RhoA mRNA Expression Research Articles

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.

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.

RhoA/rock signaling mediates peroxynitrite-induced functional impairment of Rat coronary vessels.

BackgroundDiabetes-induced vascular dysfunction may arise from reduced nitric oxide (NO) availability, following interaction with superoxide to form peroxynitrite. Peroxynitrite can induce formation of 3-nitrotyrosine-modified proteins. RhoA/ROCK signaling is also involved in diabetes-induced vascular dysfunction. The study aimed to investigate possible links between Rho/ROCK signaling, hyperglycemia, and peroxynitrite in small coronary arteries.MethodsRat small coronary arteries were exposed to normal (NG; 5.5 mM) or high (HG; 23 mM) D-glucose. Vascular ring constriction to 3 mM 4-aminopyridine and dilation to 1 μM forskolin were measured. Protein expression (immunohistochemistry and western blot), mRNA expression (real-time PCR), and protein activity (luminescence-based G-LISA and kinase activity spectroscopy assays) of RhoA, ROCK1, and ROCK2 were determined.ResultsVascular ring constriction and dilation were smaller in the HG group than in the NG group (P < 0.05); inhibition of RhoA or ROCK partially reversed the effects of HG. Peroxynitrite impaired vascular ring constriction/dilation; this was partially reversed by inhibition of RhoA or ROCK. Protein and mRNA expressions of RhoA, ROCK1, and ROCK2 were higher under HG than NG (P < 0.05). This HG-induced upregulation was attenuated by inhibition of RhoA or ROCK (P < 0.05). HG increased RhoA, ROCK1, and ROCK2 activity (P < 0.05). Peroxynitrite also enhanced RhoA, ROCK1, and ROCK2 activity; these actions were partially inhibited by 100 μM urate (peroxynitrite scavenger). Exogenous peroxynitrite had no effect on the expression of the voltage-dependent K+ channels 1.2 and 1.5.ConclusionsPeroxynitrite-induced coronary vascular dysfunction may be mediated, at least in part, through increased expressions and activities of RhoA, ROCK1, and ROCK2.

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.

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.

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.

Clinicopathological Significance of RhoA Expression in Digestive Tract Cancer: A Systematic Review and Meta-Analysis

RhoA protein expression has been reported in different types of cancer. We performed an up-to-date meta-analysis to evaluate the clinicopathological characteristics of RhoA protein expression in patients with gastrointestinal cancer. We searched in several databases, including MEDLINE (PubMed) and China National Knowledge Infrastructure, to identify studies examining the association between RhoA protein and cancer. The quality of the included studies was assessed. Cochrane Collaboration's Software Review Manager 5.3 was utilized to test the heterogeneity, overall effect, and publication bias of the combined studies. The reported odds ratio and 95% confidence interval (CI) were calculated by using fixed and random effects models depending on the heterogeneity of the included studies. A total of 15 studies met the inclusion criteria of the meta-analysis. RhoA expression was significantly higher in gastrointestinal cancer than in normal tissues. RhoA protein expression in digestive system neoplasms was significantly associated with tumor clinical staging, metastatic status and differentiated degree. However, no association with gender was found. RhoA mRNA expression was no associated with clinicopathological significance. Current evidence supports the conclusion that RhoA expression is associated with clinical staging, metastatic status, and differentiated degree in digestive system neoplasms. RhoA expression may play an important role in the carcinogenesis and metastasis of gastrointestinal cancer.

Roles of Ras Homolog A in Invasive Ductal Breast Carcinoma.

Breast cancer has a poor prognosis owing to tumor cell invasion and metastasis. Although Ras homolog (Rho) A is involved in tumor cell invasion, its role in breast carcinoma is unclear. Here, RhoA expression was examined in invasive ductal carcinoma (IDC), with a focus on its relationships with epidermal-mesenchymal transition (EMT) and collective cell invasion. Forty-four surgical IDC tissue samples and two normal breast tissue samples were obtained. RhoA, E-cadherin, vimentin, and F-actin protein expression were analyzed by immunohistochemistry. RhoA, ROCK, mTOR, AKT1, and PIK3CA mRNA expression were conducted using laser microdissection and semi-nested quantitative reverse transcription-polymerase chain reaction. RhoA expression was stronger on the tumor interface of IDCs than the tumor center (P<0.001). RhoA expression was correlated with ROCK expression only in HER2-subtype IDC (P<0.05). In IDCs co-expressing RhoA and ROCK, F-actin expression was stronger on the tumor interface, particularly at the edges of tumor cells, than it was in ROCK-negative IDCs (P<0.0001). In conclusion, RhoA expression was not correlated with EMT in IDC, but enhanced F-actin expression was localized on the edge of tumor cells that co-expressed ROCK. RhoA/ROCK signaling may be associated with collective cell invasion, particularly in HER2-subtype IDC.

Absence of the Adenosine A2A Receptor Confers Pulmonary Arterial Hypertension Through RhoA/ROCK Signaling Pathway in Mice.

Numerous evidence suggests that RhoA/Rho kinase (ROCK) signaling pathway plays an important role in the pathogenesis of pulmonary arterial hypertension (PAH), but little is known about its effects on the development of PAH in mice with absence of the adenosine A2A receptor (A2AR). Eight A2AR knockout (KO) and 8 wild-type mice were used. Morphometric analysis of pulmonary arterioles included right ventricle/left ventricle plus ventricular septum (Fulton index), vessel wall thickness/total vascular diameter (WT%), and vessel wall area/total vascular area (WA%). The expression of RhoA and ROCK1 mRNA was determined by real-time polymerase chain reaction. The expression of RhoA, ROCK1, and phosphorylation of myosin phosphatase target subunit 1 proteins in pulmonary tissue was tested by Western blot. The position of ROCK1 protein was evaluated by immunohistochemistry. Compared with wild-type mice, A2AR KO mice displayed (1) increased Fulton index, WT%, and WA% (P < 0.01); (2) increased mRNA expression of RhoA and ROCK1 (each P < 0.05); (3) increased protein expression of RhoA, ROCK1, and phosphorylation of myosin phosphatase target subunit 1 (each P < 0.01); (4) increased location of ROCK1 protein in endothelial and smooth muscle cells of pulmonary artery, bronchial, and alveolar epithelial cells. Activation of RhoA/ROCK signaling pathway may cause pulmonary vascular constriction, pulmonary artery remodeling, and PAH in adenosine A2A receptor KO mice.

EphA2 Is a Potential Player of Malignant Cellular Behavior in Non-Metastatic Renal Cell Carcinoma Cells but Not in Metastatic Renal Cell Carcinoma Cells.

ObjectivesTo investigate the role of EphA2 in malignant cellular behavior in renal cell carcinoma (RCC) cells and whether FAK/RhoA signaling can act as downstream effectors of EphA2 on RCC cells.MethodsExpression of EphA2 protein in non-metastatic RCC (Caki-2 and A498), metastatic RCC cells (Caki-1 and ACHN), HEK-293 cells and prostate cancer cells (PC-3 and DU-145; positive controls of EphA2 expression) was evaluated by Western blot. Changes in mRNA or protein expression of EphA2, FAK or membrane-bound RhoA following EphA2, FAK or RhoA small interfering RNA (siRNA) transfection were determined by reverse transcription polymerase chain reaction or Western blot. The effect of siRNA treatment on cellular viability, apoptosis and invasion was analyzed by cell counting kit-8, Annexin-V and modified Matrigel-Boyden assays, respectively.ResultsIn all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; however, in HEK-293 cells, EphA2 expression was very low. Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines. For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control. In non-metastatic RCC cells, FAK siRNA significantly attenuated the invasiveness, resistance to apoptosis, as well as expression of membrane-bound RhoA protein without changing protein expression of EphA2. RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation.ConclusionsOur data provide the first functional evidence that the EphA2/FAK/RhoA signaling pathway plays a critical role in the malignant cellular behavior of RCC and appears to be functional particularly in the early stage of malignant progression of non-metastatic RCC.

Nitric oxide enhances keratinocyte cell migration by regulating Rho GTPase via cGMP-PKG signalling.

ObjectiveNitric oxide (NO) has been shown to improve wound healing, but the mechanism underlying this function is not well defined. Here, we explored the effect of NO on the migration of a human keratinocyte cell line (HaCaT) and its possible mechanism.MethodsThe effects of NO on HaCaT cells in the presence of different concentrations of the NO donor sodium nitroprusside (SNP) were evaluated in a cell migration assay. Subsequently, the cytoskeleton reorganization of cultured HaCaT cells stained with rhodamine-phalloidin was observed with a confocal laser scanning microscope. The mRNA expression and active proteins of CDC42, Rac1 and RhoA in the cultured cells were determined via RT-PCR and pull-down assays, respectively. Furthermore, the roles of various inhibitors or agonists specific to cGMP, PKG and CDC42, Rac1, RhoA in the effects of NO on HaCaT cell migration, F-actin stress fibre formation, and Rho GTPase expression were observed.ResultsIt was also found HaCaT cell migration was increased by SNP in a dose-dependent manner, and the other two NO donors either spermine NONOate or SNAP had almost the same effects on HaCat cell migrations. The formation of F-actin stress fibres in SNP-treated HaCaT cells was increased. The mRNA expression and the active proteins of CDC42, Rac1 and RhoA were found to be upregulated after SNP treatment. Similar effects were observed after the cells were treated with a cGMP or PKG agonist. Additionally, the SNP-mediated upregulation of the mRNA expression and the active proteins of CDC42, Rac1 and RhoA were inhibited by the addition of an inhibitor of cGMP or PKG. Moreover, the SNP-mediated promoting effects of migration and cytoskeleton reorganization were inhibited by treatment with inhibitors of cGMP, PKG, CDC42, Rac1 and RhoA respectively.ConclusionOur data indicated that the stimulatory effects of NO on cell migration of HaCaT cells are mediated by the cGMP signalling pathway via the upregulation of Rho-GTPase expression, which might promote cytoskeleton reorganization.

Neuroprotective effects of electroacupuncture on early- and late-stage spinal cord injury.

Previous studies have shown that the neurite growth inhibitor Nogo-A can cause secondary neural damage by activating RhoA. In the present study, we hypothesized that electroacupuncture promotes neurological functional recovery after spinal cord injury by inhibiting RhoA expression. We established a rat model of acute spinal cord injury using a modification of Allen's method. The rats were given electroacupuncture treatment at Dazhui (Du14), Mingmen (Du4), Sanyinjiao (SP6), Huantiao (GB30), Zusanli (ST36) and Kunlun (BL60) acupoints with a sparse-dense wave at a frequency of 4 Hz for 30 minutes, once a day, for a total of 7 days. Seven days after injury, the Basso, Beattie and Bresnahan (BBB) locomotor scale and inclined plane test scores were significantly increased, the number of apoptotic cells in the spinal cord tissue was significantly reduced, and RhoA and Nogo-A mRNA and protein expression levels were decreased in rats given electroacupuncture compared with rats not given electroacupuncture. Four weeks after injury, pathological tissue damage in the spinal cord at the site of injury was alleviated, the numbers of glial fibrillary acidic protein- and neurofilament 200-positive fibers were increased, the latencies of somatosensory-evoked and motor-evoked potentials were shortened, and their amplitudes were increased in rats given electroacupuncture. These findings suggest that electroacupuncture treatment reduces neuronal apoptosis and decreases RhoA and Nogo-A mRNA and protein expression at the site of spinal cord injury, thereby promoting tissue repair and neurological functional recovery.

Zuo-Gui and You-Gui pills, two traditional Chinese herbal formulas, downregulated the expression of NogoA, NgR, and RhoA in rats with experimental autoimmune encephalomyelitis

Ethnopharmacological relevanceZuo-Gui pills (ZGPs) and You-Gui pills (YGPs) are 2 traditional Chinese herbal formulas used for treating multiple sclerosis (MS) in the clinical setting and have been shown to have neuroprotective effects in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The aim of this study was to explore the mechanisms underlying the neuroprotective functions of ZGPs and YGPs. Materials and methodsFemale Lewis rats were randomly divided into normal control, EAE model, 2g/kg ZGP-treated EAE, 3g/kg YGP-treated EAE, and prednisone acetate-treated groups. EAE model was induced by subcutaneous injection of MBP68–86 antigen. The neurological function scores were estimated. Histological structures of the brains and spinal cords were observed, and myelinated and axons imaged. NogoA, Nogo receptor (NgR), and RhoA transcript and protein levels were measured by real-time quantitative RT-PCR and western blotting on postimmunization (PI) days 14 (acute stage) and 28 (remission stage). ResultsZGPs and YGPs significantly reduced neurological functions scores and abrogated inflammatory infiltrates, demyelination, and axonal damage. Furthermore, treatment with ZGPs and YGPs inhibited NogoA, NgR, and RhoA mRNA and protein expression in rats at both the acute and remission stages. ZGPs exhibited stronger effects on NogoA and RhoA expressions, as well as neurological function, during the acute stage of EAE, while YGPs caused greater reductions in NogoA expression during the remission stage. ConclusionsOur findings suggested that ZGPs and YGPs exerted neuroprotective effects by downregulation of NogoA, NgR, and RhoA pathways, with differences in response times and targets observed between ZGPs and YGPs.

Abstract 4965: Hypoxia has different effects on proliferation, migration and metastasis of various types of tumor cells

Abstract Hypoxia promotes angiogenesis and assigns radio- and chemotherapy resistance and aggressive phenotype to tumor cells. HIF-1 alpha, the main regulator of hypoxia induced processes, is activated in areas of low oxygen supply. Rho GTPases such as Rac1, cdc42 and RhoA play an important role in actin polimerization, depolimerization and stress fiber formation during tumor morphogenesis, cell adhesion, and cell migration. In the present study cell lines of melanoma (HT168-M1), fibrosarcoma (HT1080), colon cancer (HT25, HT29), lung cancer (H1975) and a head-neck cancer (PECA-PJ15) were cultured under normoxic and hypoxic conditions containing 1% and 5% O2. Proliferation tests with sulforhodamine B, migration assays with modified Boyden chamber and time-lapse videomicroscopy were performed with all cell lines. Furthermore, investigation of the expression and localization of HIF-1alpha, Rac1, cdc42 and RhoA mRNA by quantitative RT-PCR and immunfluorescence and the activation of small GTPases were performed. In addition, we examined the role of the hypoxic condition (CoCl2 administration) and HIF blockade (chetomin administration) on the metastatic potential of tumor cells using orthotopic xenograft models. The various tumor cell lines displayed different behaviour in vitro and in vivo. In the case of the most motile cell line (HT-168-M1), proliferation and migration capacity, moreover the gene expression patterns of small GTPases and HIF-1alpha were significantly higher under hypoxic conditions compared to normoxic cells. Migration capacity of the PECA-PJ15 cell line was higher when cultured under higher oxygen concentration. The hypoxic condition was less tolerated by both human colon carcinoma cell lines. In HT29 cells RhoA translocated into the nucleus under the hypoxic condition. The in vivo experiments demonstrated that primary tumor size and the number of metastasis were the largest in the CoCl2-treated group, and the smallest in the chetomin-treated group. In conclusion, our findings support that different phenotypes of the cancer cells form under hypoxia presumably via different pathways. This work is supported by OTKA K84173. Citation Format: Enikő Tátrai, Alexandra Bartal, Zsuzsanna Fehér, Ákos Farkas, Sándor Paku, István Kenessey, József Tóvári. Hypoxia has different effects on proliferation, migration and metastasis of various types of tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4965. doi:10.1158/1538-7445.AM2014-4965

MicroRNA 340 is involved in UVB-induced dendrite formation through the regulation of RhoA expression in melanocytes.

The influence of UV irradiation on pigmentation is well established, but the molecular and cellular mechanisms controlling dendrite formation remain incompletely understood. MicroRNAs (miRNAs) are a class of small RNAs that participate in various cellular processes by suppressing the expression of target mRNAs. In this study, we investigated the expression of miRNAs in response to UVB irradiation using a microarray screen and then identified potential mRNA targets for differentially expressed miRNAs among the genes governing dendrite formation. We subsequently determined the ability of miRNA 340 (miR-340) to suppress the expression of RhoA, which is a predicted miR-340 target gene that regulates dendrite formation. The overexpression of miR-340 promoted dendrite formation and melanosome transport, and the downregulation of miR-340 inhibited UVB-induced dendrite formation and melanosome transport. Moreover, a luciferase reporter assay demonstrated direct targeting of RhoA by miR-340 in the immortalized human melanocyte cell line Pig1. In conclusion, this study has established an miRNA associated with UVB irradiation. The significant downregulation of RhoA protein and mRNA expression after UVB irradiation and the modulation of miR-340 expression suggest a key role for miR-340 in regulating UVB-induced dendrite formation and melanosome transport.

Abstract 351: Activation Of The Central Renin Angiotensin System (RAS) Causes Selective Cerebrovascular Dysfunction

Approximately 30% of hypertensive patients have low plasma renin. The deoxycorticosterone (DOCA)/salt model of hypertension exhibits low peripheral RAS activity, but activation of the central RAS. The impact of central RAS activation on the cerebrovasculature is not defined. We tested the hypothesis that activation of the central RAS would impair endothelial function in brain. We produced mild hypertension in male C57BL/6 mice using subcutaneous DOCA and 0.9% salt in their drinking water for 3 weeks (no nephrectomy was performed) followed by studies of isolated arteries in vitro . Blood pressure was elevated modestly after DOCA/salt treatment (79±2 vs 95±3 mmHg). Endothelial function was severely impaired in cerebral arteries from mice treated with DOCA/salt (e.g. 10 μM acetylcholine: 33±3% vs 4±4%) but was unaffected in similar-sized mesenteric arteries. Because microvascular dysfunction is a key feature of small vessel disease in brain that increases the risk of stroke and cognitive impairment, we examined responses of small cerebral arterioles (baseline diameter ~30 μm) in vivo using a cranial window. Endothelium-dependent dilation to acetylcholine in cerebral arterioles was reduced by ~75% following DOCA/salt treatment (e.g. 10 μM acetylcholine: 26±5% vs 6±3%). In contrast, endothelium-independent vasodilation to nitroprusside (NO donor) or nifedipine (Ca 2+ channel blocker) was similar between groups. Microvascular endothelial dysfunction was not affected by a scavenger of superoxide but was restored to normal by local inhibition of Rho kinase (ROCK) with Y27632. Activation of AT1 receptors by angiotensin II can increase ROCK signaling. Local inhibition of AT1 receptors with losartan or inhibition of mineralocorticoid receptors (MR) with eplerenone improved endothelium-dependent dilation in DOCA/salt treated mice to normal levels. RhoA and ROCK2 mRNA expression and total endothelial NO synthase protein expression was similar between groups. These findings suggest that activation of the central RAS profoundly but selectively impairs endothelial function in brain. Our findings identify AT1 receptors, MR and ROCK as key contributors to endothelial microvascular dysfunction in this model of mild DOCA/salt-induced hypertension.

Study the effect of Pingchuan formula on Rho/Rock signaling pathway in the mice of asthmatic model

Objective To study the effect of Pingchuan Formula（PCF）on Rho/Rock signaling pathway in the mice of asthmatic model, explore the mechanism of PCF on asthma. Methods Forty BALB/C mice were randomly divided into four groups：a control group, a model group,a dexamethasone group and a PCF group. After treated with medicine for 4 weeks, albumen expression of RhoA and RockI in lung tissues were detected by using Western blot, mRNA expressions of RhoA and RockI were detected by using Real-time PCR. Results The albumen and the mRNA expression of RhoA and RockI in the model group were（1.05± 0.20）,（1.06±0.08）,（6.60±1.09）, （6.53±1.84）, the control group were（0.76±0.08）, （0.84±0.14）, （3.82± 1.77）, （3.65±1.46）,there was a significant difference between two groups（P0.05）. Conclusion The expressions of Rho/Rock signaling pathway were controlled significantly by PCF, and the curative effect was similar to dexamethasone. Key words: Asthma; Pingchuan Formula; The Rho/Rock signaling pathway

Antenatal taurine improves neuronal regeneration in fetal rats with intrauterine growth restriction by inhibiting the Rho-ROCK signal pathway.

The Rho-ROCK signal pathway is an important mediator of inhibitory signals that blocks central nervous cell regeneration. Here, we investigated whether antenatal taurine improved neuronal regeneration in fetal rats with intrauterine growth restriction (IUGR) by inhibiting this pathway. Thirty pregnant rats were randomly divided into three groups: control, IUGR, and IUGR + antenatal taurine supplementation (taurine group). The mRNA levels of Ras homolog gene A (Rho A), Rho-associated coiled-coil forming protein kinase 2 (ROCK2), and proliferating cell nuclear antigen (PCNA) were detected using real-time quantitative PCR. RhoA, ROCK2 and PCNA-positive cells were counted using immunohistochemistry. Antenatal taurine supplementation decreased RhoA and Rock2 mRNA expression, increased PCNA mRNA expression, and significantly decreased RhoA, ROCK2-positive and increased PCNA-positive cell counts in IUGR fetal rat brain tissues (p < 0.05). Thus, antenatal taurine supplementation inhibited the expression of key Rho-ROCK signal molecules and improved IUGR fetal brain development.

Activation of hepatocyte growth factor promotes apoptosis of hepatic stellate cells via the Rho pathway

To investigate the role of activated hepatocyte growth factor (HGF) in apoptosis of hepatic stellate cells (HSCs) and in modulating the Rho signaling pathway. HSCs were divided into the following groups: blank control, consisting of HSCs without treatment; two treatment controls, consisting of HSCs exposed to exogenous HGF at 50 ng/ml and HSCs exposed to exogenous HGF activator (HGFA) at 70 ng/ml; three experimental groups, consisting of HSCs exposed to both exogenous HGF and HGFA, HSCs pre-incubated with the HGF inhibitor c-met at 500 ng/ml for 6 hours and then exposed to exogenous HGF and HGFA, and HSCs pre-incubated with the Rho pathway inhibitor Y-27632 at 10 ng/ml and then exposed to exogenous HGF and HGFA. Activation status of the cultured HSCs was determined by change in expression of alpha-smooth muscle actin (SMA). The optimal intervention concentration of Y-27632 was determined by MTT assay. The apoptotic status of HSCs was determined by flow cytometry. Expression of the HGF-alpha chain was detected by immunofluorescence. The expression of RhoA was evaluated by PCR (for mRNA) and by immunohistochemical staining and Western blot analysis (for protein). Exposure to 10 mumol/L Y-27632 led to obvious growth inhibition of HGF + HGFA-induced HSCs, compared with the other concentrations tested (P less than 0.05). HGF + HGFA induced the expression of the HGF-alpha chain in a time-dependent manner (P less than 0.01); however, the increases in expression of HGF-alpha chain induced by HGF alone and HGFA alone were not significantly different from the level in the blank controls (P more than 0.05). Exposure to HGF alone and HGFA alone led to a time-dependent increase in apoptosis (24 h, 48 h, 72 h) but exposure to HGF + HGFA led to the highest levels of apoptosis (P less than 0.05). Exposure to HGF + HGFA led to a time-dependent decrease in RhoA mRNA and protein expression (P less than 0.01). Activation of hepatocyte growth factor promotes apoptosis of hepatic stellate cells by suppressing RhoA expression and down-regulating the Rho signaling pathway.