OBJECTIVEWhite matter damage (WMD) in preterm infants is a well-recognized serious complication of prematurity. The collapse of cell skeleton of growth cone after hypoxia-ischemia (HI) is considered as the basic neuropathologic change of the long-term residuals of premature white matter damage. F-actin is the major component of cell skeleton and maintains the normal form of cells, its function and potential mechanism of WMD have not been reported. In this study, changes of F-actin and its influencing factor RhoA were investigated.

METHODSTotally 184 Sprague-Dawley (SD) rats (age 2 days, body weight 6 to 8 grams) were randomly divided into 14 groups: 7 different time WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d, 14 d, 28 d) and 7 corresponding control groups. The 2 day-old SD rats were subjected to ligation of right carotid artery (ischemia), and then they were put into a box full with 6% oxygen and 94% nitrogen for 4 hours (hypoxia). The light microscopy was used to observe the brain pathological changes and the electron microscopy was used to detect the brain ultrastructural changes after hypoxia and ischemia. Eighty SD rats were used for flurescent-immunohistochemical method to detect the distribution of F-actin in cell membrane and cytoplasm of both WMD groups and the control groups at 12 h, 24 h, 48 h, 72 h, 7 d after HI respectively. The distribution of F-actin was reflected by the percentage of non-integrity cells. Another 80 SD rats were used for real time RT-PCR to detect the expression of RhoAmRNA in the white matter tissue of both WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d) and the control groups.

RESULTS(1) Necrosis of lateral ventricle tissue was observed by 72 h after HI. Dilatation of ventricle and formation of capsular space beneath white matter had been observed by 14 d after HI. (2) Disregulation, pyknosis, mitochondrion swelling and chromatin agglutination were observed in WMD groups. The maldevelopment of myelins in WMD groups was detected at 1 h after HI. (3) The fluorescent stains decreased on cellular membrane, but increased in cytoplasm with time. The percentage of non-integrity cells was significantly higher (P < 0.05) in HI groups (0.32 +/- 0.04, 0.43 +/- 0.04, 0.56 +/- 0.03, 0.65 +/- 0.04, 0.87 +/- 0.03) than the controls (0.02 +/- 0.01, 0.02 +/- 0.01, 0.01 +/- 0.01, 0.02 +/- 0.01, 0.02 +/- 0.01). (4) The expression of RhoA mRNA was significantly increased (P < 0.05) in HI groups (1.205, 2.415, 4.830, 1.500) in the white matter tissue compared with the controls (0.300, 0.375, 0.375, 0.530) at 12 h, 24 h, 48 h, 72 h after HI. The expression of RhoA mRNA reached the peak value at HI 48 h, and then gradually decreased. The expression of RhoA mRNA at HI 7 d in WMD group (0.500) was not significantly different from the control (P > 0.05).

CONCLUSION(1) The pathological and ultrastructural changes of white matter in WMD groups after HI suggest that the WMD model was successfully set up in premature 2 days SD rats. (2) F-actin is redistributed within cells after HI: expression in membrane is decreased and expression in plasma was increased. The redistribution possibly results in the collapse and retraction of cells. (3) The expression of RhoA mRNA is increased significantly after HI, which may lead to the redistribution of F-actin. (4) The increase of the expression of RhoA mRNA is not persistent, but the redistribution of F-actin is continued, which suggests that RhoA may not be the only factor affecting the redistribution of F-actin.

Actins ; genetics ; metabolism ; Animals ; Humans ; Hypoxia-Ischemia, Brain ; metabolism ; Infant ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; rhoA GTP-Binding Protein ; genetics ; metabolism

OBJECTIVETo investigate the effect of exogenous nitric oxide (NO) on the migration of HaCaT cell and its possible mechanism.

METHODSSodium nitroprusside (SNP) was used as the donor of NO. Different concentrations of SNP (0.1, 1.0, 10.0, 100.0, 1000.0 micromol/L) were added into nutrient culture medium of HaCaT cells. Cell migration rate was observed and calculated at post scratching hour (PSH) 0 (immediately after scratching), 6, 12, 24, 48. The most suitable concentration of SNP and culture duration were selected as stimulation condition. Cytoskeletons of HaCaT cells were observed under confocal laser scanning microscope. The expressions of integrin beta 1, RhoA, Rac1 and Cdc42 of cells in experiment group (cultured with 10.0 micromol/L SNP for 24 hours) and negative control group were determined at mRNA and protein levels with RT-PCR and Western blot respectively. Data were processed with one-way analysis of variance (ANOVA) and repeated measure ANOVA.

RESULTSMigration rate of HaCaT cells in each group increased gradually as time after scratching went on. There were significant differences between PSH 6-48 and PSH 0 in cells cultured with 10.0 micromol/L SNP (F = 31.002, P values all below 0.05). Pili were rarely observed in negative control group with slender stress fibers in cells. In comparison, the amount of pili amount increased obviously in experiment group with thickened stress fibers. Compared with those of cells in control group (RhoA protein expression = 0.64 +/- 0.04), integrin beta 1 expression decreased obviously (F = 8.25, P = 0.015), RhoA (0.92 +/- 0.04), Cdc42 and Rac1 were up-regulated at both protein (with F value respectively 7.25, 14.10, 6.50, P values all below 0.05) and mRNA levels (with F value respectively 23.67, 10.39, 9.52, P values all below 0.05).

CONCLUSIONSExogenous NO in suitable concentration can promote the proliferation and migration of HaCaT cell, suggesting it exerts significant effect in wound repair. The changed cytoskeletons and the down-regulated integrin beta 1 expression may be involved in this process.

Cell Line ; Cell Movement ; drug effects ; Cytoskeleton ; drug effects ; metabolism ; Humans ; Nitric Oxide ; pharmacology ; RNA, Messenger ; genetics ; rhoA GTP-Binding Protein ; genetics ; metabolism

OBJECTIVETo study the expression and possible function of RhoA in human gastric cancer cell lines.

METHODSThe expression of RhoA in human gastrointestinal cancer cell lines was detected by Western blot. Antisense plasmid of RhoA was constructed by pGEFL and transferred into gastric cancer cell line AGS by lipofectamine. Cell survival was examined by MTT assays, and cell cycle was detected by flow cytometry.

RESULTSThe expression of RhoA protein in 10 different kinds of human cancer cell lines was much higher than that in immortalized human intestinal epithelial cell line. After being transfected with antisense RhoA, with the decrease in RhoA protein expression, the growth rate of AGS was inhibited, and the number of cells in S phase was increased by 14%.

CONCLUSIONRhoA is overexpressed in many human cancer cell lines. Some of the malignant characteristics of a gastric cancer cell line can be partially reversed by inhibiting RhoA expression.

Antisense Elements (Genetics) ; pharmacology ; Cell Cycle ; Cell Line, Tumor ; Genetic Therapy ; Humans ; Stomach Neoplasms ; chemistry ; pathology ; therapy ; rhoA GTP-Binding Protein ; analysis ; antagonists & inhibitors ; physiology

OBJECTIVETo investigate the expression of RhoA, RhoC and their effector ROCK-1 in four ovarian cancer cell lines in vitro and their correlation with invasiveness.

METHODSExpression of RhoA, RhoC and ROCK-1 mRNA and protein in four ovarian cancer cell lines SW626, Skov-3, A2780 and Caov-3 was detected by RT-PCR and Western blot assay. Invasion assay was done in Boyden chamber.

RESULTSThe expression levels of RhoA, RhoC and ROCK-1 mRNA and protein varied in the four different cell lines examined. The expression level of RhoC, but not RhoA and ROCK-1, was significantly correlated with the invasive capability of these cells in vitro (r = 0.95, P < 0.01). Expression of RhoA at the level of transcription was not correlated with that at the translation level. The expression of RhoA and RhoC did not correlate with that of ROCK-1.

CONCLUSIONExpression level of RhoC may serve as an independent parameter in evaluating metastasis and become a new target in inhibiting ovarian cancer metastasis.

Cell Line, Tumor ; Cell Movement ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Intracellular Signaling Peptides and Proteins ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Phenotype ; Protein Biosynthesis ; Protein-Serine-Threonine Kinases ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transcription, Genetic ; rho GTP-Binding Proteins ; biosynthesis ; genetics ; rho-Associated Kinases ; rhoA GTP-Binding Protein ; biosynthesis ; genetics ; rhoC GTP-Binding Protein

BACKGROUND: Microribose nucleic acids (miRNAs) are implicated in the progression of lung adenocarcinoma. MicroRNA-345-5p (miR-345-5p) is a recently identified anti-oncogene in some human cancers, but its functional role and possible molecular mechanism in lung adenocarcinoma remain unknown. This study aimed to identify the biological function and underlying mechanism of miR-345-5p in lung adenocarcinoma cells. METHODS: In this study, lung adenocarcinoma tissues and adjacent tissues were collected in the First Affiliated Hospital of Anhui Medical University between April 2016 and February 2017. The expression of miR-345-5p and ras homolog family member A (RhoA) in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines (A549, H1650, PC-9, and H441) was detected by reverse transcription quantitative polymerase chain reaction analysis. Functional assays including colony formation, flow cytometry analysis, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of lung adenocarcinoma cells. In addition, RNA pulldown and luciferase reporter assays were conducted to evaluate the relationship between miR-345-5p and RhoA. Difference between the two groups was analyzed with Student's t test, while that among multiple groups was analyzed with one-way analysis of variance. RESULTS: MiR-345-5p expression displayed lower level in lung adenocarcinoma tissues (0.241 ± 0.095 vs.1.000 ± 0.233, t = 19.247, P < 0.001) and cell lines (F = 56.992, P < 0.001) than control tissues and cells. Functional experiments demonstrated that upregulation of miR-345-5p inhibited the malignant phenotypes of lung adenocarcinoma cells via suppressing cell proliferation, migration, invasion, and facilitating cell apoptosis. Additionally, RhoA was verified to be the downstream target of miR-345-5p. Expression of RhoA was downregulated by overexpression of miR-345-5p in PC-9 (0.321 ± 0.047 vs. 1.000 ± 0.127, t = 8.536, P < 0.001) and H1650 (0.398 ± 0.054 vs. 1.000 ± 0.156, t = 4.429, P = 0.011) cells. Rescue assays revealed that overexpression of RhoA rescued the suppressive effects of miR-345-5p upregulation on proliferation, migration, and invasion of lung adenocarcinoma cells. Further, miR-345-5p was found to regulate the Rho/Rho-associated protein kinase (ROCK) signaling pathway by downregulation of RhoA in lung adenocarcinoma cells. CONCLUSIONS MiR-345-5p plays a tumor suppressor role in lung adenocarcinoma cells by downregulating RhoA to inactivate the Rho/ROCK pathway.
Adenocarcinoma of Lung/genetics* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/genetics* ; MicroRNAs/genetics* ; Up-Regulation/genetics* ; rho-Associated Kinases/genetics* ; rhoA GTP-Binding Protein/genetics*

RhoA is one of the main members of RhoGTPase family involved in cell morphology, smooth muscle contraction, cytoskeletal microfilaments and stress fiber formation. It has been demonstrated that RhoA modulates endothelial cell permeability by its effect on F-actin rearrangement, but the molecular mechanism of rearrangement of actin cytoskeleton remains unclear. Recent studies prove that RhoA/Rho kinase regulates smooth muscle specific actin dynamics by activating serum response factor (SRF)-dependent transcription. To further investigate the molecular mechanism of the rearrangement of vascular endothelial cell actin cytoskeleton, we explored the relationship between the activation of SRF and F-actin rearrangement induced by RhoA in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with the constitutively active forms of RhoA (Q63LRhoA) or the dominant negative forms of RhoA(T19NRhoA) using retrovirus vector pLNCX-Q63LRhoA or pLNCX-T19NRhoA, the positive clone was obtained by G418 selection. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-free medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. Infection of PLNCX-Q63LRhoA induced F-actin rearrangement and stress fiber formation in HUVECs, as well as enhanced the expression of SRF in the nuclei. In contrast, the cells infected with T19NRhoA showed no distinct changes. With serum deprivation, the expression of SRF increased obviously in both normal and infected HUVECs, but the subcellular localization of SRF was evidently different. In HUVECs, the localization of SRF was in the nuclei after 3 d with serum deprivation, but it was redistributed outside the nuclei after 5 d with serum deprivation. In cells infected with Q63LRhoA, the immunolocalization of SRF was always in the nuclei compared with HUVECs infected with T19NRhoA, which was almost always localized in the cytoplasm. In HUVECs, the rearrangement of F-actin and formation of stress fiber increased after 3 d with serum deprivation, but appeared decreased and unpolymerized after 5 d with serum deprivation. The polymerization of F-actin and the formation of stress fiber in HUVECs infected with Q63LRhoA kept during the period of serum-free culture, whereas the rearrangement of F-actin in cells infected with T19NRhoA was not found. These results suggest that RhoA influences endothelial F-actin rearrangement in part by regulating the expression and subcellular localization of SRF.
Actins ; biosynthesis ; genetics ; Cytoskeleton ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; Protein-Serine-Threonine Kinases ; metabolism ; Serum Response Factor ; biosynthesis ; genetics ; Umbilical Veins ; cytology ; rho-Associated Kinases ; rhoA GTP-Binding Protein ; physiology

Previously, we demonstrated that the p190 Rho guanine nucleotide exchange factor (p190RhoGEF) was induced following CD40 stimulation of B cells. In this study, we examined whether p190RhoGEF and a downstream effector molecule RhoA are required for B cell differentiation. Expression of p190RhoGEF positively correlated with the expression of surface markers and transcriptional regulators that are characteristic of mature B cells with plasma cell (PC) phenotypes. Moreover, either the overexpression of p190RhoGEF or the expression of a constitutively active RhoA drove cellular differentiation toward PC phenotypes. B cell maturation was abrogated in cells that overexpressed p190RhoGEF and a dominant-negative form of RhoA simultaneously. CD40-mediated maturation events were also abrogated in cells that overexpressed either dominant-negative p190RhoGEF or RhoA. Together, these data provide evidence that p190RhoGEF signaling through RhoA in CD40-activated B cells drives the induction of the PC differentiation.
Animals ; B-Lymphocytes/*cytology/*metabolism ; Cell Differentiation/genetics/*physiology ; Cell Line ; Cells, Cultured ; Female ; Guanine Nucleotide Exchange Factors/genetics/*metabolism ; Humans ; Lymphocyte Activation/genetics/*physiology ; Mice ; Mice, Inbred BALB C ; Plasma Cells/*cytology/*metabolism ; rhoA GTP-Binding Protein/genetics/metabolism

OBJECTIVERecent studies have indicated that the signal pathway of NgR-P75NTR- RhoA plays a key role in nerve injury and remodeling, but its exact mechanism and the role of the downstream molecule RhoA regulated by P75NTR remain unclear in hypoxia-ischemia (HI) neonatal animals. The present study was designed to assess the expression of P75NTR protein and RhoA mRNA in neonatal white matter and to investigate their relationship in newborn rats with white matter damage (WMD).

METHODSThe rat WMD model was established by the ligation of right common carotid artery, followed by 6% hypoxia exposure for 4 hrs. The control group was sham-operated, without HI treatment. The histological changes of brain tissue were observed under light and electron microscopes. Expression of P75NTR protein and RhoA mRNA in the brain white matter after 12, 24, 48 and 72 hrs and 7 days of HI were detected by RT-PCR and immunohistochemistry, respectively.

RESULTSPeriventricular white matter damage was observed by 48 hrs of HI. Expression of P75NTR protein increased in the striatum and callosum zones at 12 hrs, peaked at 48 hrs, and remained at a higher level than control until 72 hrs of HI in the WMD group (P < 0.01). After 7 days of HI expression of P75NTR protein was no longer statistically different from controls. The RhoA mRNA was higher in the WMD group for the first 72 hrs and then declined to control values.

CONCLUSIONSIncreased P75NTR protein might mediate apoptosis of nerve cells and inhibit the regeneration of neuron axons. The subsequent decline back to control value may be correlated with the aggregation of necrosis of nerve cells after HI. The patterns of RhoA mRNA expression were consistent with those of P75NTR protein, suggesting that the increased P75NTR level may promote RhoA mRNA expression.

Animals ; Animals, Newborn ; Brain ; pathology ; ultrastructure ; Female ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; Immunohistochemistry ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Nerve Growth Factor ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; rhoA GTP-Binding Protein ; genetics

OBJECTIVETo investigate the role of small G-protein RhoA in neointimal formation following rat carotid artery balloon injury and related mechanisms.

METHODSMale 3-4-month-old Sprague-Dawley rats were used in the present study (10 rats per group). Group A: control; Group B: carotid artery balloon injury; Group C: injury + Ad-CMV-eGFP + Pluronic F-127; Group D: injury + Ad-CMV-N19RhoA-eGFP + Pluronic F-127; Group E: non injury + Ad-CMV-eGFP + Pluronic F-127. Perivascular gene transfer of an adenovirus co-expressing N19RhoA was performed to rat carotid artery following balloon injury and the effect on neointimal formation and the expressions of PCNA and α-SM-actin examined. Rats were killed after 14 days.

RESULTSThe protein expression of RhoA in group B was significantly higher than in group A (P = 0.001), and the positive cells rate of PCNA and α-SM-actin which were assessed by immunohistochemistry in group C (45.2% and 75.6%) was significantly higher than in group D (28.4% and 51.9%, all P < 0.01). The area of neointima was significantly smaller [(0.14 ± 0.08) mm(2) vs. (0.23 ± 0.10) mm(2), P < 0.01], the luminal area was significantly larger [(0.47 ± 0.11) mm(2) vs. (0.31 ± 0.06) mm(2), P < 0.01] in group D than in group C.

CONCLUSIONGene transfer of N19RhoA attenuates neointimal formation after balloon injury in rat carotid arteries possibly related to the modulating capacities of small G-protein RhoA on the proliferation, phenotypic differentiation and migration of vascular adventitial fibroblasts.

Adenoviridae ; genetics ; Animals ; Carotid Arteries ; metabolism ; Carotid Artery Injuries ; metabolism ; pathology ; Genetic Vectors ; Male ; Muscle, Smooth, Vascular ; metabolism ; Neointima ; Rats ; Rats, Sprague-Dawley ; Transfection ; rhoA GTP-Binding Protein ; genetics

To explore the angiotensin peptide [Ang (1-7)]-mediated inhibition of Ang II in human hepatic stellate cells (HSCs) and determine the involvement of the ACE2-Ang (1-7)-Mas axis. The human HSC line, LX2, was used in all experiments, and divided into control (unstimulated) and Ang II-stimulated (10-6 mol/L) groups. The Ang II-stimulated cells were further divided among several pre-treatment (prior to Ang II) groups: ROCK-inhibited (Y27632 blocking agent, 10-6 mol/L); irbesartan-inhibited (AT-1 receptor antagonist, 10-6 mol/L); and Mas receptor-inhibited (A779 Mas receptor antagonist, 10-6 mol/L). To explore the potential inhibitory effects of various Ang family members, the Ang II-stimulated and pre-treated LX2 cells were exposed to Ang (1-7) (10-6 mol/L) for 24 h. Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and QuantiGene assay were used to assess changes in protein and mRNA expression levels of RhoA, ROCK, and connective tissue growth factor (CTGF). Compared with the control group, Ang II-stimulated cells showed significantly increased levels of RhoA protein (0.337+/-0.074 vs. 0.870+/-0.093), ROCK2 mRNA (0.747+/-0.061 vs. 0.368+/-0.023), and CTGF mRNA (0.262+/-0.007 vs. 0.578+/-0.028) (all, P less than 0.01). Pre-treatment with irbesartan or Y27632 eliminated these responses. Ang (1-7) inhibited the Ang II-stimulated up-regulation of RhoA, ROCK, and CTGF. Ang (1-7) can inhibit the Ang II-stimulated up-regulation of RhoA, ROCK and CTGF in hepatic stellate cells, indicating that the ACE2-Ang (1-7)-Mas axis, an important branch of the renin-Ang-aldosterone system is involved in the occurrence and development of liver fibrosis.
Angiotensin I ; pharmacology ; Angiotensin II ; pharmacology ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Humans ; Peptide Fragments ; pharmacology ; RNA, Messenger ; genetics ; Signal Transduction ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism