Rho-associated Protein Kinase 1 Research Articles

Secreted Wnt6 mediates diabetes-associated centrosome amplification via its receptor FZD4.

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of β-catenin. The treatment also activated β-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or β-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-β-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of β-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.

Effects of β‑hydroxybutyric acid and ghrelin on the motility and inflammation of gastric antral smooth muscle cells involving the regulation of growth hormone secretagogue receptor.

Ghrelin is an orexigenic hormone that is produced by gastric cells. Ghrelin stimulates food intake and increases gastric movement. In rat model, injected β-hydroxybutyric acid (β-HB) leads to a decrease in body weight. It has been reported that patients with gastric erosions are slower to evacuate the stomach. The aim of the present study was to investigate the effects of ghrelin and β-HB on motility and inflammation in rat gastric antral smooth muscle cells (GASMCs). GASMCs were extracted from rat gastric antrum. Cell viability was determined using the Cell Counting Kit-8 assay. A reactive oxygen species (ROS) assay kit was used to analyze the levels of ROS using flow cytometry. Protein levels were determined using western blotting, and the expression levels of mRNAs were evaluated using reverse transcription-quantitative PCR. β-HB inhibited the expression of myosin regulatory light polypeptide 9 (MYL9), myosin light chain kinase (MLCK), transforming protein RhoA (RhoA), Rho-associated protein kinase-1 (ROCK-1) and growth hormone secretagogue receptor (GHS-R). By contrast, ghrelin increased the expression of MYL9, MLCK, RhoA, ROCK-1 and GHS-R in β-HB-treated GASMCs. β-HB increased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and ROS, and decreased the levels of manganese (Mn) superoxide dismutase (SOD), copper/zinc (Cu/Zn)SOD and catalase. Ghrelin decreased the expression of TNF-α, IL-6, ROS and catalase, whereas ghrelin promoted the expression of MnSOD and Cu/ZnSOD in β-HB-treated GASMCs. Short interfering RNA targeting GHS-R inhibited the expression of MYL9, MLCK, RhoA and ROCK-1, and increased the levels of TNF-α, IL-6 and ROS in β-HB-treated or ghrelin-treated GASMCs. The present study provided preliminary evidence that β-HB inhibits the motility of GASMCs and promotes inflammation in GASMCs, whereas ghrelin decreases these effects. GHS-R acted as a primary regulator of motility and inflammation in GASMCs treated with β-HB and ghrelin.

Valsartan inhibits RhoA-ROCK2-MYL pathway in rat model of alcoholic cardiomyopathy.

The present study aimed to investigate variations in the Ras homolog gene family, member A (RhoA)-Rho-associated protein kinase 2 (ROCK2)-myosin light chain (MYL) pathway in a rat model of alcoholic cardiomyopathy (ACM) and the role of angiotensin-converting enzyme inhibitor drugs. Rat models of ACM were established via alcoholic gavage + free access to alcohol. The structural and functional changes of the heart were analyzed by hematoxylin-eosin staining, Masson's trichrome staining, immunohistochemistry staining, western blotting and fluorescence quantitative polymerase chain reaction. A total of 16 weeks later, a decreased ejection fraction and left ventricular fractional shortening in the alcohol group compared with the control group were demonstrated resulting in an increased left ventricular end diastolic diameter. These adverse effects were ameliorated following treatment with valsartan. In addition, the alcohol group revealed a disorganized arrangement of myocardial filaments, which was improved upon treatment with valsartan. RhoA and ROCK2 protein expression significantly increased in myocardial cells in the alcohol compared with the control group. Following drug intervention with valsartan, expression of RhoA and ROCK2 proteins were inhibited in the alcohol group. Furthermore, significantly elevated RhoA and ROCK2 and decreased MYL protein and mRNA expression in the alcohol group was demonstrated compared with the control group. Administration of valsartan reversed the expression profile of RhoA, ROCK and MYL in ACM. Expression of RhoA and ROCK were elevated with downregulation of MYL resulting in heart failure. However, the angiotensin receptor antagonist diminished the expression of RhoA and ROCK and enhanced the expression of MYL. The results of the present study suggest a curative effect of valsartan in ACM.

Effect of HongJing I in Treating Erectile Function and Regulating RhoA Pathway in a Rat Model of Bilateral Cavernous Nerve Injury.

HongJing I (HJI), a traditional Chinese herbal formula, has been confirmed to be effective for the clinical treatment of erectile dysfunction (ED). However, the mechanism of action of HJI remains unclear. Here, we aimed to investigate the effect and underlying mechanisms of HJI against ED in a rat model of bilateral cavernous nerve injury (BCNI). Rats were divided into five groups: normal control (NC), BCNI-induced ED model (M), M + low-dose HJI (HL), M + medium-dose HJI (HM), and M + high-dose HJI (HH). All groups were treated with normal saline or the relevant drug for 28 consecutive days after inducing BCNI-ED. At the end of the treatment period, the intracavernous pressure (ICP) was recorded, and histological examination was conducted using Masson's trichrome staining. Immunofluorescence staining and western blotting were applied to detect the changes in fibrosis protein and Ras homolog A (RhoA), Rho-associated protein kinase 1 (ROCK1), and ROCK2 expression. We found that HJI effectively improved the ICP in the treatment groups. In addition, RhoA, ROCK1, and ROCK2 expression levels were increased upon BCNI-ED induction, and HJI successfully inhibited cavernosum fibrosis and the activation of RhoA/ROCK2 signaling. Overall, these results suggest that the effects of HJI in attenuating ED may be caused, at least in part, by the suppression of RhoA/ROCK2 signaling and alleviation of fibrosis. However, the precise mechanism surrounding this requires further investigation in future studies.

ROCK1 promotes migration and invasion of non‑small‑cell lung cancer cells through the PTEN/PI3K/FAK pathway.

Rho-associated protein kinase 1 (ROCK1), a member of the ROCK family, serves an important function in cell migration and invasion in neoplasms. ROCK1 has been found to be overexpressed in several types of cancers. However, the role of ROCK1 in non-small-cell lung cancer (NSCLC) is poorly understood. In the present study, ROCK1 was found to be overexpressed in NSCLC cells and tissues, and it was associated with poor survival of NSCLC patients. Subsequently, ROCK1 knockdown NSCLC cell lines were established using shRNA. ROCK1 knockdown significantly reduced the migration and invasion ability in the cell monolayer scratching and Transwell assays. ROCK1 knockdown was also found to markedly inhibit cell adhesion ability. Moreover, the phosphorylation of focal adhesion kinase (FAK) was inhibited by ROCK1 knockdown, reducing NSCLC cell migration and invasion ability. This mechanistic study revealed that ROCK1 significantly enhanced cell migration and invasion by inhibiting the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/FAK pathway. More importantly, the interruption of the PTEN/PI3K/FAK pathway markedly rescued the inhibition of cell migration and invasion mediated by ROCK1 knockdown. Taken together, these results suggest a novel role for ROCK1 in cell migration and invasion by inhibiting cell adhesion ability, and indicate that ROCK1 may be of value as a therapeutic target for the treatment of NSCLC.

Neuregulin‑1 protects cardiac function in septic rats through multiple targets based on endothelial cells.

The primary mechanism underlying sepsis-induced cardiac dysfunction is loss of endothelial barrier function. Neuregulin-1 (NRG-1) exerts its functions on multiple targets. The present study aimed to identify the protective effects of NRG-1 in myocardial cells, including endothelial, anti-inflammatory and anti-apoptotic effects. Subsequent to lipopolysaccharide (LPS)-induced sepsis, rats were administered with either a vehicle or recombinant human NRG-1 (rhNRG-1; 10 µg/kg/day) for one or two days. H9c2 cardio-myoblasts were subjected to LPS (10 µg/ml) treatment for 12 and 24 h with or without rhNRG-1 (1 µg/ml). Survival rates were recorded at 48 h following sepsis induction. The hemo-dynamic method was performed to evaluate cardiac function, and myocardial morphology was observed. Von Willebrand Factor levels were detected using an immunofluorescence assay. Serum levels of tumor necrosis factor α, interleukin-6, intercellular cell adhesion molecule-1 and vascular endothelial growth factor were detected using an enzyme-linked immuno-sorbent assay; the reductase method was performed to detect serum nitric oxide levels. Apoptosis rates were determined using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Ras homolog family member A (RhoA) and Rho-associated protein kinase 1 (ROCK1) protein levels were assessed using western blotting. Transmission electron microscopy was used to observe endothelial cells and myocardial ultrastructure changes. Results revealed that NRG-1-treated rats displayed less myocardial damage compared with sham rats. NRG-1 administration strengthened the barrier function of the vasculature, reduced the secretion of endothelial-associated biomarkers and exerted anti-inflammatory and anti-apoptotic effects. In addition, NRG-1 inhibited RhoA and ROCK1 signaling. The results revealed that NRG-1 improves cardiac function, increases the survival rate of septic rats and exerts protective effects via multiple targets throughout the body. The present results contribute to the development of a novel approach to reverse damage to myocardial and endothelial cells during sepsis.

LncRNA HAND2-AS1 inhibits cancer cell proliferation, migration and invasion by downregulating ROCK1 in HPV-positive and negative cervical squamous cell carcinoma.

Long non-coding (lnc)RNA HAND2 antisense RNA 1 (HAND2-AS1) exhibited tumor suppression activity in different types of cancer. However, its role in cervical squamous cell carcinoma (CSCC), which is frequently diagnosed among females, has not been elucidated. The current study revealed that lncRNA HAND2-AS1 was downregulated, while Rho-associated protein kinase 1 (ROCK1) was upregulated in serum of human papillomavirus (HPV)-positive and -negative patients with CSCC compared with healthy controls. Correlation analysis revealed that the expression levels of lncRNA HAND2-AS1 were negatively correlated with the expression levels of ROCK1 in HPV-positive and -negative patients with CSCC but not in healthy controls. Downregulation of lncRNA HAND2-AS1 distinguished patients with CSCC from healthy controls. Additionally, lncRNA HAND2-AS1 overexpression led to reduced expression levels of ROCK1 in HPV-positive and -negative human CSCC cell lines but not in normal cervical cell lines. ROCK1 overexpression did not significantly affect the expression of lncRNA HAND2-AS1 in all the cell lines investigated. lncRNA HAND2-AS1 overexpression inhibited, while ROCK1 overexpression promoted the proliferation, migration and invasion of HPV-positive and -negative human CSCC cell lines but not normal cervical cell lines. ROCK1 overexpression attenuated the effects of lncRNA HAND2-AS1 overexpression on cancer cell proliferation, migration and invasion. lncRNA HAND2-AS1 may inhibit cancer cell proliferation, migration and invasion by downregulating ROCK1 in HPV-positive and -negative CSCC.

High expression of long intergenic non-coding RNA LINC00662 contributes to malignant growth of acute myeloid leukemia cells by upregulating ROCK1 via sponging microRNA-340-5p

Long non-coding RNAs (lncRNAs) have emerged as crucial regulatory factors in diverse pathological processes, especially in tumorigenesis. Accumulating evidence has demonstrated that long intergenic non-coding RNA 00662 (LINC00662) is overexpressed in multiple cancers and facilitates cancer development and progression. However, whether LINC00662 is involved in acute myeloid leukemia (AML) remains unknown. This study was aimed to explore the expression, biological function and regulatory mechanism of LINC00662 in AML. Here, we found that LINC00662 was significantly increased in AML tissues and cell lines. Knockdown of LINC00662 significantly reduced the growth of AML cells and upregulated AML cell apoptosis. In contrast, overexpression of LINC00662 promoted AML cell growth. MicroRNA-340-5p (miR-340-5p) was predicted as a target of LINC00662. Luciferase reporter assays and RNA pull-down assays confirmed that LINC00662 directly interacted with miR-340-5p. Expression of miR-340-5p was downregulated in AML and silencing of LINC00662 upregulated miR-340-5p expression in AML cells. Moreover, overexpression of miR-340-5p inhibited cell growth and increased apoptosis in AML cells. Inhibition of miR-340-5p significantly reversed the inhibitory effect of LINC00662 silencing on AML cell growth. In addition, Rho-associated protein kinase 1 (ROCK1) was verified as a target gene of miR-340-5p in AML cells. Restoration of ROCK1 expression partially reversed LINC00662 silencing or miR-340-5p overexpression-mediated inhibitory effect on AML cell growth. Overall, our results demonstrate that LINC00662 contributes to the malignant growth of AML cells by upregulating ROCK1 via sponging miR-340-5p, highlighting the important role of the LINC00662/miR-340-5p/ROCK1 axis in regulating the malignant behavior of AML cells.

LncRNA HAND2-AS1 overexpression inhibits cancer cell proliferation in melanoma by downregulating ROCK1.

Long non-coding (lnc)RNA heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1), an lncRNA antisense transcript adjacent to the heart and neural crest derivatives expressed 2 protein (HAND2), inhibits the development of several types of cancer; however, its role in melanoma is currently unknown. In the present study, it was revealed that lncRNA HAND2-AS1 was downregulated, whereas Rho-associated protein kinase 1 (ROCK1) mRNA was upregulated in tumor tissues when compared with the healthy tissues of patients with melanoma. Expression levels of lncRNA HAND2-AS1 and ROCK1 were inversely correlated in tumor tissues, but not in healthy tissues. LncRNA HAND2-AS1 expression levels were affected by tumor thickness, but not by tumor metastasis. LncRNA HAND2-AS1 overexpression led to inhibited melanoma cancer cell proliferation and an increased expression level of ROCK1. ROCK1 overexpression caused no evident effects on lncRNA HAND2-AS1 expression, but promoted cancer cell proliferation and decreased the effects of lncRNA HAND2-AS1 overexpression on cancer cell proliferation. Thus, it is possible that lncRNA HAND2-AS1 overexpression leads to inhibited cancer cell proliferation in melanoma cells through the downregulation of ROCK1.

Serum ROCK1 mRNA is of great diagnostic value for glioma patients.

The study aimed to measure the presence of rho-associated protein kinase 1 (ROCK1) mRNA in serum samples collected from glioma and investigate its diagnostic significance in glioma.The presence of ROCK1 mRNA was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between ROCK1 mRNA and clinical characteristics was analyzed via Chi-square test. The criteria of diagnosis evaluation, including sensitivity, specificity, optimal cutoff point, and area under the curve (AUC) were determined through the receiver operating characteristic (ROC) curve analysis.ROCK1 mRNA was significantly increased in serum samples collected from glioma patients compared to the controls (P <.05). Besides, high ROCK1 mRNA expression was tightly related with Karnofsky Performance Status (KPS) score (P = .024) and World Health Organization (WHO) grade (P = .029). However, there was no association between ROCK1 expression and gender, neurological disorders, family history and cigarette smoking (all, P >.05). In addition, the optimal cutoff point was 3.025, with the sensitivity and specificity of 88.89% and 79.25%, respectively. The AUC was 0.881, indicating that ROCK1 was a diagnostic biomarker for glioma patients (P <.0001, 95% CI = 0.829–0.933).Serum ROCK1 mRNA is significantly up-regulated in glioma cases compared to healthy controls. ROCK1 may be a potential diagnostic biomarker in glioma.

MicroRNA-145 inhibits proliferation and promotes apoptosis of HepG2 cells by targeting ROCK1 through the ROCK1/NF-κB signaling pathway.

Hepatocellular carcinoma (HCC) is a malignant cancer with a high fatality rate, and the expression of microRNA-145 (miR-145) is significantly low in HCC tissue. Therefore, the effect of miR-145 on HCC was explored. Primary hepatocellular carcinoma samples and corresponding normal samples, and HepG2 cells were analyzed using flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Real-time quantitative reverse transcription-polymerase chain reaction, Western blotting, and dual-luciferase reporter assay. miR-145 expression was significantly downregulated in HCC tissue and HepG2 cells as compared to normal liver tissue. After HepG2 cells were transfected with miR-145 mimics, miR-145 expression was recovered, accompanied by a significantly lower cell number, inhibition of the G1/S phase transition, and promotion of the apoptosis of HepG2 cells, as well as changes in levels of G1/S-specific cyclin-E1 (CCNE1) and activated caspase-3. Furthermore, the rho-associated protein kinase 1 (ROCK1) levels were opposite the levels of miR-145 expression in vivo and in vitro, and additional experiments with co-transfection of miR-145 mimics and pEGFP-N3-3'UTR provided the direct evidence that the ROCK1 gene is a target of miR-145. Moreover, a significant decrease or increase in the expression of ROCK1 was associated with nuclear factor-kB (NF-κB)(p65) activity, and lipopolysaccharide (LPS) significantly increased NF-κB(p65) activity, accompanied by recovery of the reduction in the number of HepG2 cells for miR-145 mimics. The NF-κB activity and cell number were significantly (p < 0.05, p < 0.01) increased in response to the overexpression of the ROCK1 gene in HepG2 cells. We showed that miR-145 can target and downregulate ROCK1 expression, and it controls HCC by inhibiting the cell cycle and activating apoptosis via the ROCK1/NF-κB signaling pathway. Our findings will provide a new perspective for the therapy of HCC.

MicroRNA‑122 downregulates Rho‑associated protein kinase 2 expression and inhibits the proliferation of prostate carcinoma cells.

MicroRNA‑122 (miR‑122) has been reported to be involved in the pathogenesis of several types of malignancies; however, its role in prostate carcinoma remains unknown. Thus, the current study aimed to investigate the functionality of miR‑122 in prostate carcinoma. Clinical data of 54patients with prostate carcinoma who were diagnosed and treated in Union Hospital (Wuhan, China) between January 2011 and January 2013 were retrospectively analyzed. The expression levels of miR‑122 and Rho‑associated protein kinase 2 (ROCK2) in prostate tumor and adjacent healthy tissues of patients, as well as in the serum of prostate carcinoma patients and healthy controls, were detected by reverse transcription‑quantitative polymerase chain reaction. Receiver operating characteristic curve and survival curve analyses were used to examine the diagnostic and prognostic values of serum miR‑122 for prostate carcinoma. In addition, miR‑122 mimic was transfected into prostate carcinoma cells, and the effects on cell proliferation and ROCK2 expression were explored by Cell Counting Kit‑8 and western blot assays, respectively. It was observed that miR‑122 was downregulated and ROCK2 was upregulated in tumor tissues as compared with their levels in adjacent healthy tissues. miR‑122 level in the serum was also markedly lower in prostate carcinoma patients in comparison with that in healthy controls. Furthermore, a low serum level of miR‑122 was found to effectively distinguish the prostate carcinoma patients from healthy controls and to be an indicator of poor survival. In prostate carcinoma cells, miR‑122 overexpression inhibited the proliferation and the expression of ROCK2. Taken together, miR‑122 may inhibit the proliferation of prostate carcinoma cells possibly by downregulating ROCK2 expression.

MiR‑335 promotes stress granule formation to inhibit apoptosis by targeting ROCK2 in acute ischemic stroke.

Under harmful environmental conditions, stress granules (SGs), macromolecular aggregates that are associated with cell survival and death, are produced in the eukaryotic cytoplasm. However, whether and how microRNAs (miRNAs/miRs) modulate SG formation induced by acute ischemic stroke has not been investigated. In the present study, a rat model of middle cerebral artery occlusion (MCAO) was utilized and miRNA array profiling and reverse transcription-quantitative polymerase chain reaction were performed. The results revealed that miR-335 was downregulated during acute ischemic stroke, which was concomitant with reduced SG formation, enhanced apoptosis levels and increased Rho associated protein kinase 2 (ROCK2) expression. In the MCAO rat and serum-free cell models, miR-335 treatment upregulated SG formation, alleviated the ischemia-induced infarction, and decreased ROCK2 protein expression and apoptosis levels. By contrast, when compared with miR-335 treatment, the inhibition of miR-335 resulted in reduced SG formation and higher ROCK2 expression and apoptosis levels. Target prediction analysis and luciferase 3′-untranslated region reporter assay identified ROCK2 as the direct target of miR-335. Furthermore, ROCK2 silencing enhanced SG formation and attenuated the level of apoptosis in the serum-free cell model. In addition, ROCK2 silencing markedly inhibited the effect of miR-335 on SG formation and apoptosis levels. Unexpectedly, the phosphorylation of T-cell intracellular antigen-1 was significantly inhibited by miR-335 in the MCAO rat model, which provides a reasonable explanation for the promotional effect of miR-335 on SG formation by specifically targeting ROCK2. In conclusion, these results demonstrate that miR-335 promotes SG formation and inhibits apoptosis by reducing ROCK2 expression in acute ischemic stroke, which provides a possible therapeutic target for brain injury.

Non‑canonical Wnt signaling contributes to ventilator‑induced lung injury through upregulation of WISP1 expression.

Mechanical ventilation may cause ventilator-induced lung injury (VILI). Canonical Wnt signaling has been reported to serve an important role in the pathogenesis of VILI. Bioinformatics analysis revealed that canonical and non-canonical Wnt signaling pathways were activated in VILI. However, the role of non-canonical Wnt signaling in the pathogenesis of VILI remains unclear. The present study aimed to analyze the potential role of non-canonical Wnt signaling in VILI pathogenesis. Lung injury was assessed via Evans blue albumin permeability and histological scoring, as well as by inflammatory cytokine expression and total protein concentration in bronchoalveolar lavage fluid. The relative protein expression of canonical and non-canonical Wnt signaling pathway components were examined via western blotting and immunohistochemistry. The results demonstrated that 6 h of mechanical ventilation at low tidal volume (LTV; 6 ml/kg) or moderate tidal volume (MTV; 12 ml/kg) induced lung injury in sensitive A/J mice. Ventilation with MTV increased the protein levels of Wnt-induced secreted protein 1 (WISP1), Rho-associated protein kinase 1 (ROCK1), phosphorylated (p)-Ras homolog gene family, member A and p-C-Jun N-terminal kinase (JNK). Inhibition of ROCK1 by Y27632 and JNK by SP600125 attenuated MTV-induced lung injury and decreased the expression of proteins involved in non-canonical Wnt signaling, including WISP1. In conclusion, non-canonical Wnt signaling participates in VILI by modulating WISP1 expression, which has been previously noted as critical for VILI development. Therefore, the non-canonical Wnt signaling pathway may provide a preventive and therapeutic target in VILI.

Neuroanatomical Quantitative Proteomics Reveals Common Pathogenic Biological Routes between Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD).

(1) Background: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders with an overlap in clinical presentation and neuropathology. Common and differential mechanisms leading to protein expression changes and neurodegeneration in ALS and FTD were studied trough a deep neuroproteome mapping of the spinal cord. (2) Methods: A liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the spinal cord from ALS-TAR DNA-binding protein 43 (TDP-43) subjects, ubiquitin-positive frontotemporal lobar degeneration (FTLD-U) subjects and controls without neurodegenerative disease was performed. (3) Results: 281 differentially expressed proteins were detected among ALS versus controls, while 52 proteins were dysregulated among FTLD-U versus controls. Thirty-three differential proteins were shared between both syndromes. The resulting data was subjected to network-driven proteomics analysis, revealing mitochondrial dysfunction and metabolic impairment, both for ALS and FTLD-U that could be validated through the confirmation of expression levels changes of the Prohibitin (PHB) complex. (4) Conclusions: ALS-TDP-43 and FTLD-U share molecular and functional alterations, although part of the proteostatic impairment is region- and disease-specific. We have confirmed the involvement of specific proteins previously associated with ALS (Galectin 2 (LGALS3), Transthyretin (TTR), Protein S100-A6 (S100A6), and Protein S100-A11 (S100A11)) and have shown the involvement of proteins not previously described in the ALS context (Methanethiol oxidase (SELENBP1), Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN-1), Calcyclin-binding protein (CACYBP) and Rho-associated protein kinase 2 (ROCK2)).

Irreversible Activation of Rho-activated Kinases Resulted from Evolution of Proteolytic Sites within Disordered Regions in Coiled-coil Domain.

Activation of Rho-associated protein kinase 1 (ROCK1) and myotonic dystrophy kinase-related CDC42-binding kinase alpha (MRCKα) by caspases during apoptosis in vertebrates represents a prototypical example of co-option of kinases by proteases. How caspases acquired the ability to control these proteins during evolution of vertebrates is still unknown. Here, we report a phylogenetic and molecular study on the acquisition of caspase-cleavage sites in the family of Rho-activated kinases (RaKs). We demonstrate that the acquisition of such sites has more frequently occurred in identifiable intrinsically disordered regions (IDRs) within or flanking the coiled-coil domain. Thanks to computational identification of IDRs in protein sequences of different organisms, we predicted and validated the independent evolution of two caspase-cleavage sites in ROCK of arthropods and the loss of one of the MRCKα caspase-cleavage sites in ray-finned fishes. In conclusion, we shed light on the propensity of RaKs to evolve novel proteolytic sites, causing kinase activation and uniform subcellular distribution.

Inhibition of BAP31 expression inhibits cervical cancer progression by suppressing metastasis and inducing intrinsic and extrinsic apoptosis

Cervical cancer is reported as one of the most lethal types of cancer among female. However, extensive studies of the molecular mechanisms that regulate the progression of cervical cancer are still required. B-cell associated protein (BAP)-31 is a 28-kDa integral membrane protein in the endoplasmic reticulum (ER), playing essential role in modulating various physiological processes. The present study indicated that BAP31 was a novel gene associated with cervical cancer development. Here, we demonstrated that BAP31 was significantly increased in human cervical cancer specimens, which was positively correlated to histological grade of the cancer. BAP31 knockdown suppressed cell proliferation, clonogenic ability and metastasis-associated traits in vitro, as well as carcinogenesis and pulmonary metastasis in vivo. Further studies indicated that the expression levels of transforming growth factor (TGF)-β1, matrix metalloproteinase (MMP)-2, MMP-9, Rho-associated protein kinase 1 (ROCK1), α-smooth muscle actin (α-SMA), Vimentin and N-cadherin were markedly reduced by BAP31 knockdown in cervical cancer cells. In addition, intrinsic and extrinsic apoptosis was significantly induced in BAP31 knockdown cells, as evidenced by the increased expression of cleaved Caspase-8/-9/-3 and poly (ADP-ribose) polymerases (PARP). Notably, suppressing the activities of Caspase-8/-9 and −3 obviously diminished BAP31 silence-triggered apoptosis. Together, these findings highlighted an essential role for BAP31 in the modulation of tumorigenesis and metastatic potential of cervical cancer, and demonstrated a promising application of BAP31 in cancer prevention.

Long noncoding RNA POU3F3 promotes cancer cell proliferation in prostate carcinoma by upregulating rho-associated protein kinase 1.

Long noncoding RNAs (lncRNAs) POU3F3 is overexpressed in esophageal squamous-cell carcinomas, while its role in other human cancers is unclear. In this study we found that POU3F3 and rho-associated protein kinase 1 (ROCK1) were both increased in tumor tissues than in adjacent healthy tissues of patients with prostate carcinoma. Expression levels of POU3F3 increased with increase in the diameter of tumor but were not significantly affected by lymph node metastasis or distant metastasis. Expression levels of POU3F3 and ROCK1 were positive correlated in tumor tissues but not in adjacent healthy tissues. POU3F3 and ROCK1 overexpression promoted, while ROCK1 knockdown inhibited the proliferation of prostate carcinoma cells. ROCK1 knockdown reduced the enhancing effect of POU3F3 overexpression on cancer cell proliferation. POU3F3 overexpression led to ROCK1 overexpression in prostate carcinoma cells, while ROCK1 overexpression did not significantly affect POU3F3 expression. Therefore, lncRNA POU3F3 may promote cancer cell proliferation in prostate carcinoma by upregulating ROCK1.

MicroRNA-21 increases the expression level of occludin through regulating ROCK1 in prevention of intestinal barrier dysfunction.

The aim of this study is to investigate the role of molecular mechanism of microRNA (miR)-21 on tight junction (TJ)-proteins and its protective effects on the intestinal barrier. TJ proteins and target genes expression were analyzed in miR-21 inhibition and overexpression NCM460 cell lines. To further verify the role of miR-21, the mmu-miR-21 intestinal epithelial conditional knockout (IKO) mice model was established. MiR-21 expression was detected in clinical specimens of acute stercoral obstruction patients. Rho-associated protein kinase 1 (ROCK1) were identified as target genes of miR-21. There is a negative correlation between miR-21 expression level and TJ proteins levels. TJ protein and ROCK1 were significantly decreased in miR-21 IKO mice, which presented intestinal inflammation response and intestinal barrier dysfunction (both P < 0.05). Determination of clinical samples showed consistent results with NCM460 cell line and miR-21 IKO mice. MiR-21 could be a protective factor of intestinal barrier dysfunction, which promoting the expression of TJ protein by targeting ROCK1 in vivo and in vitro.

MicroRNA-217 functions as a tumor suppressor in cervical cancer cells through targeting Rho-associated protein kinase 1.

The abnormal expression of microRNAs (miRNAs/miRs) has been widely reported in various tumor types. miR-217 was demonstrated to be aberrantly expressed in a number of tumors, including pancreatic adenocarcinoma and osteosarcoma; however, its specific expression pattern has never been investigated in cervical cancer cells. Compared with normal control, the level of Rho-associated protein kinase 1 (ROCK1) expression was markedly increased in cervical cancer tissues and cells compared with that in non-cancerous tissues and cells. The expression of miR-217 was significantly reduced in cervical cancer tissues and cell lines. Overexpression of miR-217 could suppress colony formation and the cell invasion capacity of SiHa and HeLa cells. Flow cytometry indicated that miR-217 significantly increased cell apoptosis in SiHa and HeLa cells. Dual-luciferase reporter assays demonstrated that ROCK1 was a target gene of miR-217. In addition, overexpression of ROCK1 also led to an increased invasion capacity in SiHa cells, even when miR-217 was inhibited, indicating that the anti-invasive effects of miR-217 were mediated through ROCK1. In summary, the results of the present study indicated that miR-217 functions as a tumor suppressor in cervical cancer cells, primarily by targeting ROCK1.