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Purpose To investigate the corr-elation between Rap1 GAP expression in colon cancer tissues and clinicopatho-logical features and prognosis.Methods Immunohistochemistry was used to detect Rap1 GAP protein expression in 125 cases of colon cancer,and its correlation with clinicopathological features and prognosis was analyzed.Rap1 GAP protein expression in co-lon cancer LOVO,HCT116,SW480 cells and normal colon epi-thelial HCoEPiC cells was detected by Western blot.The expres-sion of Rap1 GAP was down-regulated and up-regulated in LO-VO,HCT116 and SW480 cells by lentivirus transfection,and di-vided into no-load group(sh-NON,LV-NON),sh-Rap1 GAP group(low expression Rap1 GAP)and LV-Rap1 GAP group(overexpression Rap1 GAP)according to different treatments.The transfection efficiency was verified by Western blotting.MTT assay and Transwell assay were used to detect cell proliferation,invasion and migration in each group.Results In 125 colon cancer samples,83 cases(66.4％)had the loss of Rap1 GAP expression,which was higher than that in paracancer control(7.2％,P＜0.001).The rate of loss of Rap1 GAP expression was correlated with the degree of tumor differentiation(x2=6.152,P=0.011)and the presence of mucinous adenocarcino-ma(x2=4.908,P=0.028),but not with gender,age,tumor location,tumor stage,or lymph node metastasis(P＞0.05).Western blotting results showed that compared with HCoEPiC(0.189±0.081)cells,Rap1 GAP protein expression was in-creased in colon cancer LOVO(0.238±0.008)cells.Rap1 GAP protein expression was decreased in HCT116(0.064± 0.002)and SW480(0.152±0.026)cells(F=159.6,P＜0.05).After LOVO cells were transfected with Rap1 GAP low expression lentivirus,the expression level of Rap1 GAP in sh-Rap1 GAP-1 group(0.733±0.071)and sh-Rap1 GAP-2 group(0.559±0.136)and sh-Rap1 GAP-3 group(0.606±0.037)was significantly lower than that in LOVO group(1.880± 0.129)(F=49.57,P＜0.05).Compared with sh-NON(1.260±0.109)group,the proliferation ability of sh-Rap1 GAP-2(1.569±0.059)and sh-Rap1 GAP-3(1.548±0.087)cells was significantly increased at 72 h(F=28.36,P＜0.05).Its invasion and migration ability were significantly increased(P＜0.05).After HCT116 cells transfected with overexpression lentivirus,the expression of Rap1 GAP protein in LV-Rap1 GAP group(1.395±0.137)was relatively higher than that in LV-NON group(0.485±0.097)(P＜0.05).The results of MTT assay showed that compared with LV-NON(0.652±0.047)group,the proliferation ability of cells in LV-Rap1 GAP(1.212 ±0.038)group was decreased,and the invasion and migration ability were significantly decreased(P＜0.05).The transfection results,proliferation,invasion and migration of SW480 cells were consistent with those of HCT116 cells.Conclusion The loss rate of Rap1 GAP expression is related to the differentiation degree of colon cancer and whether it is accompanied by mucin-ous adenocarcinoma.The up-regulation of Rap1 GAP expression can inhibit the proliferation,invasion and migration of colon cancer cells,providing a theoretical basis for exploring the occur-rence and development of colon cancer.
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Objective To study the cervical HPV infection in relations with Rap1GAP gene and protein level change. Methods A total of 70 cases of cervical cancer patients in our hospital were selected as the observation group ,chose 70 cases of normal cervical tissues as the control group. Respectively for 2 set of objects of cervical tissue DNA extrac-tion, total RNA extraction, amplification cDNA synthesis, PCR reaction. And detection of HPV infection, Rap1GAP gene exon E11 and E19 loss rate, Rap1GAP Rap1GAP mRNA expression and protein expression. Results The observa-tion group of patients HPV infection was significantly higher than control group (P <0.05). HPV positive and negative E11 and the E19 lack of rate comrpared in the observation group was no statistically significant difference (P > 0.05). In the observaition group, the HPV positive group organization, Rap1GAPmRNA and Rap1GAP protein expression rate and positive expression rate was significantly lower than the HPV negative group (P < 0.05). Cervical HPV infection and Rap1GAP mRNA expression were positively correlated (r = 0.578), and Rap1GAP protein positive expression were positively correlated (r = 0.554). Conclusion Cervical HPV infection and Rap1GAP mRNA expression were positively correlated, and Rap1GAP protein positive expression were positively correlated.
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The expression of Rap1 GAP protein was detected in 69 cases of papillary thyroid carcinoma and adjacent normal thyroid tissues with immunohistochemistry method.Methylation of Rapl GAP gene was analyzed by methylation-specific-PCR (MSP) in these tissues.The immunohistochemistry results indicated that the Rap1GAP protein was down-regulated in tumor tissues of 54 ( 78％ ) cases compared with normal thyroid tissues.Statistical analysis demonstrated that the decreased level of Rap1 GAP protein expression was significantly correlated with the tumor stage T according to American Joint Committee on Cancer ( AJCC,P =0.043 ).The MSP results demonstrated that 46 cases of Rap1GAP gene methylation were detected in 54 cases with down-regulated expression of Rap1GAP (66.7％),but only 1 case of methylation was found in 15 cases without obvious change of Rap1GAP expression (6.67％),showing significant difference ( P＜0.01 ).There was no methylation in normal thyroid tissues.These results suggest that raised methylation of promoter region may contribute to the low expression of Rap1GAP protein in papillary thyroid carcinoma.
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Rap 1 is expressed in human umbilical vein endothelial cells (HUVECs).Rap1-GTPase activating protein (Rap 1 GAP),with its specific target,Rap 1,has been shown to be important in the regulation of many physiological and certain pathological processes.In this study,we investigated the effect of RaplGAP expression on endothelial cell function,or,more specifically,proliferation and migration of endothelial cells.HUVECs were transfected with pcDNA3.1 (empty vector),pcDNA3.1 containing Flag-tagged-Rap1GAP or Myc-tagged-Rap1N17.The proliferation,migration and tube formation were examined and compared among the 3 groups.Expression of Rap1,Rap1GAP,extracellular signal-regulated kinase (ERK),phospho-ERK,Akt,phosphor-Akt was detected by Western blotting.The results showed that the proliferation,migration and tube formation were significantly reduced in Rap1GAP- and Rap1N17-transfected HUVECs as compared with empty vector-transfected control.These changes were coincident with increased expression of Rap 1 GAP and decreased expression of activated Rap 1,phospho-ERK and -Akt.After treatment of Rap 1 GAP-transfected HUVECs with a stimulator of Rapl guanine-nucleotide-exchange factor (Rap1GEF) 8CPT-2'OMe-cAMP,it was found that Rap1 activity was decreased as compared with empty vector-transfected control.Pretreatment of HUVECs with an ERK inhibitor PD98059 or a PI3K inhibitor LY294002 prior to stimulation not only blocked 8CPT-2'OMe-cAMP-induced phosphorylation of ERK and Akt,but also significantly reduced cell proliferation and migration.Finally,we examined the effect of vascular endothelial growth factor (VEGF) on HUVECs overexpressing Rap1GAP.VEGF-stimulated Rap1 activity,phosphorylation of ERK and Akt,cyclin D1 expression and cell proliferation were repressed in HUVECs overexpressing Rap 1 GAP as compared to empty vector-transfected control.Taken together,our findings demonstrate that Rap1GAP/Rap1 and their downstream effectors regulate proliferation and migration of HUVECs via ERK and Akt pathways.
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To find the underlying causes of primary myelodysplastic syndrome (MDS), the gene expression profiling of both CD34+ cells and bone marrow mononuclear cells from MDS patients was performed using oligonucleotide microarray and cDNA microarrays, respectively. Several candidate genes which were differentially expressed in MDS patients versus normal controls were selected and confirmed in expanding samples by quantitative real-time reverse transcription-polymerase chain reaction after clustering and bioinformatics analysis. one of these genes, RAP1GAP, was found to be expressed at a significantly higher level in patients with MDS in comparison with those suffering from other hematopoietic diseases including leukemia (P < 0.01). We propose that over-expression of RAP1GAP gene may play a role in the pathogenesis of MDS.