MiR-135a inhibits non-small cell lung cancer progression by suppressing RAB1B expression and the RAS pathway.

Lung cancer is the most common tumor in China and worldwide. Despite advances in diagnosis and therapy, it still represents the most lethal malignancy in industrialized countries. The study of regulatory noncoding RNAs has deepened our understanding of cancer on the molecular and clinical level. In this article, it showed that miR-135a was aberrantly downregulated in non-small cell lung cancer (NSCLC) cells in comparison with normal bronchial epithelial cells, and the expression of miR-135a inhibited proliferation, invasion and metastasis of NSCLC cells in vitro. Moreover, it was demonstrated that miR-135a inhibited the expression of multiple components (including RAS, Raf1, Rac1 and RhoA) of the RAS pathway via RAB1B, which was a novel target of miR-135a. The expression of miR-135a and RAB1B could effectively predict the clinical outcomes of NSCLC. In summary, miR-135a might function as a suppressor of NSCLC cells, and thus could be used as a potential therapeutic target.

MiR-1202 Exerts Neuroprotective Effects on OGD/R Induced Inflammation in HM Cell by Negatively Regulating Rab1a Involved in TLR4/NF-κB Signaling Pathway.

Recent studies have shown that the level of miR-1202 in peripheral blood is closely related to brain activity and cognitive function in patients with depression, and it is involved in glioma pathological progress. However, the correlation between miR-1202 and neuroinflammation has not been reported. The expressions of miR-1202 and small GTP-ase Rab1a at mRNA level were detected in oxygen-glucose deprivation (OGD)/reoxygenation (R) induced human microglial cells (HM cells) by RT-qPCR at different time points within 48 h. Dual luciferase report assay and immunofluorescence staining were performed to confirm whether Rab1a was the potential target of miR-1202. The toll-like receptor 4 (TLR4)/nuclear factor kappa beta (NF-κB) signal related proteins (TLR4, P65, p-P65, IκBa) and the downstream pro-inflammation factors pro-IL-1ß, pro-IL-18, as well as the inflammation factors interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) were detected by western-blotting. The expression level of TLR4 on cell surface was detected by flow cytometry. Down-regulation of miR-1202 and up-regulation of Rab1a were found in OGD/R induced HM cells. In addition, miR-1202 was identified to directly target Rab1a and down-regulate its expression. Moreover, over-expression of miR-1202 suppressed the activation of TLR4/NF-κB inflammatory signaling pathway. Rab1a can increase the expression level of TLR4 on the surface of OGD/R induced HM cells. MiR-1202 exerts neuroprotective effect by negatively regulating its target protein Rab1a, which can inactivate TLR4/NF-κB-involved inflammatory signaling pathway in OGD/R induced HM cells. These findings provide potential therapeutic targets for ischemic stroke.

MiR-655-3p inhibited proliferation and migration of ovarian cancer cells by targeting RAB1A.

OBJECTIVE: To investigate the potential effect of microRNA-655-3p (miR-655-3p) on the development of ovarian cancer (OC) and its relevant mechanism. PATIENTS AND METHODS: Expression level of miR-655-3p in OC tissues was detected. The potential target gene of miR-655-3p was firstly predicted online and subsequently verified by luciferase reporter assay and Western blot. In vitro effects of miR-655-3p on SKOV3 cells were determined as well. RESULTS: Low expression of miR-655-3p in OC was confirmed by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Ras-related protein Rab-1A (RAB1A) was a direct target of miR-655-3p in OC and was negatively regulated by miR-655-3p. Further, the effects of miR-655-3p/RAB1A axis on cell proliferation, metastasis ability, and EMT activation were emphasized. CONCLUSIONS: Our research emphasized the suppressor function of miR-655-3p in OC. By targeting RAB1A, miR-655-3p played a tumor suppressor role in OC. We affirmed the beneficial effects of miR-655-3p in OC cells for the first time, thus providing an experimental basis for the treatment of OC.

Rab29 activation of the Parkinson's disease-associated LRRK2 kinase.

Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector-binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans-Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29-mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2-mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.

Inhibition of RAB1A suppresses epithelial-mesenchymal transition and proliferation of triple-negative breast cancer cells.

RAB1A acts as an oncogene in various cancers, and emerging evidence has verified that RAB1A is an mTORC1 activator in hepatocellular and colorectal cancer, but the role of RAB1A in breast cancer remains unclear. In this investigation, RAB1A siRNA was successfully transfected in MDA-MB-231 and BT-549 human triple-negative breast cancer cells, and verified by real­time quantitative polymerase chain reaction and western blotting. Then, MTT cell proliferation, colony formation, cell invasion and wound healing assays were performed to characterize the function of RAB1A in the breast cancer cell lines. Downregulation of RAB1A inhibited cellular growth, cell migration, cell invasion and cell epithelial-mesenchymal transition. Furthermore, compared with NC siRNA transfected cells, RAB1A siRNA transfected breast cancer cells inhibited the phosphorylation of S6K1, the effector molecular of mTORC1. Collectively, our data suggested that RAB1A acts as an oncogene by regulating cellular proliferation, growth, invasion and metastasis via activation of mTORC1 pathway in triple-negative breast cancer.

miR-15b-5p induces endoplasmic reticulum stress and apoptosis in human hepatocellular carcinoma, both in vitro and in vivo, by suppressing Rab1A.

In human hepatocellular carcinoma (HCC), aberrant expression of miRNAs correlates with tumor cell proliferation, apoptosis, invasion, and migration by targeting downstream proteins. miR-15b levels are reported increased in HCC tissues; however, they negatively correlate to HCC recurrence. Our aim was to understand the reason for this phenomenon. We used the reverse transcription-polymerase chain reaction (RT-PCR) to measure miR-15b-5p expression in both HCC tissues and HCC cell lines. Our results were consistent with the report. Using bioinformatics and luciferase reporter assays, we identified Rab1A as a novel and direct target of miR-15b-5p. Inhibiting the function of Rab1A with shRab1A also inhibited the growth of HCC cells and induced endoplasmic reticulum stress (ERS) and apoptosis. Similarly, suppressing Rab1A by overexpression of miR-15b-5p also inhibited cell growth and induced ERS and apoptosis. Moreover, re-expression of Rab1A rescued the miR-15b-5p-induced ERS, apoptosis, and growth inhibition in HCC cells. In vivo assays were further performed to testify them. Taken together, our data suggest that miR-15b-5p induces ERS, apoptosis, and growth inhibition by targeting and suppressing Rab1A, acting as a tumor suppressor gene in HCC. This finding suggests a novel relation among Rabs, miRNAs, and apoptosis.

Hsc70 contributes to cancer cell survival by preventing Rab1A degradation under stress conditions.

Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology.

Selective protection of an ARF1-GTP signaling axis by a bacterial scaffold induces bidirectional trafficking arrest.

Bidirectional vesicular transport between the endoplasmic reticulum (ER) and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated in order to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E. coli secreted protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi intermediate compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. The mechanistic insights presented here establish the effectiveness of a small bacterial catalytic scaffold for studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen.

Modulation of small GTPases by Legionella.

The pathogenic bacterium Legionella pneumophila interacts intimately with signaling molecules during the infection of eukaryotic host cells. Among a diverse set of regulatory molecules, host small GTPases appear to be prominent and significant targets. Small GTPases are molecular switches that regulate cellular signaling via their respective nucleotide-bound states: When bound to GDP, they are inactive, but become activated upon binding to GTP. Legionella secretes specific bacterial proteins into the cytosol of the host cell that most prominently modulate the activities of small GTPases involved in vesicular trafficking, but probably also other G-proteins. The master regulators of vesicular trafficking, i.e., Rab and Arf proteins, are majorly targeted G-proteins of Legionella proteins, and among these, Rab1 experiences the most diverse modifications. Generally, the activities of small GTPases are modulated by GDP/GTP exchange (activation), GTP hydrolysis (deactivation), membrane recruitment, post-translational modifications (phosphocholination, adenylylation), and tight and competitive binding. Here, we discuss the consequences and molecular details of the modulation of small GTPases for the infection by Legionella, with a special but not exclusive focus on Rab and Arf proteins.

Inhibition of Rab1 GTPase and endoplasmic reticulum-to-Golgi trafficking underlies statin's toxicity in rat skeletal myofibers.

HMG-CoA reductase inhibitor statins are used for the treatment of hypercholesterolemia. However, statins have adverse effects on skeletal muscles with unknown mechanism. We have reported previously that fluvastatin induced vacuolation and cell death in rat skeletal myofibers by depleting geranylgeranylpyrophosphate (GGPP) and suppressing small GTPases, particularly Rab (FASEB J 21:4087-4094, 2007). Rab1 is one of the most susceptible Rab isoforms to GGPP depletion and is essential for endoplasmic reticulum (ER)-to-Golgi trafficking. Here, we explored whether Rab1 and ER-to-Golgi vesicle trafficking were affected by statins in cultured single myofibers isolated from flexor digitorum brevis muscles of adult rats. Western blot analysis revealed that Rab1A protein resided predominantly in membrane but not in cytosol in control myofibers, whereas it was opposite in fluvastatin-treated myofibers, indicating that fluvastatin inhibited Rab1A translocation from cytosol to membrane. GGPP supplementation prevented the effect of fluvastatin on Rab1A translocation. Brefeldin A, a specific suppressor of ER-to-Golgi trafficking, induced vacuolation and cell death in myofibers in a manner similar to that of fluvastatin. Although ER-to-Golgi traffic suppression induces unfolded protein response (UPR) and cell death in some cell types, neither fluvastatin nor brefeldin A up-regulated UPR in myofibers. Immunofluorescence study revealed that the distribution of an ER marker, calnexin, was restricted to the region around nucleus with fluvastatin, suggesting the inhibition of ER membrane traffic by fluvastatin. We conclude that suppression of Rab1 GTPase and the subsequent inhibition of ER-to-Golgi traffic are involved in statin-induced skeletal myotoxicity.