RhoA Protein Expression Research Articles

La-Related Protein 4 as a Suppressor for Motility of Ovarian Cancer Cells.

The La-related proteins (LARPs) are a family of RNA binding proteins that control the degradation and stabilization of RNAs. As emerging research reveals the biology of each LARP, it is evident that LARPs are dysregulated in some types of cancer. Upregulation of cell motility potentiates the metastatic potential of ovarian cancer cells; however, the roles of LARPs in cell motility remain unknown. In the present study, we investigated the roles of LARPs in the progression of ovarian cancer using SKOV3 human ovarian cancer cells and a public database that integrates microarray-based gene expression data and clinical data. To explore the involvement of LARPs in the cell motility, we performed RNA interference screening for LARP mRNAs in SKOV3 cells. The screening identified LARP4 as a potential suppressor of the formation of lamellipodia. Conversely, enforced expression of LARP4 suppressed the formation of lamellipodia. Moreover, cell migration was significantly increased in LARP4-depleted SKOV3 cells. Mechanistically, LARP4 depletion was associated with the decrease in RhoA protein expression. These results suggest that LARP4 may limit RhoA-dependent cell motility. In a mouse xenograft model with SKOV3 cells, LARP4 depletion potentiated peritoneal metastasis. Upon analysis of a public database of patients with ovarian cancer, the LARP4 mRNA-high expression group (n = 166) showed longer overall survival compared with the LARP4 mRNA-low expression group (n = 489), implying a positive correlation of LARP4 mRNA levels in ovarian cancer tissues with patient prognosis. Taken together, we propose that LARP4 could suppress motility and metastatic potential of ovarian cancer cells.

Effect of Piper nigrum ethanolic extract on human breast cancer cell growth and cell migration

Background: Piper nigrum (PN) is widely used as a traditional medicine, which has anti-cancer activity among others. Objective: Our study purposes to illuminate the inhibition of PN effects on breast cancer cells growth and migration along with its mechanisms. Materials and Methods: The piperine in the young fruit of PN extract was determined by the high-performance liquid chromatography method. Growth inhibition with a mechanism of PN extract was studied in the MCF-7 cells using sulforhodamine B assay, cell cycle analysis, colony formation, caspase-3 activity, and reactive oxygen species formation. Furthermore, the anti-migratory effects of PN were investigated using wound healing, matrigel migration, and gelatin zymography assay. Finally, PN mechanism was determined by reverse transcription polymerase chain reaction and Western blotting for the gene and protein levels. Results: Piperine level was showed at a high concentration in the PN extract. Further, the PN extract suppressed Rac1 mRNA expression in the mevalonate (MVA) pathway as well as repressed Rac1 and RhoA protein expression. Interestingly, PN stimulated growth inhibition in dose- and time-dependent as well as being accompanied by increasing the G1 phase arrest and inhibiting cyclin D1 and NF-κB as well as inducing caspase-3 expression. The PN extract inhibited MCF-7 cell migration by reducing matrix metalloproteinases (MMP) 9 protein expression as well as MMP 2, MMP 9, VEGFA, and ICAMP1 gene expression. Conclusion: PN could be herbal medicine for anti-cancer and anti-migratory activities with a correlation to the MVA pathway; therefore, PN maybe deserving further to study as a new candidate for treating breast cancer.

Shen'ge powder decreases the cardiomyocyte hypertrophy in chronic heart failure by activating the Rho protein/Rho-associated coiledcoil forming protein kinase signaling pathway.

The current study aimed to explore the role and the molecular mechanism of Shen'ge powder in cardiomyocyte hypertrophy in chronic heart failure (CHF). Sprague-Dawley rats were selected for the study and divided randomly into four groups: control, model, Shen'ge powder, and fasudil group. An inverted microscope was used to determine the diameter of cardiomyocytes in each group. The survival and apoptotic rate of cardiomyocytes in each group was determined by the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. The messenger RNA levels and protein expression of RhoA, Rho-associated coiled-coil forming protein kinase (ROCK), myosin light-chain phosphatase (MLCP), and myosin light-chain kinase (MLCK) were determined by quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. CHF increased the diameter and apoptotic rate of cardiomyocytes and decreased the survival rate of cardiomyocytes. The levels of RhoA, ROCK, and MLCK were increased significantly in CHF model rats, and the level of MLCP was decreased. After treating with Shen'ge powder, the expression of RhoA, ROCK, and MLCK decreased dramatically and the expression of MLCP increased. Shen'ge powder could reduce cardiomyocyte hypertrophy in CHF by regulating the Rho/ROCK signaling pathway.

Atorvastatin improves left ventricular remodeling and cardiac function in rats with congestive heart failure by inhibiting RhoA/Rho kinase-mediated endothelial nitric oxide synthase.

The aim of the present study was to investigate the effects and possible mechanisms of atorvastatin (Ato) against chronic heart failure (CHF). A rat model of CHF was established and cardiac functions were assessed using Echocardiography. The expression of RhoA/Rho kinase and endothelial nitric oxide synthase (eNOS) was assessed using western blotting and reverse transcription polymerase chain reaction following 4 weeks of treatment. The three groups assessed in the present study were as follows: The control group (no treatment), the Ato + isopropylnoradrenaline (ISO) group (subcutaneous injections of 340 mg/kg ISO + orally administered 50 mg/kg Ato dissolved in saline; administered once daily) and the ISO group (subcutaneous injections of 340 mg/kg ISO + orally administered with an equal volume of saline; administered once daily). Heart volume and weight in the ISO group were significantly increased compared with the control (C) group (P<0.01), whereas contractility was decreased. The results were reverse for the Ato group when compared with the ISO group (P<0.05). Levels of RhoA/Rho kinase protein and mRNA were significantly increased in the ISO group (P<0.01); however. The mRNA and protein expression of eNOS was significantly decreased (P<0.05) when compared with the C group. The mRNA and protein expression of RhoA/Rho kinase was significantly reduced in the Ato+ISO group compared with the ISO group (P<0.01), whereas the mRNA and protein expression of eNOS was significantly increased (P<0.05). RhoA protein expression was increased in the cytoplasm of the C group and on the cell membrane of the ISO group; however, in the Ato+ISO group, RhoA protein expression on the cell membrane was significantly downregulated when compared with the ISO group (P<0.05). The results of the present study suggest that Ato upregulates eNOS by inhibiting RhoA/Rho kinase overexpression in the myocardial tissue of rats with CHF, thus improving left ventricular remodeling and cardiac function.

RhoA and Rho-kinase inhibitors modulate cervical resistance: The possible role of RhoA/Rho-kinase signalling pathway in cervical ripening and contractility

Improper cervical function may lead premature or late-term birth. The RhoA/Rho-kinase (ROCK) signalling pathway takes part in cellular functions including smooth muscle contraction. No information is available about the cervical expression of the RhoA/ROCK system during pregnancy. Our aim was to detect the mRNA and protein expression of ROCK enzymes in rat cervices and to evaluate the effects of RhoA/ROCK inhibitors on cervical resistance.The mRNA and protein expressions of RhoA, ROCK I and II were measured in non-pregnant, pregnant and postpartum rat cervices and during parturition by Real-time qPCR and Western blot. The cervical resistance modifying effects of RhoA (simvastatin) and ROCK (fasudil, Y-27632) (10−6M) were investigated in tissue bath experiments.RhoA mRNA was increased on post-partum day 3, while the RhoA protein expression was decreased near and during parturition. ROCK I mRNA and protein expressions were fluctuating with a decrease in protein expression during parturition. ROCK II mRNA and protein expressions were sharply reduced during parturition. Simvastatin increased the cervical resistance on pregnancy days 20 and 22 while Y-27632 and fasudil reduced the resistance on pregnancy days 20.The decrease in RhoA/ROCK expression near parturition may take part in cervical ripening, especially in the final processes leading to delivery. ROCK inhibitors might be potential drug candidates to treat insufficient cervical ripening late-term pregnancies. The effect of simvastatin possibly due to its unique smooth muscle contracting activity in pregnant cervix. Compounds with simvastatin-like action might be new drug candidates for preterm cervical ripening.

RhoA/ROCK/ARHGAP26 signaling in the eutopic and ectopic endometrium is involved in clinical characteristics of adenomyosis.

ObjectiveThis study aimed to investigate RhoA, RhoA-associated coiled-coil containing protein kinase (ROCK) 1, ROCK2, and Rho GTPase-activating protein 26 (ARHGAP26) expression in the eutopic endometrium (EU) and ectopic endometrium (EC), and examine their relationships with the clinical characteristics of adenomyosis.MethodsTwenty patients with adenomyosis who underwent laparoscopy were recruited. Protein and mRNA expression of RhoA, ROCK1, ROCK2, and ARHGAP26 in EU and EC of patients with adenomyosis and in control endometrium without adenomyosis (CE) was detected.ResultsROCK1, ROCK2, and RhoA mRNA expression in EU was significantly higher than that in CE, and was highest in EC. ARHGAP26 mRNA expression in EC and EU was significantly lower than that in CE. ROCK1, ROCK2, and RhoA protein expression in EC and EU was significantly higher than that in CE. ARHGAP26 protein expression in EC and EU was significantly lower than that in CE. ROCK1, ROCK2, and RhoA gene and protein expression was positively associated and ARHGAP26 was negatively associated with the severity of menorrhagia and menstrual capacity in adenomyosis.ConclusionsRhoA, ROCK1, and ROCK2 expression is upregulated, and ARHGAP26 expression is downregulated in adenomyosis. The RhoA/ROCK-mediated signaling pathway is associated with dysmenorrhea and menstrual capacity in adenomyosis.

Polysaccharide extracts of Astragalus membranaceus and Atractylodes macrocephala promote intestinal epithelial cell migration by activating the polyamine-mediated K+ channel

Astragalus membranaceus (Radix Astragali, RA) and Atractylodes macrocephala (Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control media or media containing spermidine (5 μmol·L−1, SPD), alpha-difluoromethylornithine (2.5 mmol·L−1, DFMO), 4-Aminopyridine (40 μmol·L−1, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L−1), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca2+ ([Ca2+]cyt) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca2+]cyt and accelerated migration of IEC-6 cells, compared with the controls (P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca2+]cyt, but also restored IEC-6 cell migration to control level (P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased (P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0 : 14.1 : 0.3 : 19.9 : 181.3 : 6.3 in RA and 1.0 : 4.3 : 0.1 : 5.7 : 2.8 : 2.2 in RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.

Effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts

To investigate the effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts. Murine RAW264.7 macrophages were induced into osteoclasts by dexamethasone and 1α, 25-dihydroxyvitamin D 3, and identified by tartrate resistant acid phosphatase (TRAP) staining. Then, the osteoclasts were divided into 2 groups. The osteoclasts were treated with 400 ng/mL FTY720-P in experimental group and without FTY720-P in control group, respectively. After 48 hours of culture, the cells in 2 groups were detected by real-time fluorescent quantitative PCR, Western blot, and immunofluorescence staining. The expressions of EphA2, EphrinA2, RhoA, and the bone reconstruction associated proteins［bone morphogenetic protein 2 (BMP-2) and transform growth factor β 1 (TGF-β 1)］were analyzed and compared. RAW264.7 cells were successfully induced into osteoclasts identified by TRAP staining. Compared with control group, the relative expressions of EphA2 and EphrinA2 mRNAs and proteins in experimental group significantly decreased after 48 hours ( P<0.05), and the relative expression of RhoA protein also significantly decreased ( P<0.05). The relative expressions of BMP-2 and TGF-β 1 mRNAs were significantly increased ( P<0.05), and those protein expressions were enhanced. FTY720-P can down-regulate the expression of RhoA and promote the expressions of TGF- β 1 and BMP-2 by affecting the transduction of EphA2-EphrinA2 bidirectional signaling in osteoclasts.

Anti-nerve growth factor antibody improves airway hyperresponsiveness by down-regulating RhoA

ABSTRACTBackground: The pathogenesis of asthma is complex and continues to be considered as a challenging subject. Some studies have shown that nerve growth factor (NGF) participates in the pathogenesis of asthma, but the mechanism of airway contraction caused by NGF is still unclear. Objective: Our aim was to discuss the effect of anti-NGF antibody on RhoA expression, and further explore the role of NGF in airway hyperresponsiveness (AHR). Methods: Thirty female BALB/c mice were divided into three groups randomly: control group (group C, n = 10), asthma group (group A, n = 10) and anti-NGF antibody intervention group (group N, n = 10). The asthmatic mice were stimulated by OVA suspension, the intervention mice were given nasal instillation of anti-NGF antibody before the stimulation. Airway responsiveness, eosinophils, IL-13, IFN-γ were measured. The protein expression and mRNA level of NGF and RhoA were detected by immunohistochemical and Real Time-PCR (RT-PCR) analyses. Results: Airway responsiveness, eosinophils and IL-13 levels in group A were significantly increased compare with the other groups, and significantly decreased in group N than those in group A. IFN-γ level was significantly reduced in group A and increased in group N. Immunohistochemistry and RT-PCR analyses showed that the protein expression and mRNA level of NGF and RhoA were significantly increased in group A and significantly decreased in group N. Conclusion: NGF participates in the pathogenesis of asthma in mice. Anti-NGF antibody can inhibit airway inflammation and alleviate AHR by down-regulating the protein expression and mRNA level of RhoA.

Effect of Different Stimulating Strength of Electroacupuncture on Gastrointestinal Motility and RhoA/ROCK Signaling in Gastric Antral Smooth Muscle in Diabetic Gastroparesis Rats

To observe the effect of different strength of electroacupuncture (EA) stimulation on gastrointestinal motility and Ras homolog gene family member (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) signaling in diabetic gastroparesis (DGP) rats, so as to reveal the underlying mechanisms of EA for improving DGP. Sixty SD rats were randomly and equally divided into blank control, DGP model, weak EA, medium EA, and strong EA groups (n＝12 rats in each). The DGP model was established by intraperitoneal injection of streptozotocin (STZ, 55 mmol/kg, 2%) and high-sugar and high-fat fodder feeding for 8 weeks. EA (0.12, 0.24, 0.36 mA, 20 Hz/100 Hz) was applied to "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Liangmen" (ST 21) for 20 min, once daily for 15 successive days. Blood glucose levels were measured weekly with blood glucose meter and blood glucose test paper. Fecal phenol red excretion method was used to display gastric emptying and small intestinal propulsion function. The expression of RhoA protein in the gastric antral smooth muscle tissue was detected by immunohistochemistry and Western blot (WB), separately, and that of ROCK, myosin phosphatase target subunit 1 (MYPT 1) and phosphorylated (p)-MYPT 1 proteins in gastric antrum detected by WB. Compared with the blank control group, the gastric emptying rate and small intestine propulsion rate of the model group were significantly decreased (P<0.05), and the blood glucose level was remarkably increased (P<0.05). Moreover, the expression levels of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins in the gastric antrum were significantly down-regulated relevant to the control group (P<0.05). After administration of EA, the decreased gastric emptying rate and intestinal propulsion rate, and the down-regulated expression of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins were significantly increased in the strong, medium and weak EA stimulation groups (P<0.05). Comparison among the 3 EA groups showed that the strong stimulation was significantly superior to weak stimulation in up-regulating the expression of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins, and obviously superior to the medium stimulation in up-regulating RhoA and MYPT 1 protein levels (P<0.05), while the medium stimulation was significantly stronger than the weak stimulation in up-regulating the expression of ROCK, MYPT 1 and p-MYPT 1 proteins (P<0.05). There were no significant differences among the 3 EA groups in up-regulating the gastric emptying rate and small intestinal propulsion rate, and between the strong stimulation and medium stimulation in the expression levels of ROCK and p-MYPT 1 proteins (P>0.05). Electroacupuncture stimulation of ST 36-SP 6-ST 21 at 0.12, 0.24 and 0.36 mA can promote the gastrointestinal motility in DGP rats, which may be associated with its effects in enhancing RhoA/ROCK signaling in the gastric antral smooth muscle at different degrees.

Oxidative stress, caspase-3 activation and cleavage of ROCK-1 play an essential role in MeHg-induced cell death in primary astroglial cells

Methylmercury is a toxic environmental contaminant that elicits significant toxicity in humans. The central nervous system is the primary target of toxicity, and is particularly vulnerable during development. Rho-associated protein kinase 1 (ROCK-1) is a major downstream effector of the small GTPase RhoA and a direct substrate of caspase-3. The activation of ROCK-1 is necessary for membrane blebbing during apoptosis. In this work, we examined whether MeHg could affect the RhoA/ROCK-1 signaling pathway in primary cultures of mouse astrocytes. Exposure of cells with 10 μM MeHg decreased cellular viability after 24 h of incubation. This reduction in viability was preceded by a significant increase in intracellular and mitochondrial reactive oxygen species levels, as well as a reduced NAD+/NADH ratio. MeHg also induced an increase in mitochondrial-dependent caspase-9 and caspase-3, while the levels of RhoA protein expression were reduced or unchanged. We further found that MeHg induced ROCK-1 cleavage/activation and promoted LIMK1 and MYPT1 phosphorylation, both of which are the best characterized ROCK-1 downstream targets. Inhibiting ROCK-1 and caspases activation attenuated the MeHg-induced cell death. Collectively, these findings are the first to show that astrocytes exposed to MeHg showed increased cleavage/activation of ROCK-1, which was independent of the small GTPase RhoA.

The epigenetic factor KDM2B regulates cell adhesion, small rho GTPases, actin cytoskeleton and migration in prostate cancer cells

The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2). It has recently been shown that KDM2B co-operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. In the present study we addressed the role of KDM2B in regulating actin cytoskeleton signaling, cell-cell adhesion and migration of prostate tumor cells. We report here that KDM2B is functionally expressed in DU-145 prostate cancer cells, activated by FGF-2 and regulates EZH2. KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E-cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. RhoA and RhoB protein expression and activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. These results establish a clear functional link between the epigenetic factor KDM2B and the regulation of cell adhesion and Rho-GTPases signaling that controls actin reorganization and cell migration.

MicroRNA-141 Inhibits the Proliferation of Penile Cavernous Smooth Muscle Cells Associated with Down-Regulation of the Rhoa/Rho Kinase Signaling Pathway

The role of the RhoA/Rho kinase signaling pathway in diabetes mellitus-induced erectile dysfunction has been partially understood. In the present study, we explored the changes of the RhoA/Rho associated kinase (ROCK) signaling pathway in diabetic erectile dysfunction in vivo and the effects of microRNA-141 on the RhoA/ROCK signaling pathway in vitro. The mRNA and protein expressions of RhoA and ROCK2 were significantly increased while the expression of microRNA-141 was decreased in the penile cavernous smooth muscle cells of rats with diabetic erectile dysfunction. Moreover, increased expression of microRNA-141, decreased expressions of RhoA and ROCK2 (mRNA and protein), accelerated cell proliferation rate and reduced cell apoptosis were found in the microRNA-141 mimics group and the siRNA-Rho group. The microRNA-141 expression in the microRNA-141 inhibitors + siRNA-Rho group was significantly decreased. microRNA-141 specifically bound to Rho-3'-UTR and down-regulated the expression of Rho gene at the post transcriptional level. Decreased expression of miR-141 is associated with up-regulation of RhoA and ROCK2 in the RhoA/ROCK signaling pathway in rats with diabetic erectile dysfunction. miR-141 inhibits the growth of penile cavernous smooth muscle cells associated with down-regulation of the RhoA/ROCK signaling pathway in vitro.

LncRNA PCGEM1 Induces Ovarian Carcinoma Tumorigenesis and Progression Through RhoA Pathway

Background/Aims: Prostate cancer gene expression marker 1 (PCGEM1) is a long noncoding RNA (lncRNA) and is well known as a promoter in prostate cancer and osteoarthritis synoviocytes. However, the role PCGEM1 plays in epithelial ovarian cancer is unknown. Methods: PCGEM1 expression was examined in epithelial ovarian cancer and normal ovarian tissues using reverse transcription–PCR. Ovarian cancer cell phenotypes and genotypes were examined after PCGEM1 overexpression or downregulation in vitro; besides, the effects of PCGEM1 overexpression was also examined in vivo. Results: PCGEM1 expression level was higher in epithelial ovarian cancer tissues than in normal ovarian tissues and was positively associated with differentiation (Well vs. Mod/Poor). Upregulation of PCGEM1 induced cancer cell proliferation, migration, and invasion, but decreased cell apoptosis through upregulating RhoA, YAP (Yes-associated protein), MMP2 (matrix metalloproteinase 2), Bcl-xL, and P70S6K expression; while PCGEM1 downregulation had the opposite effect. The nude mouse xenograft assay demonstrated that PCGEM1 overexpression promoted tumor growth. Furthermore, silencing RhoA expression reversed the effect of PCGEM1 and significantly inhibited RhoA, YAP, MMP2, Bcl-xL, and P70S6K protein expression. Conclusion: In conclusion, we suggest that PCGEM1 may be an inducer in epithelial ovarian cancer tumorigenesis and progression by upregulating RhoA and the subsequent expression of YAP, P70S6K, MMP2, and Bcl-xL.

The roles of interleukin-1 and RhoA signaling pathway in rat epilepsy model treated with low-frequency electrical stimulation.

This study aims to explore the correlation between interleukin-1 (IL-1) and epilepsy in rats when treated with low-frequency electrical stimulation via the RhoA/ROCK signaling pathway. Twenty-four SD rats were elected for this study, among which six rats were assigned as the normal group. And 16 rat models with epilepsy were successfully established and assigned into the model group, the ES group and the ES + IL-8 group, with each group comprising of six rats. The seizure frequency and duration was recorded. Electroencephalogram (EEG) power was detected at α1, α2, β, θ, and δ. The mRNA expressions of IL-1β and IL-1R1 were detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR), and the protein expressions of RhoA, ROCK I and ROCK II were detected by western blotting. In comparison with the model group, the seizure frequency duration, the power of δ, θ, α1, α2, and β, the mRNA and protein expressions of IL-1β and IL-1R1, the expressions of RhoA and ROCK I proteins, and the ratio of RhoA protein between membrane and cytosol decreased in the ES group, while the expression of ROCK II increased (all P < 0.05). There was no significant difference in these indexes between the ES + IL-8 group and the model group (all P > 0.05). These findings signified that IL-1 might inhibit the efficacy of low-frequency ES for epilepsy via the RhoA/ROCK signaling pathway, which may provide a theoretical basis for clinical treatment of epilepsy.

DDEFL1 correlated with Rho GTPases activity in breast cancer.

DDEFL1 is related to maintaining a limiting amount of ARF6 in GTP-loaded form by accelerating its GTP hydrolysis activity, which has been implicated in hepatocellular cancer pathogenesis and lung cancer development. We investigated DDEFL1 expression in breast cancer and paired normal breast tissues by immunohistochemistry and found that DDEFL1 expression was significantly associated with tumor size, lymph node metastasis, high content of elastosis and TNM stage but not with menopausal status or age. We detected the mRNA and protein expression of DDEFL1 in breast cancer cell lines by Western blotting and quantitative real-time PCR (qRT-PCR). DDEFL1 was obvious in MDA-MB-435s and MDA-MB-231 but very weak in ZR-75-1. Further experiments were conducted to evaluate the effect of DDEFL1 small interfering RNA (siRNA) transfection on the biological behavior of MDA-MB-231. After transfection, the effects of DDEFL1 inhibition on expression of mRNA and protein were also analyzed by Western blotting and qRT-PCR. Increased apoptosis and invasive ability, decreased cellular proliferation was found in MDA-MB-231 with successful DDEFL1 siRNA transient transfection (p < 0.05). Western blotting and qRT-PCR results showed that the DDEFL1 inhibition up-regulated Caspase-3, Apaf-1, cytochrome c, and Bax expression and down-regulated Bcl-2 expression. The DDEFL1 inhibition also down-regulated the mRNA and protein expression of Rho, CDC42 and Rac1. Our study provided a functional linkage through DDEFL1 with breast cancer biological behaviours by Rho GTPases. Possible implication of our main finding for the DDEFL1 role in breast cancer and the downstream signaling pathways for the treatment of breast cancer.

Effects of CASZI on bronchopulmonary development of neonatal rats.

The effect of CASZI on bronchopulmonary development in neonatal rats was investigated. Forty SD rats were randomly divided into the control group (n=20) and the experimental group (n=20) using a random number table. The experimental group was treated with hyperoxia to establish the bronchopulmonary dysplasia (BPD) model, while the control group received no treatment. At 14 days of experiment, rats in each group were treated, and lung tissues were taken for hematoxylin and eosin (H&E) staining. Radial alveolar count (RAC) and morphological changes in lung tissues were observed under a microscope, and the thickness of respiratory membrane was detected via IPP6 image. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect the expression levels of CASZI, Rho kinase, RhoA and ET-A receptor in lung tissues. There were no obviously pathological changes in lung tissues of neonatal rats in the control group, while BPD could be seen in lung tissue structures of neonatal rats in the experimental group. RAC in experimental group was significantly lower than that in the control group (P<0.05), and the thickness of respiratory membrane was significantly higher than that in the control group (P<0.05). CASZI mRNA expression level in the experimental group was significantly lower than that in control group (P<0.05), and Rho kinase, RhoA and ET-A receptor mRNA expression levels were significantly higher than those in the control group (P<0.05). CASZI protein expression level in the experimental group was significantly lower than that in the control group (P<0.05), and the Rho kinase, RhoA and ET-A receptor protein expression levels were significantly higher than those in the control group (P<0.05). In conclusion, CASZI expression was downregulated during the development of BPD. CASZI downregulation may have an effect on the pulmonary microvascular regeneration through RhoA/ROCK signaling pathway, thus participating in the pathogenesis of BPD.

Modulation of chondrocyte motility by tetrahedral DNA nanostructures.

Contemporarily, a highly increasing attention was paid to nanoconstructs, particularly DNA nanostructures possessing precise organization, functional manipulation, biocompatibility and biodegradability. Amongst these DNA nanomaterials, tetrahedral DNA nanostructures (TDNs) are a significantly ideal bionanomaterials with focusing on the property that can be internalized into cytoplasm in the absence of transfection. Therefore, the focus of this study was on investigating the influence of TDNs on the chondrocytes locomotion. Tetrahedral DNA nanostructures was confirmed by 6% polyacrylamide gel electrophoresis (PAGE) and dynamic light scattering (DLS). Subsequently, the effect of TDNs on chondrocyte locomotion was investigated by real-time cell analysis (RTCA) and wound healing assay. The variation of relevant genes and proteins was detected by quantitative polymerase chain reaction (qPCR), western blotting and immunofluorescence respectively. We demonstrated that tetrahedral DNA nanostructures have positive influence on chondrocytes locomotion and promoted the expression of RhoA, ROCK2 and vinculin. Additionally, upon exposure to TDNs with the concentration of 250nmolL-1 , the chondrocytes were showed the highest motility via both RTCA and wound healing assay. Meanwhile, the mRNA and protein expression of RhoA, ROCK2 and vinculin were also significantly enhanced with the same concentration. It can be concluded that the TDNs with the optimal concentration of 250nmolL-1 could extremely promoted the chondrocytes locomotion through facilitating the expression of RhoA, ROCK2 and vinculin. These results seemed to reveal that this special three-dimensional DNA tetrahedral nanostructures may be applied to cartilage repair and treatment in the future.

Effects of geranylgeranyltransferaseⅠsilencing on the proliferation of tongue squamous cancer cells

Objective This study aims to investigate the effect of geranylgeranyltransferaseⅠ (GGTase-Ⅰ) on the proliferation and growth of tongue squamous cancer cells. Methods Three small interfering RNAs (siRNAs) were designed on the basis of the GGTase-Ⅰ sequence in GeneBank. These siRNAs were then transfected into tongue squamous cancer cells Cal-27. The mRNA and protein expression of GGTase-Ⅰ and RhoA were examined by real-time quantitative polymerase chain reaction and Western blotting, respectively. The expression of Cyclin D1 and p21 were examined by Western blotting. The proliferation and growth ability were analyzed by cell counting kit-8 assay and flow cytometry. Results The mRNA and protein expression of GGTase-Ⅰ in Cal-27 was reduced significantly after the GGTase-Ⅰ siRNAs were transfected (P<0.05). No significant difference in RhoA mRNA and protein expression was detected (P>0.05). Cyclin D1 expression decreased, whereas p21 expression increased significantly. The cell cycle was altered, and the growth-proliferative activity was inhibited (P<0.05). Conclusion GGTase-Ⅰ siRNA can inhibit the expression of GGTase-Ⅰ and the proliferative activity of tongue squamous cancer cells. GGTase-Ⅰ may be a potential target for gene therapy in tongue squamous cell cancer.

Abstract 2111: Ciclopirox inhibits tumor cell motility by suppressing protein expression of small GTPases and phosphorylation of paxillin

Abstract Ciclopirox olamine (CPX), an off-patent antifungal drug, is used for the treatment of superficial mycoses. Recent studies have demonstrated that CPX also possesses potent anticancer activity by inhibiting cell proliferation and inducing cell death in tumor cells. However, it is unknown whether CPX inhibits cell motility. In the present study, we found that CPX potently inhibited cell motility in a concentration-dependent manner in rhabdomyosarcoma (Rh30 and RD) cells, which was independent of the reduction of cell viability. As small GTPases (RhoA, Cdc42, and Rac1) and focal adhesion proteins (paxillin) play critical roles in the regulation of cell motility, we further studied whether CPX affects these proteins. Our Western blot analysis revealed that CPX did inhibit cellular protein expression of RhoA, Cdc42 and Rac1. However, by RT-PCR analysis, CPX did not alter the mRNA levels of the small GTPases. As microRNAs (miRNAs) can regulate protein expression at transcriptional and post-transcriptional levels, we investigated the effect of CPX on the expression of miRNAs. Our miRNA PCR array revealed that treatment with 10 μM of CPX for 24 hours upregulated the expression of 27 miRNAs by 2.04-6.10 fold, and downregulated the expression of 7 miRNAs by 2.00-7.89 fold in Rh30 cells. Further research is ongoing to identify whether these up/downregulated miRNAs are linked to the reduced expression of small GTPases and potentially more proteins. In addition, we found that CPX inhibited the phosphorylation of paxillin, despite no effect on the total cellular protein expression. The results suggest that CPX inhibits tumor cell motility at least by suppressing the protein expression of small GTPases and the phosphorylation of paxillin. Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA. Citation Format: Shile Huang, Tao Shen. Ciclopirox inhibits tumor cell motility by suppressing protein expression of small GTPases and phosphorylation of paxillin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2111. doi:10.1158/1538-7445.AM2017-2111