YULINK regulates vascular formation in zebrafish and HUVECs

BACKGROUND: The distinct arterial and venous cell fates are dictated by a combination of various genetic factors which form diverse types of blood vessels such as arteries, veins, and capillaries. We report here that YULINK protein is involved in vasculogenesis, especially venous formation. METHODS: In this manuscript, we employed gene knockdown, yeast two-hybrid, FLIM-FRET, immunoprecipitation, and various imaging technologies to investigate the role of YULINK gene in zebrafish and human umbilical vein endothelial cells (HUVECs). RESULTS: Knockdown of YULINK during the arterial-venous developmental stage of zebrafish embryos led to the defective venous formation and abnormal vascular plexus formation. Knockdown of YULINK in HUVECs impaired their ability to undergo cell migration and differentiation into a capillary-like tube formation. In addition, the phosphorylated EPHB4 was decreased in YULINK knockdown HUVECs. Yeast two-hybrid, FLIM-FRET, immunoprecipitation, as well as imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B or TICAM2) and markers (Clathrin and RHOB). VEGF-induced VEGFR2 internalization was also compromised in YULINK knockdown HUVECs, demonstrating to the involvement of YULINK. CONCLUSION: This study suggests that YULINK regulates vasculogenesis, possibly through endocytosis in zebrafish and HUVECs. Key points Knockdown of YULINK with morpholino in embryos of double transgenic zebrafish exhibited abnormal venous formation. Tube formation and phosphorylated EPHB4 were decreased in YULINK knockdown HUVECs. FLIM-FRET, immunoprecipitation, as well as other imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B and TICAM2) and endosome markers (Clathrin and RHOB). Knockdown of YULINK decreased the internalization of VEGF and VEGFR2 in HUVECs.

The Expression and Clinical Significance of RhoB and E-Cadherin in Non-Small-Cell Lung Cancer Tissues / 天津医药

Objective To investigate the expressions of RhoB and E-cadherin in non-small-cell lung cancer (NSCLC), and their clinical significances thereof. Methods Immunohistochemical staining was applied to detect expres-sions of RhoB and E-cadherin in 116 samples of NSCLC (NSCLC group) and 116 samples of normal lung tissues (control group). Correlations of expressions of RhoB and E-cadherin to clinical pathological parameters and prognosis were analyzed in two groups. Results The expression intensities of RhoB and E-cadherin were significantly lower in NSCLC group than those in control group (57.76%vs 87.07%,54.31%vs 85.34%,P＜0.01). There were significant differences in the expres-sion of RhoB between different pathological types, differentiation and lymph node metastasis in NSCLC group. There were significant differences in the expression of E-cadherin between different TNM stages, differentiation and lymph node metas-tasis in NSCLC group. The expression of RhoB was positively correlated with the expression of E-cadherin ( r=0.503,P＜0.01). The 3-year survival rates were significantly higher in patients with high expression of RhoB (83.93%) than those in pa-tients with low expression of RhoB (40.00%, Log-rank χ2=18.992,P＜0.01). The 3-year survival rates were significantly higher in patients with high expression of E-cadherin (85.11%) than those in patients with low expression of E-cadherin (44.93%, Log-rankχ2=16.680,P＜0.01). Further multivariate analysis suggested that both lower expressions of RhoB and E-cadherin and lymph node metastasis were prognostic indicators for NSCLC (P＜0.001). Conclusion The expressions of RhoB and E-cadherin showed a good correlation in NSCLC. Detecting the expression of RhoB combined with E-cadherin may give a clue on clinicopathological features and prognosis in patients with NSCLC.

Relationship of the Arl8a with TLR4-TRIF pathway in dendritic cells / 中华微生物学和免疫学杂志

Objective To explore the relationship of the ADP-ribosylation factor-like 8A (Arl8a)with TLR4-TRIF-GEFH1 -RhoB pathway in dendritic cells(DCs). Methods DCs were prepared from wildtype and TRIF-knockout (TRIFKO) mice. After LPS stimulation, the cells were collected for cDNA amplification. Real-time PCR method was used to detect Arl8a mRNA levels. DCs from wild-type mice were transfected with guanine nucleotide-exchange factors H1 ( GEFH1 ) small interference RNA ( siRNA ), Arl8a mRNA levels were examined with or without LPS stimulation. Then RhoB mRNA expression was analyzed in DCs transfected with the siRNA of GEFH1 and Arl8a gene, respectively. Results LPS induced the up-regulation of Arl8a mRNA in DCs from control mice but not in DCs from TRIFKO, indicating that LPS-mediated up-regulation of Arl8a was suppressed in TRIFKO DCs. In addition, siRNA of GEFH1 significantly suppressed the LPS-mediated up-regulation of Arl8a mRNA, RNAi of Arl8a and GEFH1 significantly decreased RhoB mRNA level in DCs after LPS stimulation ( P ＜ 0. 01 ). Conclusion The expression of Arl8a is involved in the TLR4-TRIF pathway in DCs, and Arl8a is closely associated with GEFH1 and RhoB at transcriptional level.

Effect of heat stress on expression of small G protein RhoB in rat livers and human prostate cancer cell line PC-3 cells / 第二军医大学学报

Objective: To investigate the changes of the small G protein RhoB expression in scalded rat livers and heat-stressed human prostate cancer cell line PC-3, so as to discuss the influence of heat stress on expression of RhoB in vitro and in vivo. Methods: Third degree burns of 30% total body surface area (TBSA) model was established with SD rats on the back. The expression of RhoB mRNA and protein in the liver was determined by RT-PCR and Western blot 2 h, 4 h, 8 h and 16 h (n=6) after scalding; the liver tissues of normal rats were taken as control (n=6). PC-3 cells were allowed to recover for 0.5 h, 1 h, 2 h, 4 h and 8 h in a cellular heat stress model and the expression of RhoB in mRNA and protein were determined by RT-PCR and Western blot, respectively; untreated PC-3 cells were taken as control. Results: The expression of RhoB mRNA in the livers peaked 4 h after scalding, being about 3.2 folds that of the control group (P<0.01); the expression began to decline 8 h after scalding. The expression of RhoB protein peaked 8 h after scalding, significantly higher than that of the control (P<0.01). RhoB mRNA level began to increase 2 h after heat stress treatment and peaked at 4 h, being about 2. 8 folds that of the control (P<0.01). The expression of RhoB protein reached its maximum at 8 h after heat stress treatment (P<0.01). Conclusion: Heat stress can up-regulate the expression of RhoB mRNA and protein in vivo and in vitro.