Silencing of circ_0078607 prevents development of gastric cancer and inactivates the ERK1/2/AKT pathway through the miR-188-3p/RAP1B axis.

The aim of this study is to explore the expression and mechanism of circ_0078607 on proliferation and apoptosis of gastric cancer. Real time PCR (RT-PCR) was performed to detect the expression of circ_0078607 in gastric cancer tumor tissues, plasma and cell lines. Cell viability was detected by cell counting Kit-8. Cell proliferation ability was assessed by cell cycle assay. The samples were analyzed by flow cytometry for the detection of apoptosis. Luciferase assay and RNA immunoprecipitation (RIP) were carried out to verify the relationship between circ_0078607 and miR-188-3p, miR-188-3p, and RAP1B. Western blot was employed to detect the protein level of RAP1B, ERK1/2 and AKT. In vivo, the effect of circ_0078607 on gastric cancer tumor growth was detected by lentivirus vector injection. Here, we found the increased level of circ_0078607 in gastric cancer tissues, gastric cancer patients plasma and cell lines. Knockdown of circ_0078607 could prevent proliferation and induce cell apoptosis in MKN-28 cells. Then we verified that circ_0078607 could interact with miR-188-3p by performed luciferase assay and RIP. Furthermore, we observed that RAP1B was a potential target of miR-188-3p. Next, we found that miR-188-3p inhibitor or overexpression of RAP1B could prevent the anti-tumor function of sh-circ_0078607. Silencing of circ_0078607 inhibited ERK1/2/AKT signal pathways via regulating miR-188-3p/RAP1B. In vivo, knockdown of circ_0078607 inhibited tumor growth. Knockdown of circ_0078607 inhibits the proliferation and induces apoptosis of gastric cancer via miR-188-3p/RAP1B signal pathway.

Rap2B promotes angiogenesis via PI3K/AKT/VEGF signaling pathway in human renal cell carcinoma.

Human renal cell carcinoma which is a highly vascular tumor is the leading cause of death from urologic cancers. Angiogenesis has a pivotal role in oncogenesis and in the viability and expansion of renal cell carcinoma. Rap2B, as a small guanosine triphosphate-binding protein of the Ras family, was first discovered in the early 1990s during the screening of a platelet complementary DNA library. Previous studies have shown that Rap2B aberrantly expressed in human carcinogenesis and promoted the development of tumors via multiple signaling pathways. However, the function of Rap2B in tumor angiogenesis that is necessary for tumor growth and metastasis remains unknown. In this study, we examined the role of Rap2B in angiogenesis in renal cell carcinoma by Western blot, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, human umbilical vascular endothelial cells growth assay, and endothelial cell tube formation assay. We found that Rap2B promoted angiogenesis in vitro and in vivo. Moreover, our data illustrated that phosphoinositide 3-kinase/AKT signaling pathway is involved in Rap2B-mediated upregulation of vascular endothelial growth factor and renal cell carcinoma angiogenesis. Taken together, these results revealed that Rap2B promotes renal cell carcinoma angiogenesis via phosphoinositide 3-kinase/AKT/vascular endothelial growth factor signaling pathway, which suggests that Rap2B is a novel therapeutic target for renal cell carcinoma anti-angiogenesis therapy.

Cytosolic proteome profiling of monocytes for male osteoporosis.

In male Caucasians with discordant hip bone mineral density (BMD), we applied the subcellular separation and proteome profiling to investigate the monocytic cytosol. Three BMD-associated proteins (ALDOA, MYH14, and Rap1B) were identified based on multiple omics evidence, and they may influence the pathogenic mechanisms of osteoporosis by regulating the activities of monocytes. INTRODUCTION: Osteoporosis is a serious public health problem, leading to significant mortality not only in aging females but also in males. Peripheral blood monocytes (PBMs) play important roles in bone metabolism by acting as precursors of osteoclasts and producing cytokines important for osteoclast development. The first cytosolic sub-proteome profiling analysis was performed in male PBMs to identify differentially expressed proteins (DEPs) that are associated with BMDs and risk of osteoporosis. METHODS: Here, we conducted a comparative proteomics analysis in PBMs from Caucasian male subjects with discordant hip BMD (29 low BMD vs. 30 high BMD). To decrease the proteome complexity and expand the coverage range of the cellular proteome, we separated the PBM proteome into several subcellular compartments and focused on the cytosolic fractions, which are involved in a wide range of fundamental biochemical processes. RESULTS: Of the total of 3796 detected cytosolic proteins, we identified 16 significant (P < 0.05) and an additional 22 suggestive (P < 0.1) DEPs between samples with low vs. high hip BMDs. Some of the genes for DEPs, including ALDOA, MYH14, and Rap1B, showed an association with BMD in multiple omics studies (proteomic, transcriptomic, and genomic). Further bioinformatics analysis revealed the enrichment of DEPs in functional terms for monocyte proliferation, differentiation, and migration. CONCLUSIONS: The combination strategy of subcellular separation and proteome profiling allows an in-depth and refined investigation into the composition and functions of cytosolic proteome, which may shed light on the monocyte-mediated pathogenic mechanisms of osteoporosis.

Rap2b promotes proliferation, migration, and invasion of lung cancer cells.

Rap2b, a member of the guanosine triphosphate-binding proteins, is widely up-regulated in many types of tumors. However, the functional role of Rap2b in tumorigenesis of lung cancer remains to be fully elucidated. In this study, we investigated the effect of Rap2b on the lung cancer malignant phenotype, such as cell proliferation and metastasis. We found that Rap2b could promote the abilities of lung cancer cell wound healing, migration, and invasion via increasing matrix metalloproteinase-2 enzyme activity. Furthermore, Rap2b overexpression could increase the phosphorylation level of extracellular signal-regulated protein kinases 1/2. In conclusion, our results suggested that Rap2b may be a potential therapeutic target for lung cancer.

miR-342-3p targets RAP2B to suppress proliferation and invasion of non-small cell lung cancer cells.

MicroRNAs (miRNAs) play critical roles in cancer development and progression. In this study, we examined the roles and molecular mechanisms of miR-342-3p in human non-small cell lung cancer (NSCLC). The results showed that miR-342-3p is downregulated in NSCLC cell lines and tissues, and its overexpression induces significant inhibition of NSCLC cell proliferation, invasion, and tumor growth in nude mice. In addition, miR-342-3p repressed RAP2B expression through interactions with its 3'-UTR region. Restoration of RAP2B expression reversed miR-342-3p-mediated inhibitory activity in NSCLC cells. Finally, analyses of miR-342-3p and RAP2B levels in NSCLCs revealed that miR-342-3p inversely correlated with RAP2B mRNA expression. Our collective findings provide preliminary evidence that miR-342-3p acts as a tumor suppressor in NSCLC through repression of RAP2B.

MicroRNA-100 regulates SW620 colorectal cancer cell proliferation and invasion by targeting RAP1B.

MicroRNAs (miRNAs) have been demonstrated to play important roles in tumorigenesis of human cancer. Fewer studies have explored the roles of miR-100 on human colorectal cancer cell proliferation and invasion. In this study, we utilized real-time PCR to verify whether miR-100 was downregulated in human colorectal cancer tissues compared with matched adjacent normal tissues. Functional studies demonstrated that ectopic expression of miR-100 inhabits cell growth and invasion and induce apoptosis, whereas knockdown of miR-100 yielded the reverse phenotype. Mechanistic studies reveal that miR-100 repressed the activity of a reporter gene fused to the 3'-untranslated region (3'-UTR) of RAP1B, whereas miR-100 silencing upregulated the expression of the reporter gene. Furthermore, we also detected that RAP1B mRNA was inversely expressed with miR-100 in colorectal cancer tissues. These data indicate that the miR-100 plays a tumor suppressor role by regulating colorectal cancer cell growth and invasion phenotype, and could serve as a potential maker for colorectal cancer therapy.

Trans-dimerization of JAM-A regulates Rap2 and is mediated by a domain that is distinct from the cis-dimerization interface.

Junctional adhesion molecule-A (JAM-A) is a tight junction-associated signaling protein that regulates epithelial cell proliferation, migration, and barrier function. JAM-A dimerization on a common cell surface (in cis) has been shown to regulate cell migration, and evidence suggests that JAM-A may form homodimers between cells (in trans). Indeed, transfection experiments revealed accumulation of JAM-A at sites between transfected cells, which was lost in cells expressing cis- or predicted trans-dimerization null mutants. Of importance, microspheres coated with JAM-A containing alanine substitutions to residues 43NNP45 (NNP-JAM-A) within the predicted trans-dimerization site did not aggregate. In contrast, beads coated with cis-null JAM-A demonstrated enhanced clustering similar to that observed with wild-type (WT) JAM-A. In addition, atomic force microscopy revealed decreased association forces in NNP-JAM-A compared with WT and cis-null JAM-A. Assessment of effects of JAM-A dimerization on cell signaling revealed that expression of trans- but not cis-null JAM-A mutants decreased Rap2 activity. Furthermore, confluent cells, which enable trans-dimerization, had enhanced Rap2 activity. Taken together, these results suggest that trans-dimerization of JAM-A occurs at a unique site and with different affinity compared with dimerization in cis. Trans-dimerization of JAM-A may thus act as a barrier-inducing molecular switch that is activated when cells become confluent.

Chemokine unresponsiveness of chronic lymphocytic leukemia cells results from impaired endosomal recycling of Rap1 and is associated with a distinctive type of immunological anergy.

Trafficking of malignant lymphocytes is fundamental to the biology of chronic lymphocytic leukemia (CLL). Transendothelial migration (TEM) of normal lymphocytes into lymph nodes requires the chemokine-induced activation of Rap1 and αLß2 integrin. However, in most cases of CLL, Rap1 is refractory to chemokine stimulation, resulting in failed αLß2 activation and TEM unless α4ß1 is coexpressed. In this study, we show that the inability of CXCL12 to induce Rap1 GTP loading in CLL cells results from failure of Rap1-containing endosomes to translocate to the plasma membrane. Furthermore, failure of chemokine-induced Rap1 translocation/GTP loading was associated with a specific pattern of cellular IgD distribution resembling that observed in normal B cells anergized by DNA-based Ags. Anergic features and chemokine unresponsiveness could be simultaneously reversed by culturing CLL cells ex vivo, suggesting that these two features are coupled and driven by stimuli present in the in vivo microenvironment. Finally, we show that failure of Rap1 translocation/GTP loading is linked to defective activation of phospholipase D1 and its upstream activator Arf1. Taken together, our findings indicate that chemokine unresponsiveness in CLL lymphocytes results from failure of Arf1/phospholipase D1-mediated translocation of Rap1 to the plasma membrane for GTP loading and may be a specific feature of anergy induced by DNA Ags.

Expression of Rap1 during germ cell development in the rat and its functional implications in 2-methoxyacetic acid-induced spermatocyte apoptosis.

OBJECTIVE: To investigate the expression pattern of Ras-related protein 1 (Rap1) during testicular development and to clarify whether its expression is developmentally regulated and whether this expression is involved in the process of germ cell apoptosis. MATERIALS AND METHODS: The expression pattern of Rap1 in the adult rat testicle was compared with that in the developing rat testicle using immunoblotting and immunohistochemical analyses. After the adult rats were treated with methoxyacetic acid (MAA), which selectively depletes primary spermatocytes, we correlated Rap1 expression with apoptotic dynamics using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), double immunofluorescent staining, and coimmunoprecipitation assays. RESULTS: During testicular development, Rap1 was expressed in the nucleus of gonocytes and in the Golgi apparatus of spermatocytes. The expression pattern of Rap1 during spermatogenesis was also shown to be stage specific. After 12 hours of MAA treatment, we found that Rap1 was translocated into the nucleus of some spermatocytes and the overlapping rate of Rap1 and NF-κB was much greater than that of Rap1 and TUNEL staining. In addition, Rap1 protein could be co-immunoprecipitated with NF-κB protein. CONCLUSION: In cooperation with NF-κB, Rap1 might be involved in the early stage of the apoptotic process occurring in the MAA-treated rat testicle. Additional characterization of this small guanosine triphosphatase in the apoptotic dynamics should provide information on the early diagnosis of MAA-induced male infertility.

cAMP-dependent oncogenic action of Rap1b in the thyroid gland.

cAMP signaling leads to activation and phosphorylation of Rap1b. Using cellular models where cAMP stimulates cell proliferation, we have demonstrated that cAMP-mediated activation, as well as phosphorylation of Rap1b, is critical for cAMP stimulation of DNA synthesis. To determine whether Rap1b stimulates mitogenesis in vivo, we have constructed a transgenic mouse where a constitutively active G12V-Rap1b, flanked by Cre recombinase LoxP sites, is followed by the dominant negative S17N mutant. Employing this novel mouse model, we have switched, in a tissue-specific (thyroid) and temporally controlled manner, the expression of Rap1b from a stimulatory to an inhibitory form. These experiments provide conclusive evidence that Rap1b is oncogenic in the thyroid in ways linked to transduction of the cAMP mitogenic signal.

Genetic and epigenetic changes in p21 and p21B do not correlate with resistance to doxorubicin or mitomycin and 5-fluorouracil in locally advanced breast cancer.

PURPOSE: The cyclin-dependent kinase inhibitor p21 acts as a main executor of p53-induced growth arrest. Recently, a second transcript, p21B, was found to code for a protein expressing proapoptotic activity. We investigated p21 and p21B for mutations and epigenetic silencing in locally advanced breast cancers treated with doxorubicin or 5-fluorouracil/mitomycin and correlated our findings with treatment response and TP53 status. EXPERIMENTAL DESIGN: We used reverse transcription-PCR to analyze p21/p21B mutation status in 73 breast cancer samples. The p21 promoter region was sequenced and analyzed for hypermethylations by methylation-specific PCR. In addition, a selection of patients were analyzed for mutations in the p21B promoter. RESULTS: The p21 gene was neither mutated nor silenced by promoter hypermethylation in any of the tumors examined. One patient harbored a novel p21 splice variant in addition to the wild-type transcript. We observed two base substitutions in the p21 transcript, C93A and G251A, each affecting six patients (8.2%). The G251A variant had not been reported previously. In 12 patients (16.4%), we observed a novel base substitution, T35C, in p21B. All three base substitutions were observed in lymphocyte DNA and therefore considered polymorphisms. The polymorphisms did not correlate with p21 staining index, treatment response to doxorubicin or 5-fluorouracil/mitomycin, or TP53 status. CONCLUSIONS: Our findings do not suggest that genetic or epigenetic disturbances in p21 or p21B cause resistance to doxorubicin or mitomycin/5-fluorouracil in breast cancer. Future studies should assess potential associations between these novel polymorphisms and breast cancer risk.

Identification of differentially expressed mRNA during pancreas regeneration of rat by mRNA differential display.

Pancreatectomy (Px) is known to cause islet hypertrophy and is a putative method to mimic hyperglycemia representing type II diabetes mellitus. Therefore, finding new genes related to pancreatectomy will help to understand the molecular mechanism of hypertrophy and hyperglycemia, and may provide new diagnostic markers of type II diabetes. To this end, mRNA differential display was used to isolate genes that show transcriptional changes in pancreas of rat after 90% partial pancreatectomy. Forty-nine candidate pancreas regeneration-associated transcripts were isolated. cDNA sequencing and subsequent database analysis revealed that 15 transcripts showed no significant sequence similarity to previously reported genes, whereas 34 transcripts showed significant similarity with genes deposited in the GenBank. The differential mRNA expression of 49 transcripts was confirmed using screening of slot blots and Northern blot analysis was performed to several genes. It was noteworthy that the Wnt-1 inducible signaling pathway protein-1 (WISP-1), Ras-associated protein 1B (Rap1B), vascular cell adhesion molecule-1 (VCAM-1), and huntingtin interacting protein genes (HIP) were observed to be over-expressed during pancreas regeneration. Several genes' expression was modified by pancreatectomy. Profiling of gene expression in response to pancreatectomy may lead to new insights into hypertrophy and hyperglycemia representing type II diabetes, as well as into the identification of novel diagnostic markers of type II diabetes.