Long Non-Coding RNA BCAR4 Promotes Oxaliplatin Resistance in Colorectal Cancer by Modulating miR-484-3p/RAB5C Expression.

BACKGROUND: Oxaliplatin-based chemotherapy resistance is a major cause of recurrence in patients with colorectal cancer (CRC). Increasing evidence indicates that lncRNA BCAR4 is involved in the occurrence and development of various cancers. However, the effect of BCAR4 on CRC chemotherapy resistance remains unclear. METHODS: Real-time quantitative PCR and Western blotting were used to detect the expression levels of gene and protein, respectively. The role of BCAR4 in drug resistance was evaluated by cell viability and apoptosis experiments. Luciferase reporter assay and Western blot analysis confirmed the relationship between BCAR4, miR-483-3p, and RAB5C. RESULTS: Luciferase reporter assay and Western blotting analysis confirmed the relationship among BCAR4, miR-483-3p, and RAB5C. The results showed that the expression levels of BCAR4 and RAB5C were increased in CRC tumor tissue. The expression levels of BCAR4 were increased in patients with chemotherapy resistance. Functional analysis showed that knockdown of BCAR4 reduced the expression levels of proteins related to stemness, decreased the activity of cells, and promoted apoptosis of CRC cells, while overexpression of RAB5C reversed these effects. Moreover, the results showed that BCAR4 promoted oxaliplatin resistance by inhibiting cell apoptosis. Mechanistically, BCAR4 sponged miR-483-3p and promoted the expression of RAB5C. Knockdown of BCAR4 reduced tumor size and enhanced cell sensitivity to oxaliplatin in vivo. CONCLUSION: The results suggested that BCAR4/miR-483-3p/RAB5C axis has the potential to be explored as a novel therapeutic target for CRC treatment.

Endocytosis-dominated membrane area decrease requires Rab5 protein in rat melanotrophs.

Eukaryotic cells internalize extracellular macromolecules by endocytosis and it was shown that Rab5 protein is required for this process. While it is clear that endocytosis consists of vesicle fission from the plasma membrane, the role of Rab5 protein in the plasma membrane surface area changes is still unclear. Here we studied whether Rab5 is required for membrane surface area changes in rat melanotrophs-cells deriving from the pituitary pars intermedia. The presence of this protein in melanotrophs was probed by immunocytochemistry and its putative role in membrane area dynamics was monitored electrophysiologically with membrane capacitance measurements as this parameter directly reflects changes in membrane surface area. We found that Rab5 protein exists in melanotrophs. At [Ca(2+)](i) < 3 microM, endocytosis-dominated membrane capacitance decrease was found to be blocked by microinjection of specific Rab5 antibody. At high [Ca(2+)](i), Rab5 antibody did not affect the steady-state increase in membrane capacitance, while it elevated the rate of membrane capacitance increase, which is consistent with an inhibition of endocytosis.

ADP-ribosylation of Rab5 by ExoS of Pseudomonas aeruginosa affects endocytosis.

Pseudomonas aeruginosa exoenzyme S (ExoS) is an ADP-ribosyltransferase that modifies low-molecular-weight GTPases. Here we studied the effect of Rab5 ADP-ribosylation by ExoS on its cellular function, i.e., regulation of early endocytic events. Coculture of CHO cells with P. aeruginosa induced a marked decrease in horseradish peroxidase (HRP) uptake compared to noninfected cells, while coculture with a P. aeruginosa mutant strain that fails to produce ExoS did not lead to any change in HRP uptake. Microinjection of recombinant ExoS into Xenopus oocytes induced strong inhibition of basal HRP uptake by oocytes. Moreover, coinjection of recombinant ExoS with Rab5 abolished the typical stimulation of HRP uptake obtained after GTPase microinjection. Cytosols prepared from injected oocytes were used in an endosome-endosome fusion assay. Cytosol from ExoS-microinjected oocytes was ineffective in promoting endosome-endosome fusion. However, in these conditions, the addition of Rab5 to the assay led to fusion recovery. Finally, we found that the interaction of Rab5 with EEA1 was markedly diminished after Rab5 ADP-ribosylation by ExoS.

Rab4 affects both recycling and degradative endosomal trafficking.

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.

Rab5 induces Rac-independent lamellipodia formation and cell migration.

Rab5 is a regulatory GTPase of vesicle docking and fusion that is involved in receptor-mediated endocytosis and pinocytosis. Introduction of active Rab5 in cells stimulates the rate of endocytosis and vesicle fusion, resulting in the formation of large endocytic vesicles, whereas dominant negative Rab5 inhibits vesicle fusion. Here we show that introduction of active Rab5 in fibroblasts also induced reorganization of the actin cytoskeleton but not of microtubule filaments, resulting in prominent lamellipodia formation. The Rab5-induced lamellipodia formation did not require activation of PI3-K or the GTPases Ras, Rac, Cdc42, or Rho, which are all strongly implicated in cytoskeletal reorganization. Furthermore, lamellipodia formation by insulin, Ras, or Rac was not affected by expression of dominant negative Rab5. In addition, cells expressing active Rab5 displayed a dramatic stimulation of cell migration, with the lamellipodia serving as the leading edge. Both lamellipodia formation and cell migration were dependent on actin polymerization but not on microtubules. These results demonstrate that Rab5 induces lamellipodia formation and cell migration and that the Rab5-induced lamellipodia formation occurs by a novel mechanism independent of, and distinct from, PI3-K, Ras, or Rho-family GTPases. Thus, Rab5 can control not only endocytosis but also actin cytoskeleton reorganization and cell migration, which provides strong support for an intricate relationship between these processes.