Rab5c promotes RSV and ADV replication by autophagy in respiratory epithelial cells.

Respiratory system diseases caused by respiratory viruses are common and exert tremendous pressure on global healthcare system. In our previous studies, we found that Long non-coding RNA NRAV (Lnc NRAV) and its target molecule Rab5c plays a significant role in respiratory virus infection. However, the mechanism by which Rab5c affects virus replication remains unclear. Rab5c, a protein mainly localized on the cell membranes and in early endosomes and phagosomes, participates in endocytosis mediated by clathrin and regulates the fusion of early endosome, maturation of early phagosomes, and autophagy. Therefore, we inferred that Rab5c impacts virus replication, which might be related to endocytosis or autophagy. We selected RSV (respiratory syncytial virus) as a representative enveloped virus and ADV (Adenovirus) as a representative non-enveloped virus to explore the possible mechanism of RSV and ADV replication promoted by Rab5c in A549 cells and in Rab5c-overexpressing mice. Here, we confirmed that the activated Rab5c promotes RSV and ADV replication and the inactivated Rab5c inhibits their replication. However, Rab5c promoting RSV and ADV replication is not mediated by endocytosis rather by autophagy in respiratory epithelial cells. Our study showed that Rab5c upregulates LC3-Ⅱ (microtubule-associated protein 1 light chain 3 beta) protein expression levels by interacting with Beclin1, a key autophagy molecule, which can induce autophagy and promote replication of ADV and RSV. This study enriches the understanding of the interaction between respiratory viruses and Rab5c, providing new insights for virus prevention and treatment.

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.

RAB5C, a new mRNA binding target of HuR, regulates breast cancer cell proliferation.

The posttranscriptional control of gene expression mediated by RNA-binding proteins (RBPs) is essential to determine tumor cell fate. HuR is an RBP with increased expression in various cancer types. This study aimed to clarify the regulatory mechanism of HuR's contribution to breast cancer (BC) cell proliferation by inducing RAB5C expression. First, we analyzed the expression level of HuR and RAB5C in BC tissues and cell lines by immunohistochemistry, qRT-PCR, and western blot. Next, to further investigate the effect of HuR on RAB5C expression, we used short hairpin RNAs (shRNAs) to silence endogenous HuR expression in BC cell lines MCF7 and MDA-MB-231. The binding site of RAB5C mRNA and HuR was confirmed by RNA immunoprecipitation. Finally, the function of RAB5C was investigated using flow cytometry, colony formation, and MTT assays. We found that the expression of HuR and RAB5C was significantly upregulated in BC tissues and MCF-7 and MDA-MB231 cell lines. Importantly, RAB5C mRNA stability was increased through binding of HuR to its 3'UTR. Inhibition of HuR expression using shRNA decreased RAB5C mRNA, suggesting that HuR plays a role in regulating RAB5C expression level. In addition, suppression of RAB5C expression reduced BC cell growth. These results suggest RAB5C functions as an oncogene in BC cells, HuR promoted BC cell survival by facilitating RAB5C expression. Our findings suggest that HuR and RAB5C play important roles in BC cell survival.

Novel Host Protein TBC1D16, a GTPase Activating Protein of Rab5C, Inhibits Prototype Foamy Virus Replication.

Prototype foamy virus (PFV) is a member of the oldest family of retroviruses and maintains lifelong latent infection in the host. The lifelong latent infection of PFV may be maintained by the restriction factors of viral replication in the host. However, the mechanisms involved in PFV latent infection are poorly understood. Here, we found that TBC1D16, a TBC domain-containing protein, is significantly down-regulated after PFV infection. Tre2/Bub2/Cdc16 (TBC) domain-containing proteins function as Rab GTPase-activating proteins (GAPs) and are participates in the progression of some diseases and many signaling pathways. However, whether TBC proteins are involved in PFV replication has not been determined. Here, we found that TBC1D16 is a novel antiviral protein that targets Rab5C to suppress PFV replication. Overexpression TBC1D16 inhibited the transcription and expression of Tas and Gag, and silencing TBC1D16 enhanced the PFV replication. Moreover, the highly conserved amino acid residues R494 and Q531 in the TBC domain of TBC1D16 were essential for inhibiting PFV replication. We also found that TBC1D16 promoted the production of PFV-induced IFN-ß and the transcription of downstream genes. These results suggest that TBC1D16 might be the first identified TBC proteins that inhibited PFV replication and the mechanism by which TBC1D16 inhibited PFV replication could provide new insights for PFV latency.

RAB5C, SYNJ1, and RNF19B promote male ankylosing spondylitis by regulating immune cell infiltration.

BACKGROUND: This study aimed to identify the key genes related to male ankylosing spondylitis (AS) and to analyze the role of immune cell infiltration in the pathological process of this disease. METHODS: The AS dataset was downloaded from the Gene Expression Omnibus (GEO) public database, and the data of male healthy controls (M_HC) and male AS patients (M_AS) were extracted. R software was used to identify differentially expressed genes (DEGs). Functional and pathway enrichment analysis of the DEGs was performed. A protein-protein interaction (PPI) network was constructed, and the hub genes were screened out. All expression profile data were analyzed by weighted correlation network analysis (WGCNA) to screen out the hub genes, which were then intersected with the hub genes from the PPI network to obtain the key genes. Finally, the difference in immune cell infiltration in the two sets of samples was evaluated with CIBERSORT, and the correlation between the key genes and infiltrating immune cells was analyzed. RESULTS: A total of 689 DEGs were obtained, of which 395 genes were up-regulated and 294 genes were down-regulated. Functional and pathway enrichment analysis showed that DEGs were mainly enriched in pathways related to immune response. Based on the PPI analysis, five clusters with high scores were selected. Through WGCNA, 14 gene modules were obtained. The green module with the highest correlation was selected and intersected with the cluster previously obtained to obtain three key genes, RAB5C, SYNJ1, and RNF19B. Immune infiltration analysis found that monocytes and gamma delta T cells may be involved in the process of AS. Also, RAB5C, SYNJ1, and RNF19B are all related to increased levels of monocytes and macrophages. CONCLUSIONS: RAB5C, SYNJ1, and RNF19B are key DEGs expressed in M_AS and may play a role in the disease's occurrence and development through regulating immune cell functions.

The endosomal RIN2/Rab5C machinery prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis.

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.

The Small GTPase Rab5c Exerts Bi-Function in Singapore Grouper Iridovirus Infections and Cellular Responses in the Grouper, Epinephelus coioides.

The small GTPase Rab5 is one of the master regulators of vesicular trafficking that participates in early stages of the endocytic pathway, such as endocytosis and endosome maturation. Three Rab5 isoforms (a, b, and c) share high sequence identity, and exhibit complex functions. However, the role of Rab5c in virus infection and cellular immune responses remains poorly understood. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected grouper spleen (GS) cells, we investigated the role of Rab5c in virus infection and host immune responses. Rab5c was cloned from the orange-spotted grouper, Epinephelus coioides, and termed EcRab5c. EcRab5c encoded a 220-amino-acid polypeptide, showing 99% and 91% identity to Anabas testudineus, and Homo sapiens, respectively. Confocal imaging showed that EcRab5c localized as punctate structures in the cytoplasm. However, a constitutively active (CA) EcRab5c mutant led to enlarged vesicles, while a dominant negative (DN) EcRab5c mutant reduced vesicle structures. EcRab5c expression levels were significantly increased after SGIV infection. EcRab5c knockdown, or CA/DN EcRab5c overexpression significantly inhibited SGIV infection. Using single-particle imaging analysis, we further observed that EcRab5c disruption impaired crucial events at the early stage of SGIV infection, including virus binding, entry, and transport from early to late endosomes, at the single virus level. Furthermore, it is the first time to investigate that EcRab5c is required in autophagy. Equally, EcRab5c positively regulated interferon-related factors and pro-inflammatory cytokines. In summary, these data showed that EcRab5c exerted a bi-functional role on iridovirus infection and host immunity in fish, which furthers our understanding of virus and host immune interactions.

MiR-145 functions as a tumor suppressor in Papillary Thyroid Cancer by inhibiting RAB5C.

Papillary thyroid carcinoma (PTC) accounts for the largest proportion of thyroid cancers; and its morbidity rate has dramatically increased in recent decades. However, the pathogenesis mechanisms of PTC are still not clear. This study aimed to reveal that miR-145 acts as an antitumor miRNA in the progression of PTC. In the present study, the expression of miR-145 was analyzed in 57 paired PTC patient samples. The relationship between clinicopathological features and miR-145 expression were also defined. The tumor suppressive function of miR-145 on PTC cell metastasis, proliferation and apoptosis were revealed in vitro. Also, we used dual luciferase reporter assay to define the relationship of miR-145 and RAB5C. RAB5C was reported to participate in cell invasion and cell motility. We found that miR-145 was downregulated in PTCs, which was negatively correlated with PTC progression and metastasis. MiR-145 inhibited PTC migration, proliferation and promoted apoptosis by directly suppresing RAB5C. In conclusion, miR-145 functions as a tumor suppressor in PTC by inhibiting RAB5C. MiR-145 and RAB5C are potential therapeutic targets in therapy of aggressive PTC cases.

Long Noncoding RNA NRAV Promotes Respiratory Syncytial Virus Replication by Targeting the MicroRNA miR-509-3p/Rab5c Axis To Regulate Vesicle Transportation.

Respiratory syncytial virus (RSV) is an enveloped RNA virus which is responsible for approximately 80% of lower respiratory tract infections in children. Current lines of evidence have supported the functional involvement of long noncoding RNA (lncRNA) in many viral infectious diseases. However, the overall biological effect and clinical role of lncRNAs in RSV infection remain unclear. In this study, lncRNAs related to respiratory virus infection were obtained from the lncRNA database, and we collected 144 clinical sputum specimens to identify lncRNAs related to RSV infection. Quantitative PCR (qPCR) detection indicated that the expression of lncRNA negative regulator of antiviral response (NRAV) in RSV-positive patients was significantly lower than that in uninfected patients, but lncRNA psoriasis-associated non-protein coding RNA induced by stress (PRINS), nuclear paraspeckle assembly transcript 1 (NEAT1), and Nettoie Salmonella pas Theiler's (NeST) showed no difference in vivo and in vitro Meanwhile, overexpression of NRAV promoted RSV proliferation in A549 and BEAS-2B cells, and vice versa, indicating that the downregulation of NRAV was part of the host antiviral defense. RNA fluorescent in situ hybridization (FISH) confirmed that NRAV was mainly located in the cytoplasm. Through RNA sequencing, we found that Rab5c, which is a vesicle transporting protein, showed the same change trend as NRAV. Subsequent investigation revealed that NRAV was able to favor RSV production indirectly by sponging microRNA miR-509-3p so as to release Rab5c and facilitate vesicle transportation. The study provides a new insight into virus-host interaction through noncoding RNA, which may contribute to exploring potential antivirus targets for respiratory virus.IMPORTANCE The mechanism of interaction between RSV and host noncoding RNAs is not fully understood. In this study, we found that the expression of long noncoding RNA (lncRNA) negative regulator of antiviral response (NRAV) was reduced in RSV-infected patients, and overexpression of NRAV facilitated RSV production in vitro, suggesting that the reduction of NRAV in RSV infection was part of the host antiviral response. We also found that NRAV competed with vesicle protein Rab5c for microRNA miR509-3p in cytoplasm to promote RSV vesicle transport and accelerate RSV proliferation, thereby improving our understanding of the pathogenic mechanism of RSV infection.

Intracellular distribution and transcriptional regulation of Atlantic salmon (Salmo salar) Rab5c, 7a and 27a homologs by immune stimuli.

Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon.

Rab5C enhances resistance to ionizing radiation in rectal cancer.

Rectal cancer represents one third of the colorectal cancers that are diagnosed. Neoadjuvant chemoradiation is a well-established protocol for rectal cancer treatment reducing the risk of local recurrence. However, a pathologic complete response is only achieved in 10-40% of cases and the mechanisms associated with resistance are poorly understood. To identify potential targets for preventing therapy resistance, a proteomic analysis of biopsy specimens collected from stage II and III rectal adenocarcinoma patients before neoadjuvant treatment was performed and compared with residual tumor tissues removed by surgery after neoadjuvant therapy. Three proteins, Ku70, Ku80, and Rab5C, exhibited a significant increase in expression after chemoradiation. To better understand the role of these proteins in therapy resistance, a rectal adenocarcinoma cell line was irradiated to generate a radiotherapy-resistant lineage. These cells overexpressed the same three proteins identified in the tissue samples. Furthermore, radiotherapy resistance in this in vitro model was found to involve modulation of epidermal growth factor receptor (EGFR) internalization by Rab5C in response to irradiation, affecting expression of the DNA repair proteins, Ku70 and Ku80, and cell resistance. Taken together, these findings indicate that EGFR and Rab5C are potential targets for the sensitization of rectal cancer cells and they should be further investigated. KEY MESSAGES: • Rab5C orchestrates a mechanism of radioresistance in rectal adenocarcinoma cell. • Rab5C modulates EGFR internalization and its relocalization to the nucleus. • In the nucleus, EGFR can modulate the expression of the DNA repair proteins, Ku70 and Ku80. • Rab5C, Ku70, and Ku80 are overexpressed in tumor tissues that contain tumor cells that are resistant to chemoradiation treatment.

The use of novel epitope-tagged arenaviruses reveals that Rab5c-positive endosomal membranes are targeted by the LCMV matrix protein.

We report the development of recombinant New World (Junín; JUNV) and Old World (lymphocytic choriomeningitis virus; LCMV) mammarenaviruses that encode an HA-tagged matrix protein (Z). These viruses permit the robust affinity purification of Z from infected cells or virions, as well as the detection of Z by immunofluorescent microscopy. Importantly, the HA-tagged viruses grow with wild-type kinetics in a multi-cycle growth assay. Using these viruses, we report a novel description of JUNV Z localization in infected cells, as well as the first description of colocalization between LCMV Z and the GTPase Rab5c. This latter result, when combined with our previous findings that LCMV genome and glycoprotein also colocalize with Rab5c, suggest that LCMV may target Rab5c-positive membranes for preassembly of virus particles prior to budding. The recombinant viruses reported here will provide the field with new tools to better study Z protein functionality and identify key Z protein interactions with host machinery.

Visualization of the lymphocytic choriomeningitis mammarenavirus (LCMV) genome reveals the early endosome as a possible site for genome replication and viral particle pre-assembly.

We report a fluorescence in situ hybridization (FISH) assay that allows the visualization of lymphocytic choriomeningitis mammarenavirus (LCMV) genomic RNAs in individual cells. We show that viral S segment genomic and antigenomic RNA, along with viral nucleoprotein, colocalize in subcellular structures we presume to be viral replication factories. These viral RNA structures are highly dynamic during acute infection, with the many small foci seen early coalescing into larger perinuclear foci later in infection. These late-forming perinuclear viral RNA aggregates are located near the cellular microtubule organizing centre and colocalize with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane-bound organelle as a site for the pre-assembly of viral components, including genomic RNA and viral glycoprotein, prior to their transport to the plasma membrane, where new particles will bud.

Identification of post-transcriptional regulatory networks during myeloblast-to-monocyte differentiation transition.

Treatment of leukemia cells with 1,25-dihydroxyvitamin D3 may overcome their differentiation block and lead to the transition from myeloblasts to monocytes. To identify microRNA-mRNA networks relevant for myeloid differentiation, we profiled the expression of mRNAs and microRNAs associated to the low- and high-density ribosomal fractions in leukemic cells and in their differentiated monocytic counterpart. Intersection between mRNAs shifted across the fractions after treatment with putative target genes of modulated microRNAs showed a series of molecular networks relevant for the monocyte cell fate determination, as for example the post-transcriptional regulation of the Polo-like kinase 1 (PLK1) by miR-22-3p and let-7e-5p.