Rho-associated Protein Kinase 1 Research Articles

Identifying Isoononin and Candidissiol as Rho-associated protein kinase 1 (ROCK1) inhibitors: a combined virtual screening and MD simulation approach

Rho-associated protein kinase 1 (ROCK1) is a member of the AGC family which plays crucial roles in inflammatory diseases and cancer progression. Elevated expression of ROCK1 has been reported in multiple cancer types, and thus it has emerged as a potential drug target for cancer therapeutics. In this study, we performed a structure-based virtual screening of the natural compounds taken from the IMPPAT database to find some potential molecules as inhibitors of ROCK1. For the first step, we selected the compounds based on the Lipinski rule of five, and then we filtered them based on their ADMET properties and PAINS value. After this, other parameters like binding affinities, docking score, biological properties and selectivity were calculated to find appropriate hits against ROCK1. Finally, we identified two natural compounds, Isoononin and Candidissiol, with appreciable binding affinity and selectivity towards ROCK1. Furthermore, all-atom molecular dynamics simulations were carried out on ROCK1 with the elucidated compounds, which suggested stability throughout the simulated trajectories of 100 ns. Taken together, Isoononin and Candidissiol could be considered as potential inhibitors of ROCK1 for developing anticancer therapeutics. Communicated by Ramaswamy H. Sarma

ROCK2 knockdown alleviates LPS-induced inflammatory injury and apoptosis of renal tubular epithelial cells via the NF-κB/NLRP3 signaling pathway.

Rho-associated protein kinase 2 (ROCK2) is an important regulator of the inflammatory response and has been reported to serve a role in sepsis. The present study aimed to investigate whether ROCK2 served a role in sepsis-associated acute kidney injury (S-AKI). HK-2 cells were stimulated with lipopolysaccharide (LPS) to simulate S-AKI in vitro. Subsequently, the change in ROCK2 expression levels were determined. ROCK2 in LPS-induced HK-2 cells was knocked down using short hairpin RNA-ROCK2, in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of NF-κB. Cell viability, cytotoxicity, inflammation and apoptosis were assessed using MTT, lactate dehydrogenase (LDH) release, reverse transcription-quantitative PCR, ELISA, TUNEL and western blotting assays. The protein expression levels of proteins involved in the NF-κB/NLR family pyrin domain containing 3 (NLRP3) signaling pathway were also assessed using western blotting. The results demonstrated that ROCK2 was upregulated in HK-2 cells upon LPS treatment. LPS also reduced cell viability, promoted LDH activity and increased TNF-α, IL-6 and IL-1β mRNA expression levels and concentrations. Apoptosis was also induced by LPS as indicated by an increase in the proportion of TUNEL-positive cells, decreased Bcl-2 protein expression levels and increased cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase protein expression levels. However, ROCK2 knockdown in LPS-induced HK-2 cells reversed cell viability damage and inhibited LDH activity, the generation of pro-inflammatory cytokines and apoptosis caused by LPS. Furthermore, ROCK2 knockdown inhibited the LPS-induced expression of phosphorylated-NF-κB p65, NLRP3, apoptosis-associated speck-like protein containing a CARD and caspase-1 p20. PMA treatment reversed all the aforementioned effects of ROCK2 knockdown on LPS-treated HK-2 cells. Therefore, ROCK2 knockdown may alleviate LPS-induced HK-2 cell injury via the inactivation of the NF-κB/NLRP3 signaling pathway.

Mechanisms of Action of a Herbal Formula Huangqi Guizhi Wuwu Tang for the Management of Post-Stroke Related Numbness and Weakness: A Computational Molecular Docking Study

Stroke-related numbness and weakness (SRNW) are resultant symptoms of post-stroke sufferers. Existing research has supported the use of Huangqi Guizhi Wuwu Tang (HGWT) particularly for SRNW; however, their mechanisms of action have not been fully elucidated. Therefore, this study aimed to investigate the mechanisms of action of HGWT components targeting SRNW-related proteins through a computational molecular docking approach. Target proteins associated with SRNW were identified through DrugBank database and Open Targets database. Chemical compounds from each herb of HGWT were identified from the Traditional Chinese Medicine Systems Pharmacology and Analysis Platform (TCMSP). Autodock Vina was utilized and the cut-off criterion applied for protein-ligand complexes was a binding affinity score of ≤ -9.5 kcal/mol; selected protein-ligand complexes were identified using 3D and 2D structural analyses. The protein targets PDE5A and ESR1 have highlighted interactions with compounds (BS040, DZ006, DZ058, DZ118, and HQ066) which are the key molecules in the management of SRNW. PDE5A have bioactivity with the amino acid residues (Val230, Asn252, Gln133 and Thr166) throughout PDE5A-cGMP-PKG pathways which involved reduction in myofilament responsiveness. ESR1 were predicted to be critical active with site residue (Leu346, Glu419 and Leu387) and its proteoglycans pathway involving CD44v3/CD44 that activates rho-associated protein kinase 1 (ROCK1) and ankyrin increasing vascular smooth muscle. In conclusion, HGWT may provide therapeutic benefits through strong interactions between herbal compounds and target proteins of PDE5A and ESR1. Further experimental studies are needed to unequivocally support this result which can be valuable to increase the quality of life of post-stroke patients. Keywords Herbal medicine, Complementary and alternative medicine, Natural product, Post-stroke, Computational analysis

The effect of transglutaminase-2 inhibitor on pulmonary vascular remodeling in rats with pulmonary arterial hypertension

ABSTRACT To investigate the relationship between transglutaminase type 2 (TG2) and pulmonary vascular remodeling in the formation of pulmonary arterial hypertension (PAH), and to investigate the effect of the inhibitor cystamine dihydrochloride on pulmonary vascular remodeling in rats with PAH. Thirty healthy male Sprague Dawley rats were randomly divided into a control group, a PAH model group, and an intervention group. The mean pulmonary artery pressure (mPAP), the right ventricular hypertrophy index (RVHI), the percentage wall thickness of the pulmonary artery (WT%), and the degree of neointimal proliferation were measured, and the pathological changes in the pulmonary tissues were observed.Messenger ribonucleic acid (mRNA) and protein expressions of TG2, 5-hydroxytryptamine transporter (5-HTT), and Rho-associated protein kinase 2 (ROCK2) in the pulmonary tissues of the three groups of rats were detected. Compared with the control group, the mPAP, RVHI, and WT% were significantly higher in the model group, the degree of neointimal proliferation was significantly increased, and the mRNA and protein expressions of TG2, 5-HTT, and ROCK2 in the pulmonary tissue were significantly increased. Compared with the model group, the mPAP, RVHI, WT%, and the degree of neointimal proliferation were significantly lower in the intervention group, as were the mRNA and protein expressions of TG2, 5-HTT, and ROCK2 in the pulmonary tissue. The TG2 inhibitor cystamine dihydrochloride can prevent the formation of PAH to some extent. This might be due to the inhibition of the TG2 activity, 5-HTT expression, and possibly the inhibition of RhoA/ROCK signaling pathway activation.

Characterisation of the Tumour Proteome in Primary Extramedullary Multiple Myeloma Identifies Key Proteins Associated with Transendothelial Migration

Introduction: Extramedullary multiple myeloma (EMM) refers to the spread of clonal plasma cells to tissues extending outside of the bone marrow microenvironment. EMM is present at the time of diagnosis in 6-10% of patients, however, this increases to 13-26% in patients with disease progression and relapse. Cancer cells are suspected to spread to new tissues and organs via the circulatory system as a result of molecular changes that allow malignant cells to escape the bone marrow (BM). For example, downregulation of CXCR4 (C-X-C Motif Chemokine Receptor 4), an important factor in cellular homing to the BM, has frequently been reported to be linked to the EMM phenotype. In addition, the majority of patients presenting with EMM have highly complex cytogenetic abnormalities and high-risk cytogenetic markers such as t(14;16). As EMM is an indicator of a more aggressive disease, more intensive treatment, including combination chemotherapy, is often recommended. Many of the underlying molecular mechanisms accompanying EMM are yet to be characterised. Our mass-spectrometry (MS)-based proteomic study provides insight into the unique molecular mechanisms associated with EMM, identifying key proteins linked to the progression of medullary multiple myeloma (MM) to EMM.Methods: Label-free liquid chromatography mass spectrometric analysis of age and gender matched medullary MM (n=8) and EMM (n=9) bone-marrow derived mononuclear cells (MNCs) was carried out using a Thermo Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were employed for the identification of peptides and proteins. For protein identification, the following search parameters were used: (i) 0.02 Da for MS/MS mass tolerance, (ii) 10 ppm for peptide mass tolerance, (iii) variable modification settings for methionine oxidation, (iv) fixed modification settings in relation to carbamido-methylation and (v) tolerance for up to two missed cleavages. Peptide probability was set to high confidence. Datasets were imported into Progenesis QI (version 2.0) software for further analysis. Data was filtered based on an ANOVA p-value of ≤0.05, fold change >1.5 between experimental groups, and proteins with ≥1 unique peptides contributing to the identification. Proteins with less than 70% valid values were removed from the analysis. G:profiler and STRING were utilized for functional enrichment and the characterisation of protein interaction patterns.Results: Our quantitative MS-based proteomic analysis identified a total of 492 proteins with significantly altered abundances between EMM and MM bone marrow MNC. Of these significant proteins, 275 were found to be increased in EMM compared to medullary MM and 217 were found to be decreased in EMM compared to medullary MM. Hierarchical clustering was performed to highlight the proteomic profile associated with extramedullary disease (Figure 1A). KEGG pathway analysis and gene ontology (GO) analysis of proteins found to be increased in EMM indicated an increase in proteins associated with cell adhesion, invasion, and migration pathways (Figure 1B). Interestingly, several proteins involved in leukocyte transendothelial migration were significantly increased in EMM indicating their potential involvement in the dissemination of MM cells from the bone marrow microenvironment to distal tissues (Figure 1C). Among the proteins found to be involved in this biological pathway was junctional adhesion molecule-A (F11R), a protein previously reported to play a role in EMD pathophysiology [1]. Other proteins involved in MM invasion and migration including Rho-associated protein kinase 2 (ROCK2), Ras-related C3 botulinum toxin substrate 1 (Rac1) and platelet endothelial cell adhesion molecule (PECAM-1) were significantly increased in EMM.Conclusion: Using high-resolution mass spectrometry to characterise the tumour proteome of MM patients with extramedullary disease, we have identified a significant increase in the abundance of proteins associated with leukocyte transendothelial invasion in primary EMM samples. Our study provides further insight into the molecular mechanisms within EMM and thus holds potential to enhance current efforts to provide a more personalised therapeutic approach for EMM patients.

Omega-3 fatty acids differentially reduced expression of neutrophil degranulation-associated proteins in endothelial cells during IL-6 exposure

Abstract Background Neutrophil degranulation contributes to atherogenesis and tissue injury. Mixed omega-3 fatty acid (n3-FA) formulations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have failed to reduce CV events compared to EPA only (REDUCE-IT), but the mechanisms are not understood. Purpose The purpose of this study was to compare the effects of EPA and DHA on expression of proteins linked to neutrophil degranulation in the vascular endothelium in vitro. Methods Human umbilical vein endothelial cells (HUVECs) were pretreated with EPA or DHA at equimolar levels (10 μM) for 2 h, then challenged with IL-6 at 12 ng/ml for 24 h. Proteomic analysis was performed using LC/MS to measure relative protein expression. Only significant (p<0.05) changes between treatment groups >1-fold were analyzed. Results In the Reactome “neutrophil degranulation” pathway, EPA and DHA downregulated 27 and 14 proteins, respectively, (p=9.97E-9 and 5.30E-4, respectively) relative to IL-6 alone. There were 12 protein changes common to both n3-FAs, including heme oxygenase-2 and ferritin light chain. EPA downregulated 15 proteins unlike DHA, including peroxiredoxin-6 and mitogen-activated protein kinase-1 (MAPK1). A combined 21 proteins downregulated by EPA and DHA versus IL-6 were upregulated by IL-6 alone relative to vehicle. EPA also increased expression of Rho-associated protein kinase-1 (ROCK-1), a protein downregulated by IL-6 alone and unaffected by DHA. Conclusions EPA and DHA differentially modulated expression of proteins linked to neutrophil degranulation. The distinct effects of EPA on protein expression may contribute to reduced inflammation in vascular injury compared to DHA. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amarin Pharma Inc., Elucida Research

Lysophosphatidic Acid Mediates Imiquimod-Induced Psoriasis-like Symptoms by Promoting Keratinocyte Proliferation through LPAR1/ROCK2/PI3K/AKT Signaling Pathway

Psoriasis is a chronic inflammatory skin disease. Recently, lysophosphatidic acid (LPA)/LPAR5 signaling has been reported to be involved in both NLRP3 inflammasome activation in macrophages and keratinocyte activation to produce inflammatory cytokines, contributing to psoriasis pathogenesis. However, the effect and molecular mechanisms of LPA/LPAR signaling in keratinocyte proliferation in psoriasis remain unclear. In this study, we investigated the effects of LPAR1/3 inhibition on imiquimod (IMQ)-induced psoriasis-like mice. Treatment with the LPAR1/3 antagonist, ki16425, alleviated skin symptoms in IMQ-induced psoriasis-like mouse models and decreased keratinocyte proliferation in the lesion. It also decreased LPA-induced cell proliferation and cell cycle progression via increased cyclin A2, cyclin D1, cyclin-dependent kinase (CDK)2, and CDK4 expression and decreased p27Kip1 expression in HaCaT cells. LPAR1 knockdown in HaCaT cells reduced LPA-induced proliferation, suppressed cyclin A2 and CDK2 expression, and restored p27Kip1 expression. LPA increased Rho-associated protein kinase 2 (ROCK2) expression and PI3K/AKT activation; moreover, the pharmacological inhibition of ROCK2 and PI3K/AKT signaling suppressed LPA-induced cell cycle progression. In conclusion, we demonstrated that LPAR1/3 antagonist alleviates IMQ-induced psoriasis-like symptoms in mice, and in particular, LPAR1 signaling is involved in cell cycle progression via ROCK2/PI3K/AKT pathways in keratinocytes.

Enhanced autophagy suppresses inflammation-mediated bone loss through ROCK1 signaling in bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) have strong proliferative ability and multi-directional differentiation potential. Osteoarthritis is a degenerative joint disease that is closely related to the loss of osteogenic differentiation function of BMSCs. Autophagy, plays a crucial role in the maintenance of cellular functions, but its regulatory mechanism during the osteogenic differentiation of BMSCs remains unclear. In this study, we analyzed the differential gene networks and pathways during BMSC osteogenesis using bioinformatics, and further validated the regulatory roles of autophagy during the osteogenic differentiation of BMSCs in inflammatory condition in vitro. We found that Tumor necrosis factor alpha (TNF-α) treatment led to actin cytoskeleton rearrangements and inhibited osteogenic differentiation in BMSCs. In addition, TNF-α enhanced Rho-associated protein kinase 1 (ROCK1) expression and decreased autophagy activation. ROCK1 knockdown reduced Endoplasmic Reticulum stress (ER stress) and promoted autophagy, resulting reversion of osteogenic differentiation in BMSCs under inflammatory condition. Rapamycin reversed the TNF-α-induced decrease in osteogenesis of BMSCs, assessed by alkaline phosphatase (ALP) activity and Alizarin staining. Autophagy treated with inhibitor 3-Methyladenine (3-MA) further increased TNF-α-induced osteogenesis inhibition of BMSCs. Collectively, these results indicate that ER stress and dysfunction of autophagy promote inflammation-induced bone loss through the activation of ROCK1 signaling in BMSCs.

Overexpression of ROCK1 promotes cancer cell proliferation and is associated with poor prognosis in human urothelial bladder cancer.

Rho-associated protein kinase 1 (ROCK1) has been reported to be overexpressed in many types of tumors, but its role in urothelial bladder cancer is poorly understood. The study aims to investigate the role of ROCK1 in urothelial bladder cancer and explored the underlying mechanism. Protein and mRNA levels of ROCK1 were detected in 64 urothelial bladder cancer patients using western blot, immunohistochemistry and qRT-PCR. Relationships between ROCK1 expression and clinicopathological factors and survival rate were analyzed. ROCK1 was silenced by shRNA in multiple urothelial bladder cancer cells to explore its function and underlying mechanism. ROCK1 expression was significantly increased in tumor tissues compared with the paired adjacent healthy tissues of patients. Higher ROCK1 expression of tumor tissues positively correlated with poor prognosis of patients (p = 0.0435). ROCK1 silence significantly inhibited cell proliferation and colony formation, and enhanced activation of apoptotic pathway in urothelial bladder cancer cells. High ROCK1 expression predicts poor prognosis of urothelial bladder cancer. ROCK1 silence inhibit cell proliferation and promote apoptosis, which may be of value as a therapeutic target for bladder cancer treatment.

The adenosine A2Areceptor alleviates postoperative delirium‐like behaviors by restoring blood cerebrospinal barrier permeability in rats

Postoperative delirium (POD) is a common postoperative complication in elderly patients that is associated with increased morbidity and mortality. However, the neuropathogenesis of this complication remains unknown. The blood cerebrospinal fluid barrier (BCB) and brain blood barrier (BBB) are composed of tight junctions between cells that form physical barriers, and BBB damage plays an important role in the neuropathogenesis of POD. Nevertheless, the role of BCB in POD remains to be elucidated. Herein, we investigated the effect of adenosine A2A receptor (A2A R), a key regulator of the permeability of barriers, on surgery-induced increased permeability of BCB and POD-like behaviors. Open field, buried food and Y maze tests were used to evaluate behavioral changes in rats after surgery. Levels of tight junction proteins, adherens junction proteins, A2A R, GTP-RhoA and ROCK2 in the choroid plexus were assessed by western blotting. The concentrations of NaFI and FITC-dextran in the cerebrospinal fluid (CSF) were detected by fluorescence spectrophotometry. Transmission electron microscopy was applied to observe the ultrastructure of the choroid plexus. Surgery/anesthesia decreased the levels of tight junction (e.g., ZO-1, occludin and claudin1) proteins, increased concentrations of NaFI and FITC-dextran in CSF, damaged the ultrastructure of choroid plexus, and induced POD-like behaviors in rats. An A2A R antagonist alleviated POD-like behaviors in rats. Furthermore, the A2A R antagonist increased the levels of tight junction proteins and restored the permeability of BCB in rats with POD. Fasudil, a selective Rho-associated protein kinase 2 (ROCK2) inhibitor, ameliorated POD-like behaviors induced by A2A R activation. Moreover, fasudil also abolished the increased levels of GTP-RhoA/ROCK2, decreased levels of tight junction proteins and increased permeability of BCB caused by A2A R activation. Our findings demonstrate that A2A R might participate in regulating BCB permeability in rats with POD via the RhoA/ROCK2 signaling pathway, which suggests the potential of A2A R as a therapeutic target for POD.

Binding between ROCK1 and DCTN2 triggers diabetes‑associated centrosome amplification in colon cancer cells.

Type 2 diabetes increases the risk various types of cancer and is associated with a poor prognosis therein. There is also evidence that the disease is associated with cancer metastasis. Centrosome amplification can initiate tumorigenesis with metastasis in vivo and increase the invasiveness of cancer cells in vitro. Our previous study reported that type 2 diabetes promotes centrosome amplification via the upregulation and centrosomal translocation of Rho-associated protein kinase 1 (ROCK1), which suggests that centrosome amplification is a candidate biological link between type 2 diabetes and cancer development. In the present study, functional proteomics analysis was used to further investigate the molecular pathways underlying centrosome amplification by targeting ROCK1 binding partners. High glucose, insulin and palmitic acid were used to induce centrosome amplification, and immunofluorescent staining was employed to visualize centrosomal alterations. Combined with immunoprecipitation, mass spectrometry-based proteomics analysis was used to identify ROCK1 binding proteins, and protein complex disruption was achieved by siRNA-knockdown. In total, 1,148 ROCK1 binding proteins were identified, among which 106 proteins were exclusively associated with the treated samples, 193 were only associated with the control samples, and 849 were found in both the control and treated samples. Of the proteins with evidence of centrosomal localization, Dynactin subunit 2 (DCTN2) was confirmed to be localized to the centrosomes. Treating the cells with high glucose, insulin and palmitic acid increased the protein levels of ROCK1 and DCTN2, promoted their binding with each other, and triggered centrosome amplification. Disruption of the protein complex by knocking down ROCK1 or DCTN2 expression partially attenuated centrosome amplification, while simultaneous knockdown of both proteins completely inhibited centrosome amplification. These results suggested ROCK1-DCTN2 binding as a signal for the regulation of centrosome homeostasis, which is key for diabetes-associated centrosome amplification, and enriches our knowledge of centrosome biology. Therefore, the ROCK1-DCTN2 complex may serve as a target for inhibiting centrosome amplification both in research or future therapeutic development.

Light-induced dynamic RGD pattern for sequential modulation of macrophage phenotypes

Due to the critical roles of macrophage in immune response and tissue repair, harnessing macrophage phenotypes dynamically to match the tissue healing process on demand attracted many attentions. Although there have developed many advanced platforms with dynamic features for cell manipulation, few studies have designed a dynamic chemical pattern to sequentially polarize macrophage phenotypes and meet the immune requirements at various tissue repair stages. Here, we propose a novel strategy for spatiotemporal manipulation of macrophage phenotypes by a UV-induced dynamic Arg-Gly-Asp (RGD) pattern. By employing a photo-patterning technique and the specific interaction between cyclodextrin (CD) and azobenzene-RGD (Azo-RGD), we prepared a polyethylene glycol-dithiol/polyethylene glycol-norbornene (PEG-SH/PEG-Nor) hydrogel with dynamic RGD-patterned surface. After irradiation with 365-nm UV light, the homogeneous RGD surface was transformed to the RGD-patterned surface which induced morphological transformation of macrophages from round to elongated and subsequent phenotypic transition from pro-inflammation to anti-inflammation. The mechanism of phenotypic polarization induced by RGD pattern was proved to be related to Rho-associated protein kinase 2 (ROCK2). Sequential modulation of macrophage phenotypes by the dynamic RGD-patterned surface provides a remote and non-invasive strategy to manipulate immune reactions and achieve optimized healing outcomes.

Circ-E2F3 acts as a ceRNA for miR-204-5p to promote proliferation, metastasis and apoptosis inhibition in retinoblastoma by regulating ROCK1 expression

BackgroundCircular RNA (circRNA) plays an important role in the malignant progression of many tumors, including retinoblastoma (RB). However, the role and regulatory mechanism of circ-E2F3 in RB have not been fully elucidated. MethodsQuantitative real-time PCR was used to measure circ-E2F3, miR-204-5p and Rho-associated protein kinase 1 (ROCK1) expression. Cell proliferation, apoptosis and metastasis were monitored by MTT, colony formation, flow cytometry, transwell and wound healing assays. Dual-luciferase reporter assay was employed to verify the relationship between miR-204-5p and circ-E2F3 or ROCK1. ROCK1 protein expression was detected by western blot assay. Mice xenograft models were built to assess the role of circ-E2F3 on RB tumor growth. ResultsCirc-E2F3 was upregulated in RB tissues and cells. Silencing of circ-E2F3 inhibited the proliferation, migration, invasion, and induced the apoptosis of RB cells in vitro, as well as reduced RB tumor growth in vivo. MiR-204-5p could be sponged by circ-E2F3, and its inhibitor reversed the suppressive effect of circ-E2F3 silencing on RB progression. In addition, ROCK1 was confirmed to interact with miR-204-5p. MiR-204-5p regulated RB progression by targeting ROCK1. Also, circ-E2F3 positively regulated ROCK1 expression by sponging miR-204-5p. ConclusionCirc-E2F3 functioned as a tumor promoter in RB through the miR-204-5p/ROCK1 axis.

Effects of blood oxygen saturation on pulmonary artery remodeling in an in vitro perfusion circuit model.

BackgroundPatients with transposition of the great arteries are likely to survive surgery despite severe pulmonary artery hypertension. However, the underlying mechanisms remain largely unknown. The present study aimed to test the hypothesis that high blood oxygen saturation may protect the pulmonary artery from remodeling.MethodsAn in vitro pulmonary artery perfusion model was successfully performed by connecting rabbit pulmonary artery to a closed perfusion circuit. Twenty-five rabbits were divided randomly into 5 groups according to exposure conditions: Normal Control (NC) group (unperfused normal pulmonary artery), High Saturation (HS) group (oxygen saturation range: 90–100%), Medium Saturation (MS) group (oxygen saturation: 65–75%); Low Saturation (LS) group (oxygen saturation: 40–50%), and anti-hypoxia inducible factor-1α (anti-HIF-1α) group (oxygen saturation range: 40–50%, and LW6, which is a novel HIF-1α inhibitor; was added). By staining and optical microscopy examination, pathological morphology was analyzed, and the protein expression levels of HIF-1α, angiotensin-II (Ang-II), endothelin-1 (ET-1), Rho-associated protein kinase-1 (Rock-1), and matrix metallopeptidase-2 (MMP-2) were determined by Western blotting.ResultsThe amounts of elastin, muscle, and collagen and the protein levels of ET-1, HIF-1α, Rock-1, and MMP-2, increased significantly with decreased oxygen saturation in the perfusion circuit. A significant improvement in pathological morphology was observed in the anti-HIF1α group. The expression of HIF-1α, ET-1, Ang-II, Rock-1, and MMP-2 in the anti-HIF1α group was also significantly lower than that in the LS group.ConclusionsIn the closed perfusion model, high blood oxygen saturation alleviated pulmonary vascular structural remodeling. Similar beneficial effects were observed when inhibiting the HIF-1α protein, suggesting a key role for HIF-1α in pulmonary artery remodeling.

RETRACTED: microRNA-148a-3p enhances the effects of sevoflurane on hepatocellular carcinoma cell progression via ROCK1 repression

BackgroundSevoflurane (SEVO) inactivates the aggressiveness of hepatocellular carcinoma (HCC) cells by mediating microRNAs (miRNAs). Hence, we delved into the functional role of miR-148a-3p mediated by SEVO in HCC. MethodsLiver cells (L02) and HCC cells (HCCLM3 and Huh7) were exposed to SEVO to detect cell viability in HCC. HCCLM3 and Huh7 cells were treated with restored miR-148a-3p or depleted Rho-associated protein kinase 1 (ROCK1) to elucidate their roles in HCC cells' biological characteristics. HCCLM3 and Huh7 cells were treated with SEVO, and/or vectors that changed miR-148a-3p or ROCK1 expression to identify their combined functions in HCC cell progression. Tumor xenograft in nude mice was performed to determine growth ability of tumor. The target relationship between miR-148a-3p and ROCK1 was verified. ResultsSEVO inhibited proliferation, invasion and migration and enhanced apoptosis of HCCLM3 and Huh7 cells. MiR-148a-3p up-regulation or ROCK1 down-regulation inhibited HCCLM3 and Huh7 cell progression. ROCK1 was determined to be target gene of miR-148a-3p. Down-regulating miR-148a-3p or overexpressing ROCK1 mitigated cell aggressiveness inhibition caused by SEVO. ConclusionOur study elucidates that microRNA-148a-3p enhances the effects of sevoflurane on inhibiting proliferation, invasion and migration and enhancing apoptosis of HCC cells through suppression of ROCK1.

Serum-derived exosomes containing NEAT1 promote the occurrence of rheumatoid arthritis through regulation of miR-144-3p/ROCK2 axis.

Background:Evidence has demonstrated that non-coding RNAs (ncRNAs) could be delivered efficiently to recipient cells using exosomes as a carrier. Additionally, long ncRNA nuclear enriched abundant transcript 1 (NEAT1) is emerging as a vital regulatory molecule in the progression of rheumatoid arthritis (RA). The aim of this study was to identify the NEAT1/miR-144-3p/Rho-associated protein kinase 2 (ROCK2) functional network regulating the WNT signaling pathway in RA.Methods:In vivo, a collagen-induced arthritis (CIA) model was established to analyze the effects of blood exosomes on the incidence, clinical score, and bone degradation of RA. In vitro, the CD4+T cells were characterized by flow cytometry and the cell activities were analyzed in the presence of exosome treatment alone or in combination with altered expression of NEAT1, miR-144-3p or Rho-associated protein kinase 2 (ROCK2). The expression of NEAT1, miR-144-3p, ROCK2, and corresponding proteins in the WNT signaling pathway was detected by RT-qPCR and western blot techniques. The binding profile of NEAT1 to miR-144-3p was evaluated via a combination approach of luciferase activity assay, RNA immunoprecipitation, and RNA pull-down experiments.Results:Blood exosomes extracted from RA patients increased the incidence of RA and bone destruction significantly. Overexpression of NEAT1 or ROCK2 promoted immune cell (CD4+T cells) proliferation, Th17 cell differentiation, and cell migration in response to stimulus, whereas knockout of the NEAT1 gene induced the expression of miR-144-3p in CD4+T cells. ROCK2 exogenous expression inhibited the expression of miR-144-3p, inducing activation of the WNT signaling pathway.Conclusion:A novel regulatory pathway NEAT1/miR-144-3p/ROCK2/WNT in RA was investigated as a potential target for RA therapy.

Ligand-induced conformational rearrangements regulate the switch between membrane-proximal and distal functions of Rho kinase 2

Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Its functions can be divided into two categories: membrane-proximal and membrane-distal. A recent study concluded that membrane-distal functions require the fully extended conformation, and this conclusion was supported by electron microscopy. The present solution small-angle X-ray scattering (SAXS) study revealed that ROCK2 population is a dynamic mixture of folded and partially extended conformers. Binding of RhoA to the coiled-coil domain shifts the equilibrium towards the partially extended state. Enzyme activity measurements suggest that the binding of natural protein substrates to the kinase domain breaks up the interaction between the N-terminal kinase and C-terminal regulatory domains, but smaller substrate analogues do not. The present study reveals the dynamic behaviour of this long, dimeric molecule in solution, and our structural model provides a mechanistic explanation for a set of membrane-proximal functions while allowing for the existence of an extended conformation in the case of membrane-distal functions.

Synthesis of New Thiazole-Pyridine Hybrids and Their Anticancer Activity

A series of new thiazole incorporated pyridine derivatives containing the phenoxyacetamide moiety as a linking bridge has been synthesized. The synthetic strategy involves condensation of 2-(4-formylphenoxy)-N-(thiazol-2-yl)acetamide with cyanoacetic hydrazide followed by heterocyclization with acetylacetone, treatment of the produced acrylamides with malononitrile and substituted acetophenones, then heating the generated chalcones with mononitrile in acetic acid and ammonium acetate. In vitro anticancer activity of the newly synthesized thiazole-pyridine hybrids has been evaluated against prostate (PC3), liver (HepG2), laryngeal (Hep-2), and breast (MCF-7) cancer cell lines. One of thiazole-pyridine compounds 8c demonstrates higher activity (IC50 5.71 μM) against breast cancer than 5-fluorouracil used as a reference (IC50 6.14 μM). Molecular docking procedure has provided valuable information on the binding sites of the synthesized compounds with rho-associated protein kinase 1 (ROCK-1).

Long non‑coding RNA AWPPH interacts with ROCK2 and regulates the proliferation and apoptosis of cancer cells in pediatric T‑cell acute lymphoblastic leukemia

The long non-coding (lnc)RNA associated with poor prognosis of hepatocellular carcinoma (AWPPH) serves as an oncogene in several cancers, such as liver and bladder cancers, however, to the best of our knowledge, its function in T-cell acute lymphoblastic leukemia is unknown. The results of the present study revealed that the expression levels of lncRNA AWPPH and Rho-associated protein kinase 2 (ROCK2) were upregulated in the bone marrow of patients with pediatric T-cell acute lymphoblastic leukemia compared with healthy controls. Expression levels of lncRNA AWPPH and ROCK2 were positively correlated with each other. lncRNA AWPPH and ROCK2 overexpression promoted the proliferation and inhibited the apoptosis of Loucy cells, an acute lymphoblastic leukemia cell line. Overexpression of lncRNA AWPPH resulted in upregulation of ROCK2 expression in Loucy cells. Similarly, ROCK2 overexpression also resulted in upregulation of lncRNA AWPPH in Loucy cells, suggesting an element of reciprocity in the function of lncRNA AWPPH and ROCK2. It was concluded that lncRNA AWPPH promoted the proliferation and inhibited the apoptosis of cancer cells in pediatric T-cell acute lymphoblastic leukemia possibly through interactions with ROCK2.

MiR-142-3p Reduces the Size, Migration, and Contractility of Endometrial and Endometriotic Stromal Cells by Targeting Integrin- and Rho GTPase-Related Pathways That Regulate Cytoskeletal Function.

Downregulated microRNA-142-3p signaling contributes to the pathogenesis of endometriosis, an invasive disease where the lining of the uterus grows at ectopic locations, by yet incompletely understood mechanisms. Using bioinformatics and in vitro assays, this study identifies cytoskeletal regulation and integrin signaling as two relevant categories of miR-142-3p targets. qPCR revealed that miR-142-3p upregulation in St-T1b cells downregulates Rho-associated protein kinase 2 (ROCK2), cofilin 2 (CFL2), Ras-related C3 botulinum toxin substrate 1 (RAC1), neural Wiskott-Aldrich syndrome protein (WASL), and integrin α-V (ITGAV). qPCR and Western-blotting showed miR-142-3p effect on WASL and ITGAV was significant also in primary endometriotic stroma cells. Luciferase reporter assays in ST-T1b cells then confirmed direct regulation of ITGAV and WASL. On the functional side, miR-142-3p upregulation significantly reduced ST-T1b cell size, the size of vinculin plaques, migration through fibronectin-coated transwell filters, and the ability of ST-T1b and primary endometriotic stroma cells to contract collagen I gels. These results suggest that miR-142-3p has a strong mechanoregulatory effect on endometrial stroma cells and its external administration reduces the invasive endometrial phenotype. Within the limits of an in vitro investigation, our study provides new mechanistic insights into the pathogenesis of endometriosis and provides a perspective for the development of miR-142-3p based drugs for inhibiting invasive growth of endometriotic cells.