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*

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, SPD), alpha-difluoromethylornithine (2.5 mmol·L, DFMO), 4-Aminopyridine (40 μmol·L, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca ([Ca]) 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 [Ca] 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 [Ca], 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.
Animals ; Astragalus propinquus ; chemistry ; Atractylodes ; chemistry ; Cell Line ; Cell Movement ; drug effects ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Intestines ; cytology ; drug effects ; Kv1.1 Potassium Channel ; genetics ; metabolism ; Polyamines ; metabolism ; Polysaccharides ; chemistry ; isolation & purification ; pharmacology ; Rats ; Rhizome ; chemistry ; Signal Transduction ; drug effects ; rhoA GTP-Binding Protein ; metabolism

OBJECTIVETo study the possible effect of antepartum taurine supplementation in regulating the activity of Rho family factors and promoting the proliferation of neural stem cells in neonatal rats with fetal growth restriction (FGR), and to provide a basis for antepartum taurine supplementation to promote brain development in children with FGR.

METHODSA total of 24 pregnant Sprague-Dawley rats were randomly divided into three groups: control, FGR, and taurine (n=8 each ). A rat model of FGR was established by food restriction throughout pregnancy. RT-PCR, immunohistochemistry, and Western blot were used to measure the expression of the specific intracellular markers for neural stem cells fatty acid binding protein 7 (FABP7), Rho-associated coiled-coil containing protein kinase 2 (ROCK2), ras homolog gene family, member A (RhoA), and Ras-related C3 botulinum toxin substrate (Rac).

RESULTSThe FGR group had significantly lower OD value of FABP7-positive cells and mRNA and protein expression of FABP7 than the control group, and the taurine group had significantly higher OD value of FABP7-positive cells and mRNA and protein expression of FABP7 than the FGR group (P<0.05). The FGR group had significantly higher mRNA expression of RhoA and ROCK2 than the control group. The taurine group had significantly higher mRNA expression of RhoA and ROCK2 than the control group and significantly lower expression than the FGR group (P<0.05). The FGR group had significantly lower mRNA expression of Rac than the control group. The taurine group had significantly higher mRNA expression of Rac than the FGR and control groups (P<0.05). The FGR group had significantly higher protein expression of RhoA and ROCK2 than the control group. The taurine group had significantly lower protein expression of RhoA and ROCK2 than the FGR group (P<0.05).

CONCLUSIONSAntepartum taurine supplementation can promote the proliferation of neural stem cells in rats with FGR, and its mechanism may be related to the regulation of the activity of Rho family factors.

Animals ; Animals, Newborn ; Body Weight ; drug effects ; Brain ; drug effects ; Cell Proliferation ; drug effects ; Fatty Acid-Binding Protein 7 ; analysis ; Female ; Fetal Growth Retardation ; drug therapy ; Male ; Neural Stem Cells ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Taurine ; pharmacology ; rho-Associated Kinases ; analysis ; genetics ; rhoA GTP-Binding Protein ; analysis ; genetics

Skeletal fluorosis is a chronically metabolic bone disease with extensive hyperostosis osteosclerosis caused by long time exposure to fluoride. Skeletal fluorosis brings about a series of abnormal changes of the extremity, such as joint pain, joint stiffness, bone deformity, etc. Differentiation and maturation of osteoblasts were regulated by osteoclasts via Sema4D/Plexin-B1 signaling pathway. Furthermore, the differentiation and maturation of osteoclasts are conducted by osteoblasts via RANKL/RANK/OPG pathway. Both of these processes form a feedback circuit which is a key link in skeletal fluorosis. In this study, an osteoblast-osteoclast co-culture model in vitro was developed to illustrate the mechanism of skeletal fluorosis. With the increase of fluoride concentration, the expression level of Sema4D was decreased and TGF-β1 was increased continuously. OPG/RANKL mRNA level, however, increased gradually. On the basis of that, the inhibition of Sema4D/Plexin-B1/RhoA/ROCK signaling pathway caused by fluoride promoted the level of TGF-β1 and activated the proliferation of osteoblasts. In addition, osteroprotegerin (OPG) secreted by osteoblasts was up-regulated by fluoride. The competitive combination of OPG and RANKL was strengthened and the combination of RANKL and RANK was hindered. And then the differentiation and maturation of osteoclasts were inhibited, and bone absorption was weakened, leading to skeletal fluorosis.
Animals ; Antigens, CD ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Feedback, Physiological ; Fetus ; Fluorides ; pharmacology ; GTPase-Activating Proteins ; genetics ; metabolism ; Gene Expression Regulation, Developmental ; Osteoblasts ; drug effects ; metabolism ; pathology ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteogenesis ; drug effects ; genetics ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism ; Receptors, Cell Surface ; genetics ; metabolism ; Semaphorins ; genetics ; metabolism ; Signal Transduction ; Transforming Growth Factor beta1 ; genetics ; metabolism ; rho-Associated Kinases ; genetics ; metabolism ; rhoA GTP-Binding Protein ; genetics ; metabolism

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid involved in numerous physiological responses. However, the expression of LPA receptors and the role of the Hippo signaling pathway in epithelial cells have remained elusive. In this experiment, we studied the functional expression of LPA receptors and the associated signaling pathway using reverse transcriptase-PCR, microspectrofluorimetry, western blotting and immunocytochemistry in salivary gland epithelial cells. We found that LPA receptors are functionally expressed and involved in activating the Hippo pathway mediated by YAP/TAZ through Lats/Mob1 and RhoA/ROCK. Upregulation of YAP/TAZ-dependent target genes, including CTGF, ANKRD1 and CYR61, has also been observed in LPA-treated cells. In addition, based on data suggesting that tumor necrosis factor (TNF)-alpha induces cell apoptosis, LPA upregulates TNF-induced caspase-3 and cleaved Poly(ADP-ribose)polymerase (PARP). However, small interfering RNA treatment to Yes-associated protein (YAP) or transcriptional co-activator with a PDZ-binding motif (TAZ) significantly decreased TNF-alpha- and LPA-induced apoptosis, suggesting that YAP and TAZ modulate the apoptotic pathway in salivary epithelial cells.
Adaptor Proteins, Signal Transducing/genetics/metabolism ; *Apoptosis ; Cell Line ; Epithelial Cells/*cytology/metabolism ; Gene Expression Regulation ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Lysophospholipids/*metabolism ; Phosphoproteins/genetics/metabolism ; Protein-Serine-Threonine Kinases/*metabolism ; RNA Interference ; RNA, Small Interfering/genetics ; Receptors, Lysophosphatidic Acid/genetics/*metabolism ; Salivary Glands/*cytology/metabolism ; *Signal Transduction ; Tumor Necrosis Factor-alpha/metabolism ; rho-Associated Kinases/metabolism ; rhoA GTP-Binding Protein/metabolism

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

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

OBJECTIVETo explore the effects of K-ras gene mutation on colon cancer cell line Caco-2 metastasis by regulating E-cadherin/beta-catenin/p120 protein complex formation and RhoA protein activity.

METHODSK-ras wild-type colon cancer cell line Caco-2 was transiently transfected by phr-GFP vector (control group), transfected by mutant K-ras gene phr-K-ras (Val12) vector (transfection group), transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific MAPK pathway inhibitor PD98059 (MAPK inhibition group), or transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific PI-3K pathway inhibitor LY294002 (PI-3K inhibition group), respectively. Cell migration was tested by Transwell experiment. E-cadherin and beta-catenin protein expression and intracellular location were detected by cell immunofluorescence method. Intracellular p120 protein expression was detected by Western blot. beta-catenin protein level which combined with E-cadherin was detected by immunoprecipitation. RhoA activity was analyzed by Pull-down assay.

RESULTSThe Caco-2 cell migration rate was (19.8 +/- 5.6) % in transfection group, which was significantly higher than that in control group [(14.0 +/- 4.2) %] (P = 0.001) and in MAPK inhibition group [(15.8 +/- 1.2) %] (P = 0.044), but was not significantly different from that in PI-3K inhibition group [(17.5 +/- 2.8) %] (P = 0.095). Immunofluorescence method showed that the E-cadherin and beta-catenin stain located in the cell membrane decreased in transfection group. Western blot showed that the total intracellular p120 protein decreased in transfection group and PI-3K inhibition group. Immunoprecipitation data showed that beta-catenin protein level combined with E-cadherin decreased in transfection group and PI-3K group. Pull-down test showed that RhoA protein activity was up-regulated in transfection group.

CONCLUSIONK-ras gene mutation stimulates the migration of colon cancer cell Caco-2, which may be achieved by decreasing the E-cadherin/beta-catenin/p120 protein complex formation via MAPK pathway and increasing the RhoA protein activity.

Caco-2 Cells ; Cadherins ; metabolism ; Catenins ; metabolism ; Cell Movement ; Colonic Neoplasms ; metabolism ; pathology ; Genes, ras ; genetics ; Humans ; Multiprotein Complexes ; metabolism ; Mutation ; Neoplasm Metastasis ; Transfection ; beta Catenin ; metabolism ; rhoA GTP-Binding Protein ; 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

BACKGROUNDAlthough the migration of hepatic stellate cells (HSCs) is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases (especially RhoA) in platelet-derived growth factor (PDGF)-BB-induced migration of HSCs.

METHODSThe migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases (RhoA, Rac1 and Cdc42) within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active (CA, Q63L) or dominant negative (DN, T19N) RhoA mutants. Data were analyzed using SPSS 16.0 software. Student's t test was used to analyze differences between two groups and one-way analysis of variance (ANOVA) was used among multiple groups.

RESULTSRapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic (direct) and chemotactic (indirect) stimulation by PDGF-BB. PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Rac1 and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls.

CONCLUSIONSPDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the determinants in PDGF-BB-induced HSC migration.

Animals ; Blotting, Western ; Cell Line ; Cell Movement ; drug effects ; genetics ; Cells, Cultured ; Fluorescent Antibody Technique ; Glutathione Transferase ; genetics ; metabolism ; Hepatic Stellate Cells ; drug effects ; metabolism ; Male ; Platelet-Derived Growth Factor ; pharmacology ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Sprague-Dawley ; rhoA GTP-Binding Protein ; genetics ; metabolism