LncRNA OIP5-AS1 facilitates ox-LDL-induced endothelial cell injury through the miR-98-5p/HMGB1 axis

Cerebrovascular diseases have a high mortality and disability rate in developed countries. Endothelial cell injury is the main cause of atherosclerosis and cerebrovascular disease. Long non-coding RNA (lncRNA) has been proved to participate in the progression of endothelial cell. Our study aimed to develop the function of lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury. The expression of OIP5-AS1, miR-98-5p and High-mobility group protein box-1 (HMGB1) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry were used to detect the cell proliferation and apoptosis. The levels of cyclinD1, Bcl-2 Associated X Protein (Bax), Cleaved-caspase-3, Toll like receptors 4 (TLR4), phosphorylation of p65 (p-P65), phosphorylation of nuclear factor-kappa B inhibitor α (p-IκB-α) and HMGB1 were measured by Western blot. The concentrations of Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and Tumor necrosis factor-α (TNF-α) were detected by Enzyme-linked immunosorbent assay (ELISA). The production of Reactive oxygen species (ROS), Superoxide Dismutase (SOD) and malondialdehyde (MDA) was detected by the corresponding kit. The targets of OIP5-AS and miR-98-5p were predicted by starBase 3.0 and TargetScan and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The expression of OIP5-AS1 was upregulated, while miR-98-5p was downregulated in ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Functionally, knockdown of OIP5-AS1 induced proliferation and inhibited apoptosis, inflammatory injury and oxidative stress injury in ox-LDL-induced HUVEC cells. Interestingly, miR-98-5p was a target of OIP5-AS1 and miR-98-5p inhibition abolished the effects of OIP5-AS1 downregulation on ox-LDL-induced HUVECs injury. More importantly, miR-98-5p directly targeted HMGB1, and OIP5-AS1 regulated the expression of HMGB1 by sponging miR-98-5p. Finally, OIP5-AS1 regulated the TLR4/nuclear factor-kappa B (NF-κB) signaling pathway through miR-98-5p/HMGB1 axis. LncRNA OIP5-AS1 accelerates ox-LDL-induced endothelial cell injury through regulating HMGB1 mediated by miR-98-5p via the TLR4/NF-κB signaling pathway.

     

MicroRNA-328 ameliorates oxidized low-density lipoprotein-induced endothelial cells injury through targeting HMGB1 in atherosclerosis

Atherosclerosis has been recognized as a chronic inflammatory disease, which can harden the vessel wall and narrow the arteries. MicroRNAs exhibit crucial roles in various diseases including atherosclerosis. However, so far, the role of miR-328 in atherosclerosis remains barely explored. Therefore, our study concentrated on the potential role of miR-328 in vascular endothelial cell injury during atherosclerosis. In our current study, we observed that oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) apoptosis and inhibited cell viability dose-dependently and time-dependently. In addition, indicated dosage of ox-LDL obviously triggered HUVECs inflammation and oxidative stress process. Then, it was found that miR-328 in HUVECs was reduced by ox-LDL. HUVECs apoptosis was greatly repressed and cell survival was significantly upregulated by overexpression of miR-328. Furthermore, mimics of miR-328 rescued cell inflammation and oxidative stress process induced by ox-LDL. Oppositely, inhibitors of miR-328 strongly promoted ox-LDL-induced endothelial cells injury in HUVECs. By using bioinformatics analysis, high-mobility group box-1 (HMGB1) was predicted as a downstream target of miR-328. HMGB1 has been reported to be involved in atherosclerosis development. The correlation between miR-328 and HMGB1 was validated in our current study. Taken these together, it was implied that miR-328 ameliorated ox-LDL-induced endothelial cells injury through targeting HMGB1 in atherosclerosis.     

Circ_0003423 Alleviates ox-LDL-Induced Human Brain Microvascular Endothelial Cell Injury via the miR-589-5p/TET2 Network

Brain microvascular endothelial cells (BMECs) injury is one of the main causes of cerebrovascular diseases. Circular RNA (circRNA) has been found to be involved in the regulation of cerebrovascular diseases progression. However, the role and mechanism of circ_0003423 in cerebrovascular diseases is still unclear. In our study, oxidized low density lipoprotein (ox-LDL)-induced HBMEC-IM cells were used to construct cerebrovascular cell injury model in vitro. Quantitative real-time PCR was used to determine the expression levels of circ_0003423, miR-589-5p and Ten-eleven translocation 2 (TET2). The interactions between miR-589-5p and circ_0003423 or TET2 were confirmed by dual-luciferase reporter assay, RIP assay and RNA pull-down assay. Cell viability, angiogenesis and apoptosis were measured using cell counting kit 8 assay, tube formation assay and flow cytometry. Cell oxidative stress was evaluated by detecting the levels of reactive oxygen species and lactate dehydrogenase. The protein levels were examined by western blot analysis. Our results showed that circ_0003423 was a downregulated circRNA in ox-LDL-induced HBMEC-IM cells. In the terms of mechanism, circ_0003423 was found to be a sponge of miR-589-5p. Function analysis showed that circ_0003423 overexpression could relieve ox-LDL-induced HBMEC-IM cell injury, and this effect could be reversed by miR-589-5p mimic. In addition, TET2 was confirmed to be a target of miR-589-5p, and its overexpression could alleviate ox-LDL-induced HBMEC-IM cell injury. Moreover, the rescue experiments also confirmed that TET2 silencing could abolish the inhibition effect of anti-miR-589-5p on ox-LDL-induced HBMEC-IM cell injury. In summary, our data showed that circ_0003423 alleviated ox-LDL-induced HBMEC-IM cells injury through regulating the miR-589-5p/TET2 axis.

     

miR-216b-5p regulates proliferation and apoptosis of ox-LDL-stimulated VSMCs and HUVECs via IGF2

Atherosclerosis (AS) is one of the principal causes of cardiovascular disorder. Reportedly, vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) play key roles in AS development, and microRNAs (miRNAs) regulate their functions. The function of miR-216b-5p in AS remains unknown. Human VSMCs and human HUVECs were treated with ox-LDL to establish the in vitro model of AS. MiR-216b-5p and IGF2 expressions in VSMCs and HUVECs were probed by qRT-PCR and western blot. The viability, cell cycle progression, and apoptosis of VSMCs and HUVECs were evaluated by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine, and flow cytometry assays, respectively. The binding sites between IGF2 3′UTR and miR-216b-5p were validated by dual-luciferase reporter assay. miR-216b-5p expression was declined in ox-LDL-induced VSMCs and HUVECs. In VSMCs, miR-216b-5p overexpression inhibited excessive proliferation and induced apoptosis. MiR-216b-5p could markedly restrain the viabiblity of VSMCs induced by ox-LDL and enhanced the viability of HUVECs. Additionally, IGF2 was confirmed as the direct target of miR-216b-5p and transfection of IGF2 overexpression plasmids rescued the effects of miR-216b-5p on VSMCs and HUVECs. miR-216b-5p alleviates the dysfunction of VSMCs and HUVECs caused by ox-LDL via repressing IGF2, and exerts protective functions to block the development of AS.     

Rac3, but not Rac1, promotes ox-LDL induced endothelial dysfunction by downregulating autophagy

The endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis, which can lead to oxidative stress and inflammation. The role of autophagy in the process of atherosclerosis has drawn increasing attention. The human umbilical vein endothelial cells (HUVECs), whose Ras-related C3 botulinum toxin substrate 1 (Rac1) and Rac3 was knockdown, were used to detect whether the possible molecular mechanisms of Rac1 and Rac3 for anti-inflammatory in endothelial cells was effected by downregulation of autophagy. The HUVECs were incubated with ox-LDL. The inflammatory factors and autophagy proteins were evaluated to ascertain and compare the effect of Rac1 and Rac3 on autophagy. Then, 3-methyladenine (3-MA) as an inhibiter of autophagy was used to detect whether the effect of Rac1 and Rac3 was related to autophagy. ox-LDL-induced cell dysfunction in HUVECs was determined by testing the formation of foam cells, the expression of nuclear factor (NF)-κB and nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 and NF-κB p65 and other inflammatory factors, the release of reactive oxygen species by oxidative stress and the dysfunction of the cytomembrane. And ApoE^−/− mice on a high-fat diet were used as an animal model to detect the effect of Rac1 and Rac3 in vivo. The results showed that when Rac1 and Rac3 were decreased in HUVECs, the cell dysfunction caused by ox-LDL was inhibited. If 3-MA was used to inhibit autophagy in Rac1 and Rac3 knockdown cells, the injury induced by ox-LDL on the cells was recovered. These results indicated that the effect of Rac1 and Rac3 was combined with ox-LDL, which was related to inhibition of autophagy. The effect of Rac3 was more significant than that of Rac1.     

MiR-296-5p ameliorates deep venous thrombosis by inactivating S100A4

Deep venous thrombosis is one of the most common venous thromboembolic diseases and has a low cure rate and a high postoperative recurrence rate. Furthermore, emerging evidence indicates that microRNAs are involved in deep venous thrombosis. miR-296-5p is an important microRNA that plays a critical role in various cellular functions, and S100A4 is closely related to vascular function. miR-296-5p is downregulated in deep venous thrombosis patients, and its predicted target S100A4 is upregulated in deep venous thrombosis patients. Therefore, it was hypothesized that miR-296-5p may play a vital role in the development of deep venous thrombosis by targeting S100A4. An Ox-LDL-stimulated HUVEC and deep venous thrombosis mouse model was employed to detect the biological functions of miR-296-5p and S100A4. Dual luciferase reporter assays and pull-down assays were used to authenticate the interaction between miR-296-5p and S100A4. ELISA and Western blotting were employed to detect the protein levels of thrombosis-related factors and the endothelial-to-mesenchymal transition (EndMT)-related factors. The miR-296-5p levels were reduced, while the S100A4 levels were enhanced in deep venous thrombosis patients, and the miR-296-5p levels were negatively correlated with the S100A4 levels in deep venous thrombosis patients. miR-296-5p suppressed S100A4 expression by targeting the 3ʹ UTR of S100A4. MiR-296-5p knockdown accelerated ox-LDL-induced HUVEC apoptosis, oxidative stress, thrombosis-related factor expression, and EndMT, while S100A4 knockdown antagonized these effects in ox-LDL-induced HUVECs. S100A4 knockdown reversed the effect induced by miR-296-5p knockdown. Moreover, the in vivo studies revealed that miR-296-5p knockdown in deep venous thrombosis mice exacerbated deep venous thrombosis formation, whereas S100A4 knockdown had the opposite effect. These results indicate that elevated miR-296-5p inhibits deep venous thrombosis formation by inhibiting S100A4 expression. Both miR-296-5p and S100A4 may be potential diagnostic markers and therapeutic targets for deep venous thrombosis.     

microRNA-338-3p promotes ox-LDL-induced endothelial cell injury through targeting BAMBI and activating TGF-β/Smad pathway

microRNAs (miRNAs) have been revealed to participate in the pathological process of atherosclerosis (AS). However, the exact role of miR-338-3p, a target miRNA of BMP and activin membrane-bound inhibitor (BAMBI), and its possible molecular mechanism in AS remain unidentified. In this study, we found that BAMBI was significantly decreased, whereas miR-338-3p increased in patients with AS and oxidized low-density lipoprotein (ox-LDL)-induced HUVEC cells. Furthermore, overexpression of miR-338-3p significantly decreased cell viability and elevated cell apoptosis, whereas its inhibition significantly promoted cell viability and inhibited cell apoptosis in ox-LDL-induced HUVEC cells. Moreover, miR-338-3p overexpression increased TGF-β/Smad pathway activation in ox-LDL-induced HUVEC cells. A dual-luciferase reporter assay confirmed the direct interaction between miR-338-3p and the 3′-untranslated region of BAMBI messenger RNA. Furthermore, the suppression of BAMBI ameliorated the effect of miR-338-3p inhibition against ox-LDL-induced HUVEC cell injury. In conclusion, our study thus suggests that miR-338-3p promoted ox-LDL-induced HUVEC cell injury by targeting BAMBI and activating the TGF-β/Smad pathway, which may provide a novel and promising therapeutic target for AS.     

MiR-130a-5p contributed to the progression of endothelial cell injury by regulating FAS

MicroRNAs (miRNAs) play critical roles in the development of vascular diseases. However, the effects of miR- 130a-5p and its functional targets on atherosclerosis (AS) are still largely unknown. In this regard, our aim is to explore the potentially important role of miR-130a-5p and its target gene during the progression of endothelial cell injury. We first found oxidized low-density lipoprotein (ox-LDL) induced FAS and cell apoptosis in HUVECs. Subsequently, miR-130a-5p expression was verified to be downregulated after ox-LDL treatment and negatively correlated with FAS, and FAS was identified as substantially upregulated in the ox-LDL-treated HUVEC cells. After that, the knockdown of FAS and overexpression of miR-130a-5p together were observed to aggregate ox-LDL-induced reduction of cell viability and apoptosis, cell cycle progression, cell proliferation, cell migration and invasion. In conclusion, we detected that miR-130a-5p contributed to the progression of endothelial cell injury by regulating of FAS, which may provide a new and promising therapeutic target for AS.Key words: Atherosclerosis, ox-LDL, miR-130a-5p, FAS     

Paeoniflorin attenuates oxidized low-density lipoprotein-induced apoptosis and adhesion molecule expression by autophagy enhancement in human umbilical vein endothelial cells

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction is recognized as a driving force in the development of atherosclerosis (AS). Paeoniflorin (Pae), a typical traditional herbal medicine, possesses anti-inflammatory, antioxidative, antihyperglycaemic, and antiapoptotic properties. Our study aimed to investigate the effects of Pae on ox-LDL-induced injury of the human umbilical vein endothelial cells (HUVECs) and to explore its molecular mechanism. We found that ox-LDL stimulation inhibited cell viability, activated autophagy, and induced apoptosis and adhesion molecule expression in HUVECs. Pae rescued ox-LDL-induced viability reduction and enhanced the ox-LDL-induced autophagy activation in HUVECs. Pae inhibited ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement in HUVECs. In addition, inhibition of SIRT1 by EX-527 abolished the promoting effect of Pae on autophagy and restored the inhibitory effect of Pae on apoptosis and adhesion molecule expression in the presence of ox-LDL. In conclusion, Pae attenuated ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement via upregulation of SIRT1 in HUVECs, shedding light on the mechanism underlying the protective effect of Pae on ox-LDL-induced injury of HUVECs.     

IL-1β promotes hypoxic vascular endothelial cell proliferation through the miR-24-3p/NKAP/NF-κB axis

Purpose: Our previous data indicated that miR-24-3p is involved in the regulation of vascular endothelial cell (EC) proliferation and migration/invasion. However, whether IL-1β affects hypoxic HUVECs by miR-24-3p is still unclear. Therefore, the present study aimed to investigate the role and underlying mechanism of interleukin 1β (IL-1β) in hypoxic HUVECs.Methods: We assessed the mRNA expression levels of miR-24-3p, hypoxia-inducible factor-1α (HIF1A) and NF-κB-activating protein (NKAP) by quantitative real-time polymerase chain reaction (RT-qPCR). ELISA measured the expression level of IL-1β. Cell counting kit-8 (CCK-8) assays evaluated the effect of miR-24-3p or si-NKAP+miR-24 on cell proliferation (with or without IL-1β). Transwell migration and invasion assays were used to examine the effects of miR-24-3p or si-NKAP+miR-24-3p on cell migration and invasion (with or without IL-1β). Luciferase reporter assays were used to identify the target of miR-24-3p.Results: We demonstrated that in acute myocardial infarction (AMI) patient blood samples, the expression of miR-24-3p is down-regulated, the expression of IL-1β or NKAP is up-regulated, and IL-1β or NKAP is negatively correlated with miR-24-3p. Furthermore, IL-1β promotes hypoxic HUVECs proliferation by down-regulating miR-24-3p. In addition, IL-1β also significantly promotes the migration and invasion of hypoxic HUVECs; overexpression of miR-24-3p can partially rescue hypoxic HUVECs migration and invasion. Furthermore, we discovered that NKAP is a novel target of miR-24-3p in hypoxic HUVECs. Moreover, both the overexpression of miR-24-3p and the suppression of NKAP can inhibit the NF-κB/pro-IL-1β signaling pathway. However, IL-1β mediates suppression of miR-24-3p activity, leading to activation of the NKAP/NF-κB pathway. In conclusion, our results reveal a new function of IL-1β in suppressing miR-24-3p up-regulation of the NKAP/NF-κB pathway.     

Rapamycin Inhibits Oxidized Low Density Lipoprotein Uptake in Human Umbilical Vein Endothelial Cells via mTOR/NF-κB/LOX-1 Pathway

Background

Lectin-like oxidized low-density lipoprotein-1 (LOX-1) is the major receptor for oxidized low density lipoprotein (ox-LDL) uptake in human umbilical vein endothelial cells (HUVECs). Previously, we found that rapamycin inhibited ox-LDL accumulation in HUVECs, and this effect was related to its role in increasing the activity of autophagy-lysosome pathway. In this study, we determined whether rapamycin could also reduce ox-LDL uptake in HUVECs and investigated the underlying signaling mechanisms.

Results

Flow cytometry and live cell imaging showed that rapamycin reduced Dil-ox-LDL accumulation in HUVECs. Furthermore, rapamycin reduced the ox-LDL-induced increase in LOX-1 mRNA and protein levels. Western blotting showed that rapamycin inhibited mechanistic target of rapamycin (mTOR), p70s6k and IκBα phosphorylation triggered by ox-LDL. Flow cytometry implied that mTOR, NF-κB knockdown and NF-κB inhibitors significantly reduced Dil-ox-LDL uptake. Moreover, immunofluorescent staining showed that rapamycin reduced the accumulation of p65 in the nucleus after ox-LDL treatment for 30 h. mTOR knockdown decreased LOX-1 protein production and IκBα phosphorylation induced by ox-LDL. NF-κB knockdown and NF-κB inhibitors reduced LOX-1 protein production, but did not inhibit mTOR phosphorylation stimulated by ox-LDL.

Conclusions

These findings demonstrate that rapamycin reduce mTOR phosphorylation and subsequently inhibit NF-κB activation and suppresses LOX-1, resulting in a reduction in ox-LDL uptake in HUVECs.     

microRNA-181a represses ox-LDL-stimulated inflammatory response in dendritic cell by targeting c-Fos

Oxidized LDL (ox-LDL) activates dendritic cells (DCs), thereby initiating inflammation responses in atherosclerosis, yet the modulatory mechanisms remain unclear. MicroRNAs (miRNAs) are important regulators for DC functions. This study evaluated the regulation by miRNAs of the ox-LDL-induced DC immune response. In CD11c⁺ DCs from ApoE-deficient mice with hyperlipidemia, microRNA miR-181a was significantly up-regulated. In cultured bone marrow-derived DCs (BMDCs), ox-LDL promoted DC maturation and up-regulated miR-181a expression. Abundance of miR-181a attenuated ox-LDL-induced CD83 and CD40 expression, inhibited the secretion of interleukin (IL)-6 and TNF-α, and up-regulated IL-10, an important anti-inflammatory cytokine that was inhibited by ox-LDL. Inhibition of the endogenous miR-181a reversed the effects on CD83 and CD40 as well as the effects on IL-6 and TNF-α. The putative target genes of miR-181a were evaluated by gene ontology assessment, and the c-Fos-mediated inflammation pathway was identified. miR-181a targeted the 3′ untranslated region of c-Fos mRNA by luciferase experiments. Thus, abundance of miR-181a reduced c-Fos protein, whereas inhibition of miR-181a increased c-Fos protein in BMDCs. We therefore suggest that miR-181a attenuates ox-LDL-stimulated immune inflammation responses by targeting c-Fos in DCs.     

Degradation of heparan sulfate proteoglycans enhances oxidized-LDL-mediated autophagy and apoptosis in human endothelial cells

BackgroundCell surface heparan sulfate proteoglycans (HSPG) play an important role in atherogenesis. We hypothesized that degradation of HSPG may increase the binding of atherogenic oxidized low density lipoprotein (ox-LDL) to endothelial cells, and result in extensive HSPG degradation as well as autophagy and apoptosis.MethodsPrimary human umbilical vein endothelial cells (HUVECs) were used to study the expression of lectin-like ox-LDL receptor-1 (LOX-1), HSPG, autophagy and apoptosis in response to ox-LDL and heparinase III (Hep III).ResultsAs expected, ox-LDL treatment resulted in LOX-1 expression, ox-LDL uptake and reactive oxygen species (ROS) generation. Ox-LDL treatment also resulted in a modest degradation of HSPG and increase in autophagy (expression of LC3, beclin-1 and Atg5) and apoptosis (enhanced expression of caspases and Bax, and reduced expression of Bcl-2 and Bcl-xL). The effects of ox-LDL were blocked by pretreatment of cells with LOX-1 antibody or apocynin, an NADPH oxidase inhibitor. Hep III alone caused HSPG degradation and slightly, but significantly, increased ROS generation, and induced autophagy and caspase expression. However, autophagy and apoptosis induced by Hep III were not affected by apocynin or LOX-1 antibody. Importantly, Hep III pretreatment of cells significantly enhanced ox-LDL-induced HSPG degradation, LOX-1 expression, ox-LDL uptake and ROS generation as well as autophagy and apoptosis.ConclusionThese data demonstrate that Hep III enhances the pro-atherosclerotic characteristics in HUVECs induced by ox-LDL.     

LncRNA XIST Promoted OGD-Induced Neuronal Injury Through Modulating/miR-455-3p/TIPARP Axis

In recent years, the incidence of ischemic stroke has gradually increased, but its pathogenesis has not been fully elucidated. lncRNAs played an important role in the occurrence and regulation of disease, but the research on ischemic stroke is very limited. Therefore, the role of lncRNA in ischemic stroke needs further exploration. The mice model was built to obtain OGD-induced neuronal cells for the following experiments. The protein expression of TCDD inducible poly [ADP-ribose] polymerase (TIPARP), B-cell lymphoma-2 (Bcl-2) and Cleaved Caspase-3 (Cleaved-cas3) were detected with western blot. qRT-PCR was used to analyze expression of XIST, miR-455-3p and TIPARP. CCK-8 assay indicated the capacity of cell proliferation. Flow cytometry was applied to assess cell apoptosis rate. Moreover, dual-luciferase reporter assay and RIP assay were used to determine that the relationship among XIST, miR-455-3p and TIPARP. In this study, we found that oxygen–glucose deprivation (OGD) induced XIST expression, inhibited miR-455-3p expression and promoted TIPARP mRNA and protein expression in neurons. Furthermore, XIST could affect cell growth of OGD-induced neuronal cells. Further analysis showed that XIST could regulate TIPARP by binding to miR-455-3p, and overexpression of miR-455-3p or inhibition of TIPARP could reverse the effects of high XIST expression on OGD-induced neuronal cells. On the contrary, suppression of miR-455-3p or promotion of TIPARP could reverse the effects of low XIST expression on OGD-induced neuronal cells. XIST could affect cell proliferation and apoptosis through miR-455-3p/TIPARP axis in OGD-induced neuronal cells, providing a new regulatory network to understand the pathogenesis of hypoxia-induced neuronal injury.

     

LncRNA-XIST promotes dermal papilla induced hair follicle regeneration by targeting miR-424 to activate hedgehog signaling

BackgroundAlopecia is a highly prevalent disease characterizing by the loss of hair. Dermal papilla (DP) cells are the inducer of hair follicle regeneration, and in vitro three-dimensional (3D) culturing DP cells have been proven to induce hair follicle regeneration. However, the molecular mechanisms behind the regulation of 3D culturing DP cells remain unclear.Methods3D-cultivated DP cells were used as in vitro cell model. DP sphere xenograft to nude mice was performed for in vivo study of hair follicle regeneration. qRT-PCR, Western blotting, and immunofluorescence were used for detecting the level of XIST, miR-424 and Hedgehog pathway-related proteins, respectively. H&E staining was used to examine hair neogenesis. Cell viability, proliferation and ALP activity were measured by MTT, CCK-8 and ELISA assays, respectively. Luciferase assays were used for studying molecular regulation between XIST, miR-424 and Shh 3′UTR.ResultsXIST and Shh were up-regulated, and miR-424 was down-regulated in 3D DP cells. Molecular regulation studies suggested that XIST sponged miR-424 to promote Shh expression. Knockdown of XIST suppressed DP cell activity, cell proliferation, ALP activity and the expression of other DP markers by sponging miR-424. Knockdown of XIST suppressed Shh mediated hedgehog signaling by targeting miR-424. Moreover, the knockdown of XIST inhibited DP sphere induced in vivo hair follicle regeneration and hair development.ConclusionXIST sponges miR-424 to promote Shh expression, thereby activating hedgehog signaling and facilitating DP mediated hair follicle regeneration.     

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.