaling pathway

Proinflammatory cytokine such as interleukin (IL)-1β causes inflammation of articular cartilage. In this current study, we explored the chondroprotective effects of long noncoding RNA (lncRNA) MALAT-1 on cell proliferation, apoptosis, and matrix metabolism in IL-1β-induced inflammation in articular chondrocytes. Articular chondrocytes from knee joints of normal rats were isolated and cultured, followed by identification through observation of toluidine blue and COL II immunocytochemical stainings. The proliferation of chondrocytes at passage 2 was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The inflammatory chondrocytes induced by 10 ng/mL IL-1β were observed and identified by toluidine blue and COL II immunocytochemical stainings. pcDNA 3.1 and pcDNA-MALAT-1 were transfected in the chondrocytes. Ultrastructure of chondrocytes was observed by using a transmission electron microscope. The MTT assay was carried out to evaluate chondrocyte viability. Hoechst 33258 staining and flow cytometry were adopted to assess chondrocyte apoptosis. The chondrocytes at passage 2 with the biological characteristics of chondrocytes were used for subsequent experiments. In IL-1β-treated chondrocytes, the growth rate of chondrocytes slowed down, the cells became narrow and long, the vacuoles were seen in the cells, and the morphology of the chondrocytes was irregular. The toluidine blue staining and the immunohistochemical staining of COL II became weaker. In response to IL-1β induction, articular chondrocytes showed reduced MALAT-1 expression; moreover, obvious cartilage injury was observed with decreased chondrocyte viability and Col II expression and elevated chondrocyte apoptosis, MMP-13 expression, and p-JNK expression. With the treatment of pcDNA-MALAT-1, the cartilage injury was alleviated with increased chondrocyte viability and type II collagen (Col II) expression and reduced chondrocyte apoptosis, MMP-13 expression and p-JNK expression. Taken together these results, lncRNA MALAT-1 blocked the activation of the JNK signaling pathway; thereby, IL-1β-induced inflammation in articular chondrocytes was reduced with enhanced chondrocyte proliferation and suppressed chondrocyte apoptosis and extracellular matrix degradation.     

Overexpression of long noncoding RNA SLC26A4-AS1 inhibits the epithelial–mesenchymal transition via the MAPK pathway in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is recognized as one of the most prevalent types of thyroid cancer with poor prognosis. Long noncoding RNA (lncRNA) has undergone an intensive study for their involvement in tumor treatment. This study intends to unravel the association of lncRNA SLC26A4-AS1 with PTC. Initially, PTC-related expression profiling data (GSE33630) was utilized to screen differentially expressed lncRNAs in PTC and the underlying mechanisms involved with the mitogen-activated protein kinase (MAPK) pathway. Moreover, PTC tumor tissues and paracancerous tissues were arranged to determine expressions of TP53, SLC26A4-AS1, and genes related to epithelial–mesenchymal transition (EMT) and the MAPK pathway. Furthermore, SLC26A4-AS1 was overexpressed or underexpressed and JNK was underexpressed through cell transfection to examine the effect of SLC26A4-AS1 on PTC via MAPK pathway. Besides, tumor formation in nude mice was used to verify the fore experiment. LncRNA SLC26A4-AS1 regulating TP53 had the potential to participate in PTC by regulating the MAPK pathway. SLC26A4-AS1 was expressed poorly in PTC. Notably, SLC26A4-AS1 elevated E-cadherin expression while it reduced that of ERK and Vimentin. In addition, the overexpression of SLC26A4-AS1 inactivated the MAPK pathway by promoting TP53 and decreased cell migration, proliferation, and invasion. In addition to all these effects, the overexpression of SLC26A4-AS1 promoted apoptosis of TPC-1 cells. Additionally, the overexpression of lncRNA SLC26A4-AS1 reduced xenograft tumor volume in nude mice. Furthermore, the effect of SLC26A4-AS1 overexpression was found to be promoted after the MAPK pathway inactivation. Taken together, the overexpression of lncRNA SLC26A4-AS1 coffered anti-oncogenic effects on PTC through the inactivation of the MAPK pathway.     

PCSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax–caspase9–caspase3 pathway

This paper investigated the effects of ox-LDL on PCSK9, and the molecular mechanisms of PCSK9 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cells (HUVECs), to clarify the role of PCSK9 in atherosclerogenesis. HUVECs were incubated with ox-LDL for 24?h. The apoptosis was observed by Hoechst 33258 staining. The expression of PCSK9, LOX-1 mRNAs and proteins was detected by RT-PCR, western blot, respectively. The PCSK9 siRNAs labeled with fluorescence were transfected into HUVECs by Lipofectamine 2000. After transfection for 24?h, cells were treated with ox-LDL for 24?h, HUVECs apoptosis transfected siRNA was detected by Hoechst 33258 staining and flow cytometer. The expression of Bcl-2, Bax, caspase3, 8, 9 was detected by western blot. The activity of caspase3, 9 was detected by kits. Our results showed that apoptosis of HUVECs and the expressions of PCSK9 and LOX-1 were upregulated secondary to induction by ox-LDL in a concentration-dependent manner. However, ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression, were significantly reduced by PCSK9 siRNA. These results demonstrate a linkage between HUVEC apoptosis and PCSK9 expression. Furthermore, we detected the possible pathway involved in apoptotic regulation by PCSK9 siRNA; our results showed that the expression of Bcl-2 decreased, whereas that of Bax increased. In addition, ox-LDL enhanced the activity of caspase9 and then caspase3. Pretreatment of HUVECs with PCSK9 siRNA blocked these effects of ox-LDL. These findings suggest that ox-LDL-induced HUVECs apoptosis could be inhibited by PCSK9 siRNA, in which Bcl/Bax-caspase9-caspase3 pathway maybe was involved through reducing the Bcl-2/Bax ratio and inhibited the activation of both caspase9 and 3.     

Long noncoding RNA OIP5-AS1 causes cisplatin resistance in osteosarcoma through inducing the LPAATβ/PI3K/AKT/mTOR signaling pathway by sponging the miR-340-5p

The abnormal expression of long noncoding RNAs (lncRNAs) plays an important role in the regulation of human cancer progression and drug resistance. The lncRNA OPI5-AS1 is a crucial regulator in some cancers; however, its role in cisplatin resistance of osteosarcoma remains unclear. We found that OIP5-AS1 was significantly upregulated in cisplatin-resistant (CR) osteosarcoma cells MG63-CR and SaOS2-CR compared with the corresponding parental cells. OIP5-AS1 silencing suppressed cell growth in vitro and in vivo, and promoted apoptosis of MG63-CR and SaOS2-CR cells, indicating that knockdown of OIP5-AS1 significantly decreased cisplatin resistance in MG63-CR and SaOS2-CR cells. This conclusion was supported by the decreased expression of the drug resistance-related factors multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp) upon OIP5-AS1 silencing. In addition, OIP5-AS1 downregulation suppressed the PI3K/AKT/mTOR signaling pathway. Importantly, we demonstrated that OIP5-AS1 functions as a competing endogenous RNA of miR-340-5p and regulates the expression of lysophosphatidic acid acyltransferase (LPAATβ), which is a target of miR-340-5p. Moreover, downregulation of miR-340-5p partly reversed the inhibitory effect of OIP5-AS1 knockdown on the PI3K/AKT/mTOR pathway and therefore counteracted cisplatin resistance in MG63-CR and SaOS2-CR cells. In conclusion, OIP5-AS1 causes cisplatin resistance in osteosarcoma through inducing the LPAATβ/PI3K/AKT/mTOR signaling pathway by sponging the miR-340-5p. Our results contribute to a better understanding of the function and mechanism of OIP5-AS1 in osteosarcoma cisplatin resistance.     

Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

The Hippo pathway is involved in the regulation of contact inhibition of proliferation and responses to various physical and chemical stimuli. Recently, several upstream negative regulators of Hippo signaling, including epidermal growth factor receptor ligands and lysophosphatidic acid, have been identified. We show that fibronectin adhesion stimulation of focal adhesion kinase (FAK)-Src signaling is another upstream negative regulator of the Hippo pathway. Inhibition of FAK or Src in MCF-10A cells plated at low cell density prevented the activation of Yes-associated protein (YAP) in a large tumor suppressor homologue (Lats)–dependent manner. Attachment of serum-starved MCF-10A cells to fibronectin, but not poly-d-lysine or laminin, induced YAP nuclear accumulation via the FAK–Src–phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K) signaling pathway. Attenuation of FAK, Src, PI3K, or PDK1 activity blocked YAP nuclear accumulation stimulated by adhesion to fibronectin. This negative regulation of the Hippo pathway by fibronectin adhesion signaling can, at least in part, explain the effects of cell spreading on YAP nuclear localization and represents a Lats-dependent component of the response to cell adhesion.     

Substance P inhibits high urea-induced apoptosis through the AKT/GSK-3β pathway in human corneal epithelial cells

To investigate the effect of substance P (SP) on human corneal epithelial cells (HCECs) that have been stressed by a high urea environment and to determine the relationship between SP and the protein kinase B (AKT)/glycogen synthase kinase-3β (GSK-3β) signaling pathway. An in vitro model of chronic renal failure (CRF)-related dry eye was used to study HCECs that were treated with high urea concentrations. Cell proliferation was assayed using a cell counting kit-8 test. Besides, cell apoptosis was evaluated by flow cytometry. Furthermore, the effects of SP and the AKT inhibitor perifosine on the urea-treated HCECs were examined using immunofluorescence, quantitative real time polymerase chain reaction (qRT-PCR), and Western blot analysis. SP markedly reduced the number of apoptotic HCECs and decreased the cleaved caspase-3 expression levels while contributing to increased cellular proliferation (P < 0.05). The Western blot analysis and qRT-PCR experiments revealed that SP significantly increased the expression of p-AKT and p-GSK-3β (P < 0.05); additionally, these increases were attenuated after the perifosine inhibition of the AKT signaling pathway (P < 0.05). These in vitro experiments demonstrated that SP may protect against the apoptotic damage of HCECs caused by the high urea condition. The underlying mechanism may be related to the activation of the AKT/GSK-3β signaling pathway.     

Retracted: Targeting of SPP1 by microRNA-340 inhibits gastric cancer cell epithelial–mesenchymal transition through inhibition of the PI3K/AKT signaling pathway

Gastric cancer (GC) is a common heterogeneous disease. The critical roles of microRNA-340 (miR-340) in the development and progression of GC were emphasized in accumulating studies. This study aims to examine the regulatory mechanism of miR-340 in GC cellular processes. Initially, microarray technology was used to identify differentially expressed genes and regulatory miRs in GC. After that, the potential role of miR-340 in GC was determined via ectopic expression, depletion, and reporter assay experiments. Expression of secreted phosphoprotein 1 (SPP1), miR-340, phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, and epithelial–mesenchymal transition (EMT)-related genes was measured. Moreover, to further explore the function of miR-340 in vivo and in vitro, proliferation, apoptosis, migration, invasion, and tumorigenic capacity were evaluated. SPP1 was a target gene of miR-340 which could then mediate the PI3K/AKT signaling pathway by targeting SPP1 in GC. Furthermore, miR-340 levels were reduced and SPP1 was enriched in GC tissues and cells, with the PI3K/AKT signaling pathway being activated. Inhibitory effects of upregulated miR-340 on SPP1 and the PI3K/AKT signaling pathway were confirmed in vivo and in vitro. Overexpression of miR-340 or the silencing of SPP1 inhibited GC cell proliferation, invasion, migration, and EMT process, but promoted apoptosis of GC cells. Typically, targeting of SPP1 by miR-340 may contribute to the inhibition of proliferation, migration, invasion, and EMT of GC cells via suppression of PI3K/AKT signaling pathway.     

The cardioprotective effect of dihydromyricetin prevents ischemia–reperfusion-induced apoptosis in vivo and in vitro via the PI3K/Akt and HIF-1α signaling pathways

Reperfusion therapy is widely used to treat acute myocardial infarction (AMI). However, further injury to the heart induced by rapidly initiating reperfusion is often encountered in clinical practice. A lack of pharmacological strategies in clinics limits the prognosis of patients with myocardial ischemia-reperfusion injury (MIRI). Dihydromyricetin (DMY) is one of the most abundant components in vine tea, commonly known as the tender stems and leaves of Ampelopsis grossedentata. The aim of this study was to evaluate the cardioprotection of DMY against myocardial ischemia-reperfusion (I/R) injury and to further investigate the underlying mechanism. An I/R injury was induced by left anterior descending coronary artery occlusion in adult male rats in vivo and a hypoxia–reoxygenation (H/R) injury in H9c2 cardiomyocytes in vitro. We found that DMY pretreatment provided significant protection against I/R-induced injury, including enhanced antioxidant capacity and inhibited apoptosis in vivo and in vitro. This effect correlated with the activation of the PI3K/Akt and HIF-1α signaling pathways. Conversely, blocking Akt activation with the PI3K inhibitor LY294002 effectively suppressed the protective effects of DMY against I/R-induced injury. In addition, the PI3K inhibitor partially blocked the effects of DMY on the upregulation of Bcl-2, Bcl-xl, procaspase-3, -8, and -9 protein expression and the downregulation of HIF-1α, Bnip3, Bax, Cyt-c, cleaved caspase-3, -8, and -9 protein expression. Collectively, these results showed that DMY decreased the apoptosis and necrosis by I/R treatment, and PI3K/Akt and HIF-1α plays a crucial role in protection during this process. These observations indicate that DMY has the potential to exert cardioprotective effects against I/R injury and the results might be important for the clinical efficacy of AMI treatment.     

Correction to: Isorhamnetin protects against cardiac hypertrophy through blocking PI3K–AKT pathway

Molecular and Cellular Biochemistry -