ObjectiveTo investigate the effect of different mechanical tensions on the expressions of RhoA/Rho associated protein kinases (ROCK) in rat tendon stem cells (TSCs). MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged, 2-3 months; weighing, 200-250 g) by enzymatic digestion method and cultured for 2-3 passages, then seeded on micro groovdishes. The 4% (4% stretch group) and 8% (8% stretch group) mechanical stretching was performed for 4 hours every day at 1 Hz. After 1, 2, and 3 days, the protein and mRNA expressions of RhoA and ROCK were measured by Western blot and real-time quantitative PCR. The cell proliferation was measured by cell counting kit 8. The cells were not stretched as control group. ResultsThe TSCs at passage 2 showed a cobble-stone shape and aggregation growth; TSCs seeded on micro groovdishes showed random growth, and the cells grew along the stretching direction after mechanical stretching. The mRNA expressions of RhoA and ROCK in control group, 4%, and 8% stretch groups showed an increasing tendency at 1, 2, and 3 days, showing significant difference between groups (P<0.05). The protein expressions of RhoA and ROCK in 4% and 8% stretch groups were similar to those in control group at 1 day (P>0.05), but the expressions in 4% and 8% stretch groups showed an increasing tendency at 2 and 3 days, which were significantly higher than those in control group (P<0.05). The cell proliferation of 8% stretch group was significantly lower than that of 4% stretch group and control group at each time point (P<0.05), but no significant difference was found between 4% stretch group and control group (P>0.05). ConclusionThe expressions of RhoA and ROCK of rat TSCs are positively correlated with stretch intensity. So RhoA/ROCK may be an important molecule in TSCs after mechanical stretching.
Objective To detect the expressions of RhoA and Snail in gastric cancer tissues， and explore the relati-onship of these expressions to the biological behavior of the gastric cancer. Methods The expressions of RhoA and Snail protein in the paraffin-embedded specimens of 189 gastric cancer patients were detected by immunohistochemical method. The relationships of their expressions to clinicopathologic features of gastric cancer or survival， and the relevance of RhoA expression and Snail expression were analyzed. Results ① The expressions of RhoA and Snail protein in the gastric cancer tissues were significantly higher than those in the paraneoplastic tissue （RhoA：P=0.008；Snail：P=0.000） and the normal gastric mucosa tissue （RhoA：P=0.010；Snail：P=0.000）；The expression of RhoA had no significant difference between the paraneoplastic tissue and the normal gastric mucosa tissue （P=0.782）， however， the expression of Snail in the paraneoplastic tissue was significantly higher than that in the normal gastric mucosa tissue （P=0.001）. ② The expression of RhoA in the gastric cancer tissue was associated with TNM staging and Lauren type （P＜0.05）， but it was not associated with tumor diameter， lymph node metastasis， or differentiation degree （P＞0.05）. The expression of Snail in the gastric cancer tissue was associated with tumor diameter， lymph node metastasis， differentiation degree， TNM staging， or Lauren type （P＜0.05）. The expressions of RhoA and Snail in the gastric cancer tissue were not associated with patients’ gender and age （P＞0.05）. ③ The expression of RhoA protein was significantly positi-vely correlated with Snail protein in the gastric cancer（rs=0.203， P=0.005）. ④ The TNM staging of tumor， RhoA and Snail expressions， and lymph node metastasis were all the independent prognostic factors of postoperative gastric cancer patients （P＜0.05）. Conclusions RhoA and Snail proteins express in gastric cancer tissues， and involves in gastric carcinogenesis and the development process， and RhoA/Snail signaling pathway may play an important role in invasion and metastasis of gastric cancer.
ObjectiveTo explore the mechanism by which the tumor suppressor gene Testin affects the proliferation, migration, and invasive biological activity of lung adenocarcinoma cell lines by regulating the RhoA pathway. MethodThe cbioportal tumor gene expression was used to screen for genes with high correlation with TES gene expression in lung adenocarcinoma, and the 200 genes with the highest correlation were selected for pathway enrichment analysis. Upload these 200 genes to the David gene annotation tool for GO_Biological Process pathway analysis, GO Molecular Function pathway analysis, KEGG pathway analysis, and Reactome pathway analysis. The lung adenocarcinoma cell line H1299 was cultured, and an overexpression Testin plasmid was constructed and transfected into H1299 cells. The mRNA and protein expression of RhoA, Rac1, and Cdc42 were detected using qRT PCR and western blot. On the basis of downregulating RhoA expression through overexpression of Testin, the overexpression plasmid of RhoA (TES+RhoA) was transfected simultaneously to induce a downregulation of RhoA expression, and the changes in malignant phenotype of lung adenocarcinoma cells were detected. The biological activity changes of adenocarcinoma cell lines after the above intervention were verified through CCK-8 experiment, Transwell experiment, and Matrigel experiment. Results The results of pathway analysis prediction showed that Testin may be involved in regulating the Rho GTPase signaling pathway. Overexpression of Testin did not affect the mRNA levels of RhoA, Rac1, and Cdc42 (all P>0.05), nor did it affect the protein expression levels of Rac1 and Cdc42 (all P>0.05), but it significantly reduced the protein level of RhoA (P<0.05). Knocking down RhoA in lung adenocarcinoma cell H1299 can significantly inhibit cell proliferation, migration, and invasion ability (all P<0.05). Simultaneously transfecting RhoA overexpression plasmid on the basis of overexpression of Testin can downregulate RhoA expression, but does not affect Testin expression. ConclusionsRhoA plays a pro-cancer role in lung adenocarcinoma, and Testin can inhibit RhoA expression. Overexpression of RhoA can rescue Testin's effect on lung adenocarcinoma cell proliferation, migration, and invasion. Testin exerts its anti-cancer biological activity by regulating RhoA.
ObjectiveTo investigate the effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts.MethodsMurine RAW264.7 macrophages were induced into osteoclasts by dexamethasone and 1α, 25-dihydroxyvitamin D3, 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 β1 (TGF-β1)］were analyzed and compared.ResultsRAW264.7 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-β1 mRNAs were significantly increased (P<0.05), and those protein expressions were enhanced.ConclusionFTY720-P can down-regulate the expression of RhoA and promote the expressions of TGF- β1 and BMP-2 by affecting the transduction of EphA2-EphrinA2 bidirectional signaling in osteoclasts.