RhoA mRNA Research Articles

Effects of PI3Kδ-RhoA pathway on phagocytosis defect of alveolar macrophages in a mouse model of chronic obstructive pulmonary disease

Objective: To investigate effects of Phosphoinositide3-Kinases (PI3Kδ)-Ras homolog gene family member A(RhoA) pathway on phagocytosis deficiency of alveolar macrophages (AMs) in a mouse model of chronic obstructive pulmonary disease (COPD). Methods: Twenty mice were exposed to cigarette smoking to establish the COPD model, with 20 mice as the control group. AMs were isolated from lung tissue by discontinuous density gradient centrifugation and then divided into a healthy control group, a COPD group, a healthy IC87114 group and a COPD IC87114 group. The culture of IC87114 group was mixed with a final concentration of 1 nmol/L IC87114 for 24 hours. Mean fluorescence intensity (MFI) and the positive percent of AMs engulfing flurescein isothiocyanate-labeled Escherichina coli (FITC-E.coli) (AM%) were detected by flow cytometry. Real-Time PCR(RT-PCR)and Western blot were applied to detect mRNA and protein. G-LISA RhoA Kit was used to detect the activity of RhoA, and laser scanning confocal microscopy was used to observe the cytoskeleton structure of AMs. Results: Phagocytosis of AM: MFI and AM %in the COPD group [(4 512±517), (32.2±4.6)%] were decreased than those in the healthy control group [(9 857±1 042), (68.0±4.0)%, all P<0.01]. Compared with the COPD group, MFI and AM% in the COPD IC87114 group [(6 894±472), (50.6±2.1)%] were increased (all P<0.01). The expressions of mRNA and protein of PI3Kδ in the COPD group (3.14±0.54, 0.84±0.08) were increased than those in the healthy control group (1.00±0.00, 0.57±0.07) (all P<0.01). Compared with the COPD group, the expressions of mRNA and protein of PI3Kδ in the COPD IC87114 group (1.52±0.28, 0.66±0.13) were decreased (all P<0.01). The RhoA mRNA, protein and activity in the COPD group (0.70±0.07, 0.41±0.10, 0.70±0.06) were decreased compared to those in the healthy control group (1.00±0.00, 0.56±0.09, 1.19±0.09) (all P<0.01). Compared with the COPD group, the expression of mRNA, protein and activity of RhoA in the COPD IC87114 group(0.91±0.08, 0.48±0.06, 0.86±0.06) were increased (P<0.01, P<0.05). Cytoskeleton of AM: The pseudopods of the healthy control group and the healthy IC87114 group extended well, and the ability of phagocytosing FITC-E.coli was intact, but there were some defects in the COPD group. Compared with the COPD group, the COPD IC87114 group was better, both in phagocytosing and extending of pseudopods. Negative correlations existed between the mRNA, protein of PI3Kδ with mRNA, protein and activity of RhoA. Negative correlations also existed between the mRNA, protein of PI3Kδ with MFI, but positive correlations between RhoA and MFI were observed in all groups. Conclusion: The phagocytosis of AMs in COPD mice was defective, with abnormal rearrangement of the cytoskeleton. PI3Kδ negatively regulated RhoA, while PI3Kδ over activation resulted in decreasing activity of RhoA and then induced abnormal cytoskeleton rearrangement in AMs, which led to phagocytosis deficiency.IC87114 inhibited PI3Kδ activation, improved the activity of RhoA and partly recovered phagocytosis of AMs.

Mechanisms of cytoskeleton and PI3Kδ-RhoA in fine particulate matter deteriorating phagocytosis defect of alveolar macrophage in mice with chronic obstructive pulmonary disease

Objective: To explore the mechanism of cytoskeleton and PI3Kδ-RhoA in fine particulate matter deteriorating phagocytosis defect of alveolar macrophage (AM) in chronic obstructive pulmonary disease (COPD) mice. Methods: Forty mice were randomly divided into four groups: health control group, COPD group, health PM2.5 group, COPD PM2.5 group and with ten in each group. A mouse model of COPD was established by cigarette smoke exposure, and health PM2.5 group and COPD PM2.5 group mice were given PM2.5 (588 μg/m(3)) aerosol inhalation for 90 days. AM were isolated from lung tissue by discontinuous density gradient centrifugation. Mean fluorescence intensity (MFI) and the percent of alveolar macrophage engulfing flurescein isothiocyanate-labeled Escherichia coli (FITC-E.coli) AM (AM%) were detected by flow cytometry. The mRNA and protein expression were measured by real time polymerase chain reaction (RT-PCR) and Western blot. The activity of RhoA was measured by GTPase linked immunosorbent assay (G-LISA) Kit. Cytoskeleton was observed by laser scanning confocal microscopy. Results: The MFI and the AM% in COPD group [4 512±517, (32.19±4.57)%] and health PM2.5 group [7 631±585, (50.78±4.58)%] were significantly lower than those in health control group [9 857±1 042, (68.53±2.88)%], while those in COPD PM2.5 group [3 121±393, (21.90±2.58)%] were lower than those in COPD group (all P<0.01). The mRNA and protein of PI3Kδ in COPD group (3.41±0.54, 0.84±0.08)and health PM2.5 group (1.52±0.35, 0.71±0.11) were higher than those in health control group (1.00±0.00, 0.57±0.07) (all P<0.05), and in COPD PM2.5 group (5.53±0.42, 1.17±0.25), the above parameters were remarkably increased as compared to those in COPD group (all P<0.01). The mRNA, protein and activity of RhoA in COPD group (0.70±0.07, 0.41±0.10, 0.70±0.06) and health PM2.5 group (0.84±0.06, 0.46±0.11, 0.87±0.07) were lower than those in health control group (1.00±0.00, 0.56±0.09, 1.19±0.09) (all P<0.05), and above parameters of COPD PM2.5 group (0.42±0.05, 0.31±0.06, 0.44±0.04) were significantly lower than COPD group (all P<0.01). Cytoskeleton of AM: long and dense filopodia and membrane fold could been seen clearly around the AM of health control group; in COPD group and health PM2.5 group, short and sparse filopodia and slightly deformed AM can been seen. Filopodia remarkably decreased and rigid cells with impaired capacity of engulfing FITC-E.coli can be generally observed in COPD PM2.5 group. Negative correlations were existed between PI3Kδ mRNA, protein and RhoA mRNA, protein, activity in all groups (all P<0.01). Negative correlations were existed between PI3Kδ mRNA, protein and MFI, and positive correlations were existed between RhoA mRNA, protein, activity and MFI in all groups (all P<0.05). Conclusion: Fine particulate matter (PM2.5) can deteriorate the phagocytosis of AM from COPD mice through over activating PI3Kδ and inhibiting the activity of RhoA then causing cytoskeleton abnormal rearrangement.

Antenatal taurine supplementation in fetal rats with growth restriction improves neural stem cell proliferation by inhibiting the activities of Rho family factors

Objective: To investigate whether antenatal taurine supplementation improves neural stem cell proliferation in rats with fetal growth restriction (FGR) through regulating the activity of Rho family factors.Methods: FGR models were established via food restriction throughout pregnancy. Pregnant rats were randomly divided into the control group, the FGR group (given 40% of the normal daily feeding in the control group), and the Taurine group (FGR model treated with 300 mg/kg·d taurine from gestational day seven). Expression of fatty acid binding protein-7 (FABP-7), Rho-associated coiled coil-forming protein kinase (ROCK2), Ras homolog gene family member A (RhoA), and rac in the brains of newborn rats was detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blotting (WB).Results: Relative FABP7 mRNA levels, the optical density (OD) values of FABP7-positive cells and the expression levels of the tested proteins all demonstrated that the number of neural stem cells (NSCs) in brain tissue was lower in the FGR group than in the control group but was significantly increased after antenatal taurine supplementation (p < .05). Compared with the control group, the mRNA and protein levels of RhoA and ROCK2 were higher in the FGR group but lower in the Taurine group (p < .05). In contrast, the rac mRNA level was lower in the FGR group than in the control group but was higher in the Taurine group (p < .05).Conclusions: Taurine prenatal supplementation improved neural stem cell proliferation in rats with FGR by inhibiting the activity of Rho family factors.

MiR-155-5p positively regulates CCL17-induced colon cancer cell migration by targeting RhoA.

Colorectal cancer is the second most common cause of cancer-related death, which is due to migration of tumor cells to distant sites of metastasis. Accumulating data indicate that mciroRNAs play an important role in several aspects of colon cancer cell biology. Herein, we examined the role of miR-155-5p in colon cancer cell migration induced by the CCL17-CCR4 axis in HT-29 colon cancer cells. We found that miR-155-5p knockdown in serum starved colon cancer cells decreased CCL17-induced cell chemotaxis. Moreover, knocking down miR-155-5p markedly decreased CCL17-provoked activation of RhoA in colon cancer cells. Bioinformatics analysis predicted two putative binding sites in the AU-rich element at the 3′-UTR of RhoA mRNA. MiR-155-5p binding to RhoA mRNA was verified using a target site blocker and functionally validated by RNA immunoprecipitation assays, showing that miR-155-5p-dependent regulation of RhoA mRNA is mediated by AU-rich elements present in the 3′-UTR region. Taken together, these results show that miR-155-5p positively regulates RhoA mRNA levels and translation as well as cell migration in serum starved colon cancer cells and indicate that targeting miR-155-5p might be a useful strategy to antagonize colon cancer metastasis.

Antenatal taurine supplementation improves neural axon development in fetal rats with intrauterine growth restriction by inhibiting the activity of Rho-ROCK signaling pathway

Objective To investigate the effects of prenatal taurine supplementation on the Rho-ROCK signaling pathway activity and synaptophysin (Syp) expression in brain tissues of rats with intrauterine growth restriction. Methods Eighteen pregnant Sprague-Dawley rats were randomly divided into control group, fetal growth restriction (FGR) group and taurine group, with six rats in each group. Low-protein diet was given in FGR and taurine groups to establish an FGR model. Taurine 300 mg/(kg·d) was supplemented from gestational day 12 until delivery in taurine group. The mRNA expression levels of neurite growth inhibitor-A(Nogo-A), neurite growth inhibitor receptor (NgR), Rho-A and ROCKⅡin fetal rat brain were detected using reverse transcriptase polymerase chain reaction (n=24), which are the key signaling molecules of the Rho-ROCK signal pathway. The protein expression levels of Nogo-A and NgR were detected by Western blot (n=12). The mean optical density in Nogo-A, NgR and Syp was determined by immunohistochemistry (n=18). One-way analysis of variance and LSD-t test were used for statistical analysis. Results (1) Expression of mRNA: the expression levels of Nogo-A, NgR, Rho-A and ROCKⅡ mRNA in fetal rat brain were 4.09±1.34, 3.01±0.77, 39.89±7.71 and 7.82±1.83, respectively in FGR group, and were significantly higher than in control group (1.00±0.13, 1.00±0.10, 1.02±0.30 and 1.00±0.10) (t=4.735, 5.204, 7.682 and 10.675, all P 0.05). (2) Expression of protein by Western blot: the expressions of Nogo-A and NgR protein in fetal rat brain were 1.51±0.09 and 0.31±0.05 in FGR group, 0.82±0.06 and 0.06±0.01 in taurine group, and 1.04±0.10 and 0.09±0.12 in control group. The expression was significantly higher in FGR group than in control group (t=9.644 and 5.285, both P 0.05). (3) Positive expression of protein: the positive expressions of Nogo-A and NgR protein in fetal rat brain were 0.28±0.06 and 0.11±0.02 in FGR group, 0.10±0.02 and 0.04±0.01 in taurine group, and 0.07±0.01 and 0.04±0.01 in control group. The expression was significantly higher in FGR group than in control group (t=9.778 and 7.645, both P 0.05). The positive expression of Syp protein in fetal rat brain was 0.08±0.01 in FGR group, and was significantly lower than in control group (0.16±0.04, t=4.600, P 0.05). Conclusions Prenatal taurine supplementation can improve neural axon development via down-regulating the expressions of the key molecules of Rho-ROCK signal pathway in fetal rat brain tissue. Key words: Fetal growth retardation; Taurine; Rho-associated kinases; Synaptophysin; Signal transduction; Axons; Rats, Sprague-Dawley

RhoA knockdown by cationic amphiphilic copolymer/siRhoA polyplexes enhances axonal regeneration in rat spinal cord injury model.

Spinal cord injury (SCI) results in permanent loss of motor and sensory function due to developmentally-related and injured-induced changes in the extrinsic microenvironment and intrinsic neuronal biochemistry that limit plasticity and axonal regeneration. Our long term goal is to develop cationic, amphiphilic copolymers (poly (lactide-co-glycolide)-g-polyethylenimine, PgP) for combinatorial delivery of therapeutic nucleic acids (TNAs) and drugs targeting these different barriers. In this study, we evaluated the ability of PgP to deliver siRNA targeting RhoA, a critical signaling pathway activated by multiple extracellular inhibitors of axonal regeneration. After generation of rat compression SCI model, PgP/siRhoA polyplexes were locally injected into the lesion site. Relative to untreated injury only, PgP/siRhoA polyplexes significantly reduced RhoA mRNA and protein expression for up to 4 weeks post-injury. Histological analysis at 4 weeks post-injury showed that RhoA knockdown was accompanied by reduced apoptosis, cavity size, and astrogliosis and increased axonal regeneration within the lesion site. These studies demonstrate that PgP is an efficient non-viral delivery carrier for therapeutic siRhoA to the injured spinal cord and may be a promising platform for the development of combinatorial TNA/drug therapy.

Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase II in spinal cord injury rats

Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase (ROCK) signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan (GV3), Dazhui (GV14), Zusanli (ST36) and Ciliao (BL32) and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the mRNA and protein expression of Rho-A and Rho-associated kinase II (ROCKII) of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKII. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKII. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of RhoA and ROCKII. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.

Long non-coding RNA NONMMUG014387 promotes Schwann cell proliferation after peripheral nerve injury.

Schwann cells play a critical role in peripheral nerve regeneration through dedifferentiation and proliferation. In a previous study, we performed microarray analysis of the sciatic nerve after injury. Accordingly, we predicted that long non-coding RNA NONMMUG014387 may promote Schwann cell proliferation after peripheral nerve injury, as bioinformatic analysis revealed that the target gene of NONMMUG014387 was collagen triple helix repeat containing 1 (Cthrc1). Cthrc1 may promote cell proliferation in a variety of cells by activating Wnt/PCP signaling. Nonetheless, bioinformatic analysis still needs to be verified by biological experiment. In this study, the candidate long non-coding RNA, NONMMUG014387, was overexpressed in mouse Schwann cells by recombinant adenovirus transfection. Plasmid pHBAd-MCMV-GFP-NONMMUG014387 and pHBAd-MCMV-GFP were transfected into Schwann cells. Schwann cells were divided into three groups: control (Schwann cells without intervention), Ad-GFP (Schwann cells with GFP overexpression), and Ad-NONMMUGO148387 (Schwann cells with GFP and NONMMUGO148387 overexpression). Cell Counting Kit-8 assay was used to evaluate proliferative capability of mouse Schwann cells after NONMMUG014387 overexpression. Polymerase chain reaction and western blot assay were performed to investigate target genes and downstream pathways of NONMMUG014387. Cell proliferation was significantly increased in Schwann cells overexpressing lncRNA NONMMUG014387 compared with the other two groups. Further, compared with the control group, mRNA and protein levels of Cthrc1, Wnt5a, ROR2, RhoA, Rac1, JNK, and ROCK were visibly up-regulated in the Ad-NONMMUGO148387 group. Our findings confirm that long non-coding RNA NONMMUG014387 can promote proliferation of Schwann cells surrounding the injury site through targeting Cthrc1 and activating the Wnt/PCP pathway.

Extracellular HSP70/HSP70-PCs regulate hepatocarcinoma cell migration and invasion via RhoA.

The effects of heat shock protein 70 (HSP70)/HSP70-peptide complexes (HSP70-PCs) on the invasion and metastasis ability of hepatocellular carcinoma (HCC) Huh-7 cells were investigated. Wound healing assay revealed that cells treated with HSP70/HSP70-PCs healed faster than negative control cells. HSP70/HSP70-PCs-treated cells also exhibited better migration ability and higher invasion ability than control cells. HSP70/HSP70-PCs treatment did not alter the messenger RNA (mRNA) or protein levels of matrix metalloproteinase-9; the opposite was true for Ras homolog family member A (RhoA) mRNA and protein levels. RNA interference of RhoA attenuated the migration of HSP70/HSP70-PCs-treated cells. The present findings indicate that regulation of HCC cell migration by HSP70/HSP70-PCs occurs via regulation of RhoA expression.

MiR-31 Regulates Rho-Associated Kinase-Myosin Light Chain (ROCK-MLC) Pathway and Inhibits Gastric Cancer Invasion: Roles of RhoA.

BackgroundThis study evaluated how the expression of miR-31 can be used to detect gastric cancer (GC) to help illuminate the role of miR-31 and RhoA in GC cells.Material/MethodsWe carried out our experiments using tissue specimens from 70 GC patients. The relative expression of miR-31 and RhoA mRNA in tissues and cells was detected by RT-PCR. The expression level of RhoA protein was detected by immunohistochemistry. GC cell line BGC-823 was transfected with six groups of vectors: blank group, NC (negative control) group, miR-31 mimics group, miR-31 mimics + RhoA group, miR-31 mimics + ROCK group, and miR-31 mimics + MLCK agonist group. AGS cells were also transfected with six groups of vectors: blank group, NC group, miR-31 inhibitor group, miR-31 inhibitor + RhoA siRNA group, miR-31 inhibitor + ROCK siRNA group, and miR-31 inhibitor + MLCK inhibitor group. Transwell assay was performed to detect the invasion and migration of cells. The protein expression in different transfected groups was detected using Western blotting.ResultsGC tissues exhibited significantly lower levels of miR-31 expression compared to pericarcinous tissues (p<0.01). Moreover, a significantly higher expression of RhoA in GC tissues was observed (p<0.05). MiR-31 inhibited RhoA expression by binding to 3′UTR of mRNA, whereas miR-31 mimics significantly decreased the number of invaded and migrated cells (p<0.05). The activation of RhoA, ROCK, and phosphorylation of MLC remarkably exacerbate the invasion and migration ability of GC cells (p<0.05).ConclusionsWe found miR-31 could downregulate the ROCK/MLC pathway by inhibiting the expression of RhoA in order to suppress the invasion and migration of GC cells.

Effects of RhoA silencing on proliferation of tongue squamous cancer cells

This study investigated the effect of RhoA silencing through RNA interference on proliferation and growth of tongue cancer cells, as well as explored the possible mechanisms of this effect. SSC-4 tongue cancer cells were cultured in vitro and then transfected with small interfering RNA to knock down RhoA expression. The tested cells were divided into three groups: experimental group (experimental group 1: transfected with RhoA-siRNA-1; experi-mental group 2: transfected with RhoA-siRNA-2), negative control group (transfected by random sequence NC-siRNA), and blank control group (transfected with Lipofectamine). The expression levels of RhoA mRNA were respectively measured by quantitative real-time polymerase chain reaction and western blot assay. Moreover, the expression levels of cyclin D1, p21, and p27 and RhoA protein were evaluated by Western blot assay. Proliferation and growth potentiality were analyzed through evaluation of doubling times and methyl thiazolyl tetrazolium assessment. The expression levels of RhoA gene and protein of experimental groups significantly decreased following siRNA transfection compared with those in the negative and blank control groups. The expression of cyclin D1 decreased significantly and that of p21 and p27 increased significantly. The doubling time was extended and the growth potentiality decreased. The results indicated that RhoA silencing can inhibit proliferation of tongue cancer cells, whereas RhoA affects cell proliferation by regulating the cell cycle pathway. Thus, RhoA is a potential target in gene therapy for tongue cancer.

Mechanistic target of rapamycin is activated in bovine granulosa cells after LH surge but is not essential for ovulation.

The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6hr, returning to basal levels by 12hr after GnRH treatment, RHOA mRNA levels increased at 6hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR-1 were higher 3hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH-induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.

Effect of Three Statins on Glucose Uptake of Cardiomyocytes and its Mechanism.

BackgroundThe aim of this study was to investigate the effects of different statins on glucose uptake and to confirm its mechanism in primary cultured rat cardiomyocytes after administration of atorvastatin, pravastatin, and rosuvastatin.Material/MethodsPrimary cultured rat cardiomyocytes were randomly assigned to 5 groups: normal control group (OB), insulin group (S1), statin 1-μM (S2), 5-μM (S3), and 10-μM (S4) groups for 3 different statins. The 2-[3H]-DG uptake of each group was determined and the mRNA and protein expression levels of glucose transporter type 4 (GLUT4), insulin receptor substrate (IRs), and RhoA were assessed.ResultsAfter treatment with different concentrations of statins and insulin, the 2-[3H]-DG uptake showed a significant negative correlation with the concentration of atorvastatin (P<0.05), and no significant correlation with pravastatin and rosuvastatin. The mRNA and protein expression levels of GLUT4 and IRs-1 in primary cultured cardiomyocytes were both significantly reduced by atorvastatin treatment (P<0.05). Pravastatin and rosuvastatin showed no significant effects on GLUT4 and IRs-1 expression. The mRNA and protein expression levels of RhoA both showed no significant difference when treated with the 3 statins.ConclusionsThese results confirm that atorvastatin can inhibit insulin-induced glucose uptake in primary cultured rat cardiomyocytes by regulating the PI3K/Akt insulin signal transduction pathway.

Self-Assembled Tetrahedral DNA Nanostructures Promote Adipose-Derived Stem Cell Migration via lncRNA XLOC 010623 and RHOA/ROCK2 Signal Pathway.

Self-assembled tetrahedral DNA nanostructures (TDNs) with precise sizes have been extensively applied in various fields owing to their exceptional mechanical rigidity, structural stability, and modification versatility. In addition, TDNs can be internalized by mammalian cells and remain mainly intact within the cytoplasm by escaping degradation by nucleases. Here, we studied the effects of TDNs on cell migration and the underlying molecular mechanisms. TDNs remarkably enhanced the migration of rat adipose-derived stem cells and down-regulated the long noncoding RNA (lncRNA) XLOC 010623 to activate the mRNA expression of Tiam1 and Rac1. Furthermore, TDNs highly up-regulated the mRNA and protein expression of RHOA, ROCK2, and VCL. These results indicate that TDNs suppressed the transcription of lncRNA XLOC 010623 and activated the TIAM1/RAC1 and RHOA/ROCK2 signaling pathways to promote cell migration. On the basis of these findings, TDNs show a high potential for application in tissue repair and regenerative medicine as a functional three-dimensional DNA nanomaterial.

Effects of phased joint intervention on Rho/ROCK expression levels in patients with portal hypertension.

The current study investigated the effects of phased joint intervention on clinical efficacy and Rho/Rho-associated coil protein kinase (ROCK) expression in patients with portal hypertension complicated by esophageal variceal bleeding (EVB) and hypersplenism. Patients with portal hypertension (n=53) caused by liver cirrhosis complicated by EVB and hypersplenism treated with phased joint intervention were assessed, and portal hemodynamics, blood, liver function, complications, and rebleeding incidence were analyzed. Reverse transcription-quantitative polymerase chain reaction was used to measure Rho, ROCK1 and ROCK2 mRNA expression levels in peripheral blood mononuclear cells prior to and following phased joint intervention, and western blotting was employed to determine the protein expression levels of Rho, ROCK1, ROCK2, phosphorylated (p) myosin phosphatase target subunit 1 (MYPT1) and total-MYPT1. All patients underwent an emergency assessment of hemostasis with a 100% success rate. Varicose veins were alleviated, and portal hemodynamics and liver function improved following intervention. Furthermore, preoperative and postoperative expression levels of Rho, ROCK1 and ROCK2 mRNA were higher compared with the control group. Notably, the mRNA expression levels of Rho, ROCK1 and ROCK2 in the postoperative group were significantly lower when compared with the preoperative group. Protein expression levels of Rho, ROCK1, ROCK2 and pMYPT1 in the postoperative group were lower, as compared with the preoperative group. Concentration levels of transforming growth factor-β1, connective tissue growth factor and platelet-derived growth factor in peripheral blood were significantly reduced following phased joint intervention. Therefore, the present findings demonstrated that phased joint intervention is able to effectively treat EVB and hypersplenism, and improve liver function. The efficacy of phased joint intervention may be associated with its role in the regulation of the Rho-ROCK signaling pathway.

Gastrokine 1 inhibits gastric cancer cell migration and invasion by downregulating RhoA expression.

We investigated whether GKN1, a gastric tumor suppressor, contributes to the progression of gastric cancer by regulating RhoA expression. We analyzed the expression of GKN1, RhoA, miR-185, and miR-34a in 35 gastric cancer tissues, and compared their expression with T category and TNM stage. Cell migration and invasion, as well as the expression of epithelial-to-mesenchymal transition (EMT)-related proteins, were assessed in GKN1- and RhoA small interfering RNA (siRhoA)-transfected and recombinant-GKN1-treated AGS and MKN1 gastric cancer cells. Expression of RhoA protein and messenger RNA (mRNA) was increased in 15 (42.9%) and 17 (48.6%) of 35 gastric cancer tissues respectively, and was associated with higher T category and TNM stage. GKN1 expression was significantly decreased in 27 gastric cancers (77.1%) with a higher T category, and was inversely correlated with RhoA mRNA expression. In AGS and MKN1 cells, GKN1 expression increased miR-185 and miR-34a expression and reduced RhoA mRNA and protein expression. A positive relationship between GKN1 and miR-34a and miR-185 expression and an inverse relationship between miR-34a and RhoA expression were observed in gastric cancer tissues. Cell migration and invasiveness were markedly decreased in GKN1- and siRhoA-transfected cells. GKN1 expression and silencing of RhoA decreased the expression of the proteins Snail, Slug, and vimentin. Furthermore, miR-185 and miR-34a silencing in MKN1 cells transfected with GKN1 stimulated cell migration and invasion, and increased the expression of EMT-related proteins. Our data suggest that GKN1 may inhibit gastric cancer cell migration and invasion by downregulating RhoA expression in a miR-185- and miR-34a-dependent manner.

A cellular high-throughput screening approach for therapeutic trans-cleaving ribozymes and RNAi against arbitrary mRNA disease targets

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets.To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N = G,C,A,U; H = C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization.

Lentivirus-mediated PGC-1α overexpression protects against traumatic spinal cord injury in rats

Peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) is a crucial neuronal regulator in the brain. However, its role in the spinal cord and the underlying regulating mechanisms remain poorly understood. Our previous study demonstrated that PGC-1α is significantly down-regulated following acute spinal cord injury (SCI) in rats. The current study aimed to explore the effects of PGC-1α overexpression on the injured spinal cord by establishing a contusive SCI model in adult Sprague–Dawley rats, followed by immediate intraspinal injection of lentiviral vectors at rostral and caudal sites 3mm from the lesion epicenter. Hindlimb motor function was monitored using the Basso–Beattie–Bresnahan Locomotor Rating Scale (BBB scores), and cords were collected. Transfection efficiency analysis showed that lentivirus successfully induced enhanced PGC-1α expression. This resulted in attenuated apoptotic changes and a greater number of surviving spinal neurons, as determined by transmission electron microscopy and Nissl staining, respectively. Western blot and immunofluorescence analyses revealed increased growth-associated protein 43 and 5-hydroxytryptamine expression, two key markers of axonal regeneration. Importantly, BBB scores showed improved hindlimb motor functional recovery. Moreover, quantitative real-time polymerase chain reaction analysis demonstrated significantly inhibited RhoA, ROCK1, and ROCK2 mRNA expression, revealing a potential mechanism of PGC-1α overexpression following traumatic SCI. Altogether, these results suggest that gene delivery of PGC-1α exerts a significant neuroprotective effect following traumatic SCI, which could serve as a promising treatment for repair of the injured cord, and RhoA–ROCK pathway inhibition may partially underlie this neuroprotection.

Role of the RhoA/ROCK pathway in high-altitude associated neonatal pulmonary hypertension in lambs.

Exposure to high-altitude chronic hypoxia during pregnancy may cause pulmonary hypertension in neonates, as a result of vasoconstriction and vascular remodeling. We hypothesized that susceptibility to pulmonary hypertension, due to an augmented expression and activity of the RhoA/Rho-kinase (ROCK) pathway in these neonates, can be reduced by daily administration of fasudil, a ROCK inhibitor. We studied 10 highland newborn lambs with conception, gestation, and birth at 3,600 m in Putre, Chile. Five highland controls (HLC) were compared with 5 highland lambs treated with fasudil (HL-FAS; 3 mg·kg(-1)·day(-1) iv for 10 days). Ten lowland controls were studied in Lluta (50 m; LLC). During the 10 days of fasudil daily administration, the drug decreased pulmonary arterial pressure (PAP) and resistance (PVR), basally and during a superimposed episode of acute hypoxia. HL-FAS small pulmonary arteries showed diminished muscular area and a reduced contractile response to the thromboxane analog U46619 compared with HLC. Hypoxia, but not fasudil, changed the protein expression pattern of the RhoA/ROCKII pathway. Moreover, HL-FAS lungs expressed less pMYPT1(T850) and pMYPT1T(696) than HLC, with a potential increase of the myosin light chain phosphatase activity. Finally, hypoxia induced RhoA, ROCKII, and PKG mRNA expression in PASMCs of HLC, but fasudil reduced them (HL-FAS) similarly to LLC. We conclude that fasudil decreases the function of the RhoA/ROCK pathway, reducing the PAP and PVR in chronically hypoxic highland neonatal lambs. The inhibition of ROCKs by fasudil may offer a possible therapeutic tool for the pulmonary hypertension of the neonates.

MiR-124 Promote Neurogenic Transdifferentiation of Adipose Derived Mesenchymal Stromal Cells Partly through RhoA/ROCK1, but Not ROCK2 Signaling Pathway.

ObjectiveSome recent studies suggest that multiple miRNAs might regulate neurogenic transdifferentiation of mesenchymal stromal cells (MSCs). In the present study, we hypothesized that the miR-124 can repress the expression of RhoA upon the neurogenesis of adipose derived MSCs (ADMSCs).MethodsMiRNA expression dynamics during neurogenic transdifferentiation of ADMSCs were measured. The expression of neuron-specific enolase (NSE), Tuj-1 (Neuron-specific class III beta-tubulin) and glial fibrillary acidic protein (GFAP), as well as electrophysiological properties, were detected after neurogenic transdifferentiation. The targeting of miR-124 over RhoA was verified by dual luciferase assay, qRT-PCR and western blot. The functions of miR-124 and the RhoA/ROCK signaling pathway were studied using gain and loss of function experiments in vitro.ResultsMiR-124 is significantly upregulated during neurogenic transdifferentiation of ADMSCs. Knockdown of endogenous miR-124 hampered neurogenic transdifferentiation and the acquired electrophysiological properties. MiR-124 could directly target RHOA mRNA and repress its expression, through which it increased the proportion of transdifferentiated (transdiff.) cells with positive NSE, Tuj-1 and GFAP. RhoA/ROCK1, but not ROCK2 is a downstream signaling pathway of miR-124 in the process of transdifferentiation.ConclusionMiR-124 is an important miRNA modulating neurogenic transdifferentiation of ADMSCs at least partly via the miR-124/RhoA/ROCK1 signaling pathway. These findings provided some fundamental information for future use of ADMSCs as an agent for regenerative medicine and cell therapy for neurological diseases.