Expression Of Rab7 Research Articles

Impact of CD137-CD137L signaling on secretion of mouse vascular smooth muscle cells-derived exosomes: role of Rab7 pathway

Objective: To investigate whether CD137-CD137L signaling could affect the secretion of mouse vascular smooth muscle cells (VSMCs) -derived exosomes through autophagy mediated Rab7 pathway. Methods: Primary thoracic aorta VSMCs from C57BL/6J mouse were obtained by tissue block adherence method. VSMCs between the third to fifth passages were used and VSMCs were divided into 4 groups: control group, CD137 agonist group, lentivirus control group, Rab7 lentiviral interference group. VSMCs in CD137 agonist group were treated with recombinant protein of CD137L (10 μg/ml), VSMCs in lentivirus control group were treated with lentiviral followed by recombinant protein of CD137L (10 μg/ml), VSMCs in Rab7 lentiviral interference group were treated with Rab7 lentiviral intervention followed by recombinant protein of CD137L (10 μg/ml). Western blot was used to detect the protein expression of LC3Ⅱ, p62, Rab7, CD9, CD81 and Hsc70. Fluorescence microscopy was used to track the changes of autophagy in cells infected with mRFP-GFP-LC3. Transmission electron microscope was used to observe the morphology and size of VSMCs-derived exosomes. The nanoparticle tracking analysis(NTA) was used to detect the concentration and size of exosomes in each group. Results: (1) The expressions of Rab7, LC3Ⅱ and p62 protein in VSMCs of CD137 activation group were significantly higher than those in control group (all P<0.05). The expressions of Rab7, LC3Ⅱ and p62 protein in Rab7 lentivirus interference group was lower than in CD137 activation group (all P<0.05), while the expressions were similar between the lentivirus control group and the CD137 activation group (all P>0.05). (2) The total number of fluorescent spots and yellow fluorescent spots in the VSMCs of the CD137 activation group were higher than those in the control group (all P<0.05), and the number of yellow fluorescent spots was higher than that of the red fluorescent spots in the VSMCs of the CD137 activation group ((50.3±0.9) vs. (10.3±1.5)/cell). The total numbers of fluorescent spots and yellow fluorescent spots in VSMCs of Rab7 lentivirus interference group were lower than those of CD137 activation group (both P<0.05), and the number of red fluorescent spots in VSMCs was higher than that of yellow fluorescent spots ((40.7±4.0) and (10.7±1.2)/cell) in the Rab7 lentiviral interference group. The total numbers of fluorescent spots and yellow fluorescent spots in the VSMCs were similar between the lentivirus control group and the CD137 activation group (all P>0.05). (3) Under transmission electron microscopy, the size of the VSMCs-derived exosomes was about 30-150 nm. The exosome markers (CD9, CD81) could be detected in vesicles by Western blot. NTA results showed that the concentration of VSMCs-derived exosomes was significantly higher in the CD137-activated group than in the control group (P<0.05), which was significantly lower in the Rab7 lentiviral interference group than in the CD137-activation group (P<0.05) and was similar between the lentivirus control group and the CD137 activation group (P>0.05). The expression of Hsc70 protein in exosomes secreted by CD137 activation group was higher than that in the control group (P<0.05). The expression of Hsc70 protein in exosomes was lower in Rab7 lentivirus interference group than in the CD137 activation group (P<0.05), which was similar between the lentivirus control group and the CD137 activation group (P>0.05). The expression of LC3Ⅱ protein in exosome was higher in CD137 activation group than in control group (P<0.05), which was lower in Rab7 lentivirus interference group than in CD137 activation group (P<0.05), which was similar between the lentivirus control group and the CD137 activation group (P>0.05). Conclusion: The CD137-CD137L signaling may affect the secretion of mouse VSMCs-derived exosomes through modulating the Rab7 pathway mediated autophagy.

LncRNA OSER1-AS1 acts as a ceRNA to promote tumorigenesis in hepatocellular carcinoma by regulating miR-372-3p/Rab23 axis

Long non-coding RNAs (lncRNAs) are crucial regulators of tumorigenesis and progression in human cancer, including hepatocellular carcinoma (HCC). However, the role of most lncRNAs that are dysregulated in HCC remains to be elucidated. Here, we investigated the role of OSER1-AS1 in the progression of HCC. The results of database and qRT-PCR analysis demonstrated that OSER1-AS1 was highly expressed in HCC tissues and the high expression of OSER1-AS1 was closely associated with larger tumor size, advanced tumor stages, lower disease free survival and overall survival of HCC patients. OSER1-AS1 knockdown significantly inhibited the proliferation, invasion and migration of HCC cells, and induced the apoptosis. In addition, the dual luciferase reporter assay directly demonstrated that OSER1-AS1 functioned as a molecular sponge for miR-372-3p to promote Rab23 expression. Moreover, the results of immunohistochemistry and western blot analysis showed that Rab23 was highly expressed in HCC tissues, and the high expression of Rab23 was closely associated with the poor overall survival of HCC patients. Immunofluorescence assay also found the subcellular localization of Rab23 in HCC cells. Rab23 was obviously downregulated in cells that were transfected with miR-372-3p mimics. MiR-372-3p mimics significantly inhibited the proliferation and invasion of HCC cells). Rab23 restoration partially reversed miR-372-3p-induced tumor suppressive effects on HCC cells. In conclusion, we found that OSER1-AS1 acted as a ceRNA to sponge miR-372-3p, thereby positively regulating the Rab23 expression and ultimately acting as a tumor suppressor gene in HCC progression.

Long Non-Coding RNA LINC00152 Regulates Cell Proliferation, Migration And Invasion In Esophageal Squamous Cell Carcinoma Via miR-107/Rab10 Axis.

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common malignant tumor in East Asia. Emerging evidence indicated that long intergenic non-coding RNA 152 (LINC00152) acts as an oncogene in many types of cancers including ESCC. This study aims to identify the biological mechanisms of LINC00152 in ESCC, hinting for new therapeutic target for ESCC patients.MethodsThe levels of LINC00152, microRNA-107 (miR-107) and Ras-related protein Rab-10 (Rab10) were measured in ESCC tissues and cells using qRT-PCR. The protein level of Rab10 was measured by Western blot assay. The putative target of LINC00152 or miR-107 was searched using starBase v2.0 and TargetScan online databases, and dual-luciferase reporter assay was conducted to detect the interaction between miR-107 and LINC00152 or Rab10. The cell viability was monitored by CCK8 assay, and the abilities of migration and invasion were assessed by Transwell assay, respectively. The mice model experiments were constructed to affirm the biological role of LINC00152 in vivo.ResultsLINC00152, Rab10 was significantly upregulated, and miR-107 was strikingly down-regulated in ESCC tissues and cell lines (TE-1 and KYSE30). LINC00152 was verified as a sponge for miR-107, and Rab10 was a direct target of miR-107. LINC00152 depletion decreased cell viability and abilities of migration and invasion by regulating miR-107 in vitro and blocked xenograft tumor growth in vivo. The overexpression of miR-107 reduced cell viability and the abilities of migration and invasion by modulating Rab10. LINC00152 positively regulated Rab10 expression by sponging miR-107.ConclusionIn this study, we found that LINC00152 modulated Rab10 to promote cell proliferation, migration and invasion in ESCC by sponging miR-107. This new regulatory network may provide a novel therapeutic target for ESCC patients.

Chronic sleep fragmentation shares similar pathogenesis with neurodegenerative diseases: Endosome-autophagosome-lysosome pathway dysfunction and microglia-mediated neuroinflammation.

AimsInsufficient sleep has been found to result in varying degrees of cognitive impairment and emotional changes. Sleep was reported probably responsible for cleaning metabolic wastes in brain by increasing extracellular bulk flow. Herein, we propose that chronic sleep insufficiency in young adult wild‐type mice is also linked with dysfunction of intracellular protein degradation pathways and microglia‐mediated neuroinflammation, which are potentially important mechanisms in the initiation of neurodegeneration.MethodsWe applied the chronic sleep fragmentation (CSF) model to induce chronic sleep insufficiency in wild‐type mice. After 2 months of CSF, cognitive function, amyloid‐β accumulation, dysfunction of endosome‐autophagosome‐lysosome pathway, and microglia activation were evaluated.ResultsFollowing CSF, impairment of spatial learning and memory, and aggravated anxiety‐like behavior in mice were identified by behavioral experiments. Increased intracellular amyloid‐β accumulation was observed in cortex and hippocampus. Mechanistically, CSF could significantly enhance the expression of Rab5 (early endosome marker), Rab7 (late endosome marker), as well as LC3B (autophagosome marker), and autophagy‐positive regulatory factors in brain detected by immunofluorescent staining and Western blot. In addition, activation of microglia was evident by enhanced CD68, CD16/32, and CD206 levels after CSF treatment.ConclusionsChronic sleep fragmentation could initiate pathogenetic processes similar to the early stage of neurodegeneration, including dysfunction of endosome‐autophagosome‐lysosome pathway and microglia‐mediated neuroinflammation. Our findings further strengthen the link between chronic sleep insufficiency and the initiation of neurodegeneration even if lack of genetic predisposition.

RETRACTED: Downregulation of Rab23 inhibits proliferation, invasion, and metastasis of human ovarian cancer

Previously, we reported that the expression of human epididymis protein (HE4) was correlated with the expression of RAB23 in ovarian cancer cells. Rab23 is a member of the Ras-related small GTPase superfamily, which plays a key role in the Sonic Hedgehog (Shh) signaling pathway. However, the function of Rab23 in ovarian cancer remains unclear. In this study, we explored the location and expression of Rab23 in ovarian cancer tissues and cells (CaoV3 and A2780), and further investigated the function and potential mechanism of Rab23 in malignant biological behaviors including the epithelial-mesenchymal transition (EMT) process in ovarian cancer for the first time. Rab23 is highly expressed in ovarian cancer tissues and associated with advanced stage, and shortened overall survival time of ovarian cancer patients. We are the first to report that human epididymis protein (HE4) can regulate the expression of the Rab23 protein, and that knockdown of RAB23 decreases the proliferation, invasion, and migration abilities as well as inhibits the epithelial-mesenchymal transition (EMT) process in ovarian cancer cells. Furthermore, downregulation of Rab23 significantly inhibited Shh-Gli1 and PI3K-AKT signaling pathways. Collectively, our results indicate that Rab23 plays a critical role in the malignant biological behavior of ovarian cancer and may serve as a potential biomarker and therapeutic target for ovarian cancer.

Tumor-Derived Extracellular Vesicles Breach the Intact Blood-Brain Barrier via Transcytosis.

The restrictive nature of the blood-brain barrier (BBB) creates a major challenge for brain drug delivery with current nanomedicines lacking the ability to cross the BBB. Extracellular vesicles (EVs) have been shown to contribute to the progression of a variety of brain diseases including metastatic brain cancer and have been suggested as promising therapeutics and drug delivery vehicles. However, the ability of native tumor-derived EVs to breach the BBB and the mechanism(s) involved in this process remain unknown. Here, we demonstrate that tumor-derived EVs can breach the intact BBB in vivo, and by using state-of-the-art in vitro and in vivo models of the BBB, we have identified transcytosis as the mechanism underlying this process. Moreover, high spatiotemporal resolution microscopy demonstrated that the endothelial recycling endocytic pathway is involved in this transcellular transport. We further identify and characterize the mechanism by which tumor-derived EVs circumvent the low physiologic rate of transcytosis in the BBB by decreasing the brain endothelial expression of rab7 and increasing the efficiency of their transport. These findings identify previously unknown mechanisms by which tumor-derived EVs breach an intact BBB during the course of brain metastasis and can be leveraged to guide and inform the development of drug delivery approaches to deliver therapeutic cargoes across the BBB for treatment of a variety of brain diseases including, but not limited to, brain malignancies.

Effect of Autophagy Regulated by Sirt1/FoxO1 Pathway on the Release of Factors Promoting Thrombosis from Vascular Endothelial Cells.

Factors promoting thrombosis such as von Willebrand factor (vWF) and P-selectin are essential for the development of atherosclerosis (AS) and arterial thrombosis. The processing, maturation and release of vWF are regulated by autophagy of vascular endothelial cells. The Sirt1/FoxO1 pathway is an important pathway to regulate autophagy of endothelial cells, therefore the Sirt1/FoxO1 pathway may be an important target for the prevention of thrombosis. We investigated the role of ox-LDL in the release of vWF and P-selectin and the expression of Sirt1 and FoxO1 by Western Blot, Flow Cytometry, ELISA, and tandem fluorescent mRFP-GFP-LC3. We found that vWF and P-selectin secretion increased and Sirt1/FoxO1 pathway was depressed in human umbilical vein endothelial cells (HUVEC) when treated with ox-LDL. Moreover, the expression of autophagy-related protein LC3-II/I and p62 increased. Then, we explored the relationship between autophagy regulated by the Sirt1/FoxO1 pathway and the secretion of vWF and P-selectin. We found that Sirt1/FoxO1, activated by the Sirt1 activators resveratrol (RSV) and SRT1720, decreased the secretion of vWF and P-selectin, which can be abolished by the autophagy inhibitor 3-MA. The expression of Rab7 increased when Sirt1/FoxO1 pathway was activated, and the accumulation of p62 was decreased. Autophagy flux was inhibited by ox-LDL and Sirt1/FoxO1 pathway might enhance autophagy flux through the promotion of the Rab7 expression. Taken together, our data suggest that by enhancing autophagy flux and decreasing the release of vWF and P-selectin, the Sirt1/FoxO1 pathway may be a promising target to prevent AS and arterial thrombosis.

Circular RNA MYLK promotes hepatocellular carcinoma progression by increasing Rab23 expression by sponging miR-362-3p

BackgroundCircRNA myosin light chain kinase (circRNA MYLK) has been shown to promote the progression of various tumor diseases. The purpose of this study was to explore the potential molecular mechanism of circMYLK in hepatocellular carcinoma (HCC).MethodsThe quantitative Real-Time PCR (qRT-PCR) was used to measure the expressions of circMYLK, miR-362-3p and Rab23 in HCC tissues and cell lines. Huh7 and Hep3B cells were selected to explore the role of circMYLK in proliferation, invasion and migration of HCC cells in vitro. The interaction among circMYLK, miR-362-3p and Rab23 was investigated by biological information and dual luciferase gene reporter assay. The effect of circMYLK on HCC tumor growth in vivo was studied in a tumor xenograft model in mice.ResultsCircMYLK was highly expressed in HCC tissues and cell lines, which was associated with poor prognosis in HCC patients. In addition, knockdown of circMYLK remarkably inhibited the proliferation, invasion, and migration of Huh7 and Hep3B cells. MiR-362-3p was a direct target of circMYLK, and Rab23 was a direct target gene of miR-362-3p. Meanwhile, circMYLK was negatively correlated with the expression of miR-362-3p and positively correlated with Rab23 expression. Moreover, either overexpressed miR-362-3p or silencing Rab23 could observably suppress the enhanced proliferation, invasion, and migration induced by circMYLK in Huh7 and Hep3B cells. Finally, knockdown of circMYLK and overexpressed miR-362-3p could suppress the expression of Rab23, thus inhibiting the growth and proliferation of Hep3B cells in vivo.ConclusioncircMYLK promotes the occurrence and development of HCC by regulating the miR-362-3p/Rab23 axis, which provides a novel direction and theoretical basis for the early diagnosis and treatment of HCC.

Focal adhesion kinase–dependent activation of the early endocytic protein Rab5 is associated with cell migration

Focal adhesion kinase (FAK) is a central regulator of integrin-dependent cell adhesion and migration and has recently been shown to co-localize with endosomal proteins. The early endocytic protein Rab5 controls integrin trafficking, focal adhesion disassembly, and cell migration and has been shown to be activated upon integrin engagement by mechanisms that remain unclear. Because FAK is a critical regulator of integrin-dependent signaling and Rab5 recapitulates FAK-mediated effects, we evaluated the possibility that FAK activates Rab5 and contributes to cell migration. Pulldown assays revealed that Rab5-GTP levels are decreased upon treatment with a pharmacological inhibitor of FAK, PF562,271, in resting A549 cells. These events were associated with decreased peripheral Rab5 puncta and a reduced number of early endosome antigen 1 (EEA1)-positive early endosomes. Accordingly, as indicated by FAK inhibition experiments and in FAK-null fibroblasts, adhesion-induced FAK activity increased Rab5-GTP levels. In fact, expression of WT FAK and FAK/Y180A/M183A (open conformation), but not FAK/Arg454 (kinase-dead), augmented Rab5-GTP levels in FAK-null fibroblasts and A549 cells. Moreover, expression of a GDP-bound Rab5 mutant (Rab5/S34N) or shRNA-mediated knockdown of endogenous Rab5 prevented FAK-induced A549 cell migration, whereas expression of WT or GTP-bound Rab5 (Rab5/Q79L), but not Rab5/S34N, promoted cell migration in FAK-null fibroblasts. Mechanistically, FAK co-immunoprecipitated with the GTPase-activating protein p85α in a phosphorylation (Tyr397)-dependent manner, preventing Rab5-GTP loading, as shown by knockdown and transfection recovery experiments. Taken together, these results reveal that FAK activates Rab5, leading to cell migration.

Rab23 contributes to the progression of colorectal cancer via protein kinase B and extracellular signal-regulated kinase signaling pathways.

The role of Ras-related protein Rab23 in tumors has attracted increasing attention in recent years; however, whether it can function as an oncogenic protein remains under debate, and its role in colorectal cancer (CRC) is currently unknown. In the present study, high expression of Rab23 in CRC tissues was confirmed using immunohistochemistry, and high expression of Rab23 in CRC cells (SW1116 and HT29) was confirmed using reverse transcription-polymerase chain reaction and western blot analysis. A positive association of Rab23 with tumor size and advanced clinical stage was confirmed by χ2 analysis. In addition, the positive association of Rab23 with poor disease-free survival was confirmed by survival analysis. Cell experiments further demonstrated that overexpression of Rab23 increased the expression of the proliferation marker Ki-67 and the proliferative ability in SW1116 and HT29 cells. Molecular mechanism research revealed that the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways contributed to the high expression of Ki-67 and increased the proliferative ability induced by Rab23 in CRC cells. In conclusion, the study confirmed the high expression of Rab23 in CRC, and its positive association with CRC progression and poor prognosis. Furthermore, the data demonstrated that Rab23 increased the proliferation of CRC cells via the ERK and AKT signaling pathways. These results suggest that Rab23 may be used as a protein for diagnosis and prognosis prediction in patients with CRC, and is proposed to be a novel therapeutic target for improving the patient outcome.

Rab7 delays intervertebral disc degeneration through the inhibition of the p38MAPK pathway

The objective of this study is to determine whether Rab7 could delay intervertebral disc degeneration and study the mechanism. We chose old mice to established intervertebral disc degeneration model and study the effect of Rab7 on intervertebral disc by western blotting, quantitative real-time PCR, immunohistochemistry et al. Nucleus pulposus cells were cultured to study the exact mechanism. Expression of Rab7 was decreased in degenerative human nucleus pulposus tissues. With the aging of mice, the degree of intervertebral disc degeneration was aggravated and the expression of Rab7 was decreased. In addition, overexpression of Rab7 can reduce the degeneration of nucleus pulposus cells and reduce the levels of p38 and p-p38. Rab7 is a protective factor for intervertebral discs and could delays intervertebral disc degeneration through the inhibition of the p38MAPK pathway.

AUTOPHAGY REGULATION BY RUBELLA VIRUS

Aim. Some viruses can subvert host defense mechanism, autophagy, to their own benefit. We analysed the effect of Rubella virus (RV) infection on autophagy in human alveolar epithelial cells A549. Materials and methods. Cells were infected with the wild type and lab-attenuated strain, C-77w and C-77a, respectively, with a multiplicity of infection of 1.0, in parallel, the expression level of genes encoding Beclin1, Atg5, Rab7, and p62 (SQSTM1) proteins participating in different steps of autolysosome formation was measured. To investigate the role of autophagy on RV replication cycle, we measured the amount of infectious RV particles, together with the viral RNA in supernatants and cell lysates, after incubation of A549 cells with wild type or attenuated strain in the presence of the autophagy inhibitor, Bafilomycin A1, or inducer, Rapamycin. Results. The significant increase in Beclin1 and Atg5 gene expression at 24-48 (for the wild type) and 24-72 (for the attenuated type) hours after infection was observed, while significant induction of either Rab7 or SQSTM1 gene expression was not noticed. This effect was correlated with more delayed increase of IFNβ expression and IFNβ-mediated pro-apoptotic gene expression leading to apoptotic cell death 72-96 hours after infection. Moreover, Bafilomycin A1 diminished the RV infection non significantly, as evidenced by the RT-qPCR and plaque assay, while Rapamycin increased the amount of infectious RV particles released by the infected cells more dramatically with wild type comparing with attenuated strain. Conclusion. Thus, we hypothesized that RV can use an antiviral mechanism to prevent degradation and ensure its replication, differentially regulating the process of autophagy, by stimulating the initiation and suppression of later steps.

Autophagy protects murine macrophages from β-cypermethrin-induced mitochondrial dysfunction and cytotoxicity via the reduction of oxidation stress

The immunotoxicity of synthetic pyrethroid (SPs) has garnered much attention, and our previous research demonstrated that β-CYP causes immunotoxicity and oxidative stress in macrophages. Nevertheless, the underlying mechanism remains largely unknown. In this study, the murine macrophage RAW 264.7 cells and murine peritoneal macrophages (PMs) were exposed to β-CYP. The results showed that β-CYP elevated intracellular ROS levels in both RAW 264.7 cells and PMs. Exposure to β-CYP also caused mitochondrial dysfunction with reduced mitochondrial membrane potential (MMP), intracellular ATP level and mitochondrial DNA (mtDNA) content in the two cell types. In addition, exposure of RAW 264.7 cells to β-CYP for 12 h and 24 h enhanced autophagy, with elevated Beclin1, Rab7, Lamp1 and LC3-II expression levels, while 48 h of exposure attenuated autophagy. In contrast, exposure of PMs to β-CYP for 12 h promoted autophagy, whereas exposure for 24 h and 48 h impaired autophagy. Cotreatment with an antioxidant, N-acetyl-L-cysteine (NAC), partially blocked the reduced MMP, intracellular ATP level and autophagy disturbance. Moreover, cotreatment with an autophagy agonist, rapamycin (RAPA), partially blocked mitochondrial dysfunction and oxidative stress in the two cell types, whereas cotreatment with an autophagy inhibitor, 3-methyladenine (3-MA), augmented the abovementioned toxic effects. Furthermore, mitochondrial ROS levels in both RAW 264.7 cells and PMs were elevated by exposure to β-CYP, and molecular docking showed that β-CYP docked with mouse respiratory chain complex I by binding to the ND2, ND4, and ND5 subunits of the protein complex. Taken together, the data obtained in the present study demonstrate that oxidative stress partially mediates mitochondrial dysfunction and autophagy disturbance upon exposure to β-CYP in macrophages, and autophagy plays a protective role against the toxic effects.

LncRNA-SNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR-338-3p.

The incidence and death rate of colorectal cancer (CRC) is very high, which brings great need to understand the early molecular events of CRC. These studies demonstrate that long noncoding RNA (lncRNA) plays an important role in the occurrence and development of human cancer. Small nucleolar RNA host gene 15 (SNHG15) was recently identified as a cancer‐related lncRNA. In this study, we aimed to evaluate the function and mechanism of SNHG15 in CRC. The expression of SNHG15 was detected by quantitative RT‐PCR (qRT‐PCR) in CRC tissues and matched noncancerous tissues (NCTs). CCK‐8 assay, colony formation assay, flow cytometric analysis, and nude mouse xenograft mode were used to examine the tumor‐promoting function of SNHG15 in vitro and in vivo. The binding relationship between SNHG15, miR‐338‐3p and the target genes of miR‐338‐3p were screened and identified by databases, qRT‐PCR, dual luciferase reporter assay and western blot. Our results showed that SNHG15 was up‐regulated in CRC tissues compared with paired NCTs (P < 0.0001). High level of SNHG15 expression predicted poor prognosis of CRC (P = 0.0051). SNHG15 overexpression could promote cell proliferation and inhibit cell apoptosis. Animal experiments showed that up‐regulation of SNHG15 promoted tumor growth in vivo. The results of mechanism experiments showed that SNHG15 could bind to miR‐338‐3p and block its inhibition on the expression and activity of FOS or RAB14. In conclusion SNHG15 promotes cell proliferation through SNHG15/miR‐338‐3p/FOS‐RAB14 axis in CRC.

Electroacupuncture intervention relieves pain possibly by promoting MOR endocytosis in locus coeruleus in bone cancer pain rats with morphine tolerance

To observe the effect of electroacupuncture (EA) on pain behavior and expression of µ-opioid receptor (MOR) and Rab5 (an important protein molecule for internalization of MOR) in the locus coeruleus (LC) region in bone cancer pain (BCP) rats with morphine tolerance (MT), so as to explore its mechanisms underlying improvement of BCP and MT. The present study included two parts. In the first part, 23 female SD rats were randomized into sham BCP (n＝6), BCP (n＝9) and BCP＋MT (n＝8) groups, and in the second part, 61 female SD rats were randomized into 5 groups: sham BCP (n＝11), BCP (n＝11), BCP＋MT (n＝13), BCP＋MT＋EA (n＝13) and BCP＋MT＋sham EA (n＝13). The BCP morphine tolerance (BCP＋MT) model was established by injection of 10 µL of human Walker 256 breast cancer cells (MRMT-1 breast cancer cells, 1 x104 cells/µL) into the bone marrow cavity at the upper part of the left tibia and intraperitoneal injection of morphine hydrochloride (10 mg/kg, once per 12 h, for 11 successive days). On day 21 after inoculation, EA (2 Hz/100 Hz, 0.5－1.5 mA, increasing 0.5 mA every 10 min) was began to applied to bilateral "Zusanli" (ST30) and "Kunlun" (BL60) immediately after the first intraperitoneal injection of morphine. The treatment was performed for 30 min every time, once daily for 7 successive days. The paw withdrawal threshold (PWT) was detected before and 10, 11, 21, 22, 24, 26 and 28 days after inoculation. The immunoactivity of MOR and Rab5 proteins in the LC region was detected by immunofluorescence histochemistry. In the first part of the study, at the 10th day after inoculation of cancer cells, the PWT of the BCP and BCP＋MT groups was significantly lower than that of the sham BCP group (P<0.05), suggesting a success of BCP model. From day 11 to 19 after inoculation (during injection of morphine), the PWTs of the BCP＋MT group were significantly higher than those of the BCP group (P<0.01), and on day 21, the PWT of the BCP＋MT group was similar to that of the BCP group (P>0.05) but significantly lower than that of the sham BCP group (P<0.01), suggesting a success of MT. H．E. staining showed a large quantity of MRMT-1 cancer cells in the bone marrow cavity in both BCP and BCP＋MT groups. In the second part of the study, the decreased PWTs from 10th to 28th day after inoculation were significantly increased on day 22, 24, 26 and 28 in the BCP＋MT＋EA group relevant to the BCP, BCP＋MT and BCP＋MT＋sham EA groups (P<0.01). The ratios of MOR and Rab5 positive (＋) cells and MOR＋/Rab5＋ of the left LC region were significantly lower in the BCP and BCP＋MT groups than those of the sham BCP group (P<0.01), but were considerably higher in the BCP＋MT＋EA group than those in the BCP, BCP＋MT and BCP＋MT＋sham EA groups (P<0.01). The ratios of Rab5＋ and MOR＋/Rab5＋ cells of the BCP＋MT group were significantly lower than those of the BCP group (P<0.05). No significant changes were found in the ratios of MOR＋ and Rab5＋ cells and MOR＋/Rab5＋ cells after BCP＋MT＋sham EA in comparison with the BCP＋MT group (P>0.05). EA intervention can relieve pain and MT in bone cancer pain rats with MT, which may be related to its effects in increasing MOR expression and promoting endocytosis of MOR in LC region.

Rab25 promotes erlotinib resistance by activating the β1 integrin/AKT/β-catenin pathway in NSCLC.

ObjectivesEpidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI) has significant therapeutic efficacy in non‐small‐cell lung cancer (NSCLC) patients. However, acquired resistance is inevitable and limits the long‐term efficacy of EGFR‐TKI. Our study aimed to investigate the role of ras‐associated binding protein 25 (Rab25) in mediating EGFR‐TKI resistance in NSCLC.Materials and MethodsRab25 expression in NSCLC patients was measured by immunohistochemical staining. Western blotting was used to analyse the expression of molecules in the Rab25, EGFR and Wnt signalling pathways. Lentiviral vectors were constructed to knock in and knock out Rab25. The biological function of Rab25 was demonstrated by cell‐counting kit‐8 and flow cytometry. The interaction between Rab25 and β1 integrin was confirmed by co‐immunoprecipitation.ResultsRab25 overexpression induced erlotinib resistance, whereas Rab25 knockdown reversed this refractoriness in vitro and in vivo. Moreover, Rab25 interacts with β1 integrin and promotes its trafficking to the cytoplasmic membrane. The membrane‐β1 integrin induced protein kinase B (AKT) phosphorylation and subsequently activated the Wnt/β‐catenin signalling pathway, promoting cell proliferation. Furthermore, high Rab25 expression was associated with poor response to EGFR‐TKI treatment in NSCLC patients.ConclusionsRab25 mediates erlotinib resistance by activating the β1 integrin/AKT/β‐catenin signalling pathway. Rab25 may be a predictive biomarker and has potential therapeutic value in NSCLC patients with acquired resistance to EGFR‐TKI.

Chaperone-Mediated Autophagy Upregulation Rescues Megalin Expression and Localization in Cystinotic Proximal Tubule Cells.

Cystinosis is a lysosomal storage disorder caused by defects in CTNS, the gene that encodes the lysosomal cystine transporter cystinosin. Patients with nephropathic cystinosis are characterized by endocrine defects, defective proximal tubule cell (PTC) function, the development of Fanconi syndrome and, eventually, end-stage renal disease. Kidney disease is developed despite the use of cysteamine, a drug that decreases lysosomal cystine overload but fails to correct overload-independent defects. Chaperone-mediated autophagy (CMA), a selective form of autophagy, is defective in cystinotic mouse fibroblasts, and treatment with cysteamine is unable to correct CMA defects in vivo, but whether the vesicular trafficking mechanisms that lead to defective CMA in cystinosis are manifested in human PTCs is not currently known and whether PTC-specific mechanisms are corrected upon CMA upregulation remains to be elucidated. Here, using CRISPR-Cas9 technology, we develop a new human PTC line with defective cystinosin expression (CTNS-KO PTCs). We show that the expression and localization of the CMA receptor, LAMP2A, is defective in CTNS-KO PTCs. The expression of the lipidated form of LC3B, a marker for another form of autophagy (macroautophagy), is decreased in CTNS-KO PTCs indicating decreased autophagosome numbers under basal conditions. However, the autophagic flux is functional, as measured by induction by starvation or by blockage using the v-ATPase inhibitor bafilomycin A, and by degradation of the macroautophagy substrate SQSTM1 under starvation and proteasome-inhibited conditions. Previous studies showed that LAMP2A accumulates in Rab11-positive vesicles in cystinotic cells. Here, we show defective Rab11 expression, localization and trafficking in CTNS-KO PTCs as determined by confocal microscopy, immunoblotting and TIRFM. We also show that both Rab11 expression and trafficking in cystinotic PTCs are rescued by the upregulation of CMA using small-molecule CMA activators. Cystinotic PTCs are characterized by PTC de-differentiation accompanied by loss of the endocytic receptor megalin, and megalin recycling is regulated by Rab11. Here we show that megalin plasma membrane localization is defective in CTNS-KO PTCs and its expression is rescued by treatment with CMA activators. Altogether, our data support that CMA upregulation has the potential to improve PTC function in cystinosis.

The Role of Beclin-1 Acetylation on Autophagic Flux in Alzheimer's Disease.

Macroautophagy impairment plays a key role in sporadic Alzheimer's disease (sAD) neurodegenerative process. Nevertheless, the mechanism(s) that lead to a deficiency in macroautophagy in AD remains elusive. In this work, we identify, for the first time that Beclin-1 acetylation status is implicated in the alterations in autophagy observed in AD neurodegeneration. We observed that Beclin-1 is deacetylated by sirtuin 1 (SIRT1) and acetylated by p300. In addition, Beclin-1 acetylation inhibits autophagosome maturation, leading to impairment in autophagic flux. We also analyzed some proteins known to be involved in the maturation of autophagosomes such as Rab7, which participates in the fusion step with lysosomes. We observed that increased expression of Rab7 might represent a response to boost the formation of large perinuclear lysosome clusters in accordance with an increase in lysosomal biogenesis determined by increase in LAMP-2A, LAMP-1, and cathepsin D expression in AD cells. Thus, our data provides strong evidences that Beclin-1 acetylation impairs the autophagic flux, and despite lysosomal biogenesis seems to be triggered as a compensatory response, autophagosome fusion with lysosomes is compromised contributing to AD neurodegeneration.

Autophagy Activation Improves Lung Injury and Inflammation in Sepsis.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) undergoes the process of pathological event including lung tissue dysfunction, pulmonary edema, and inflammation in sepsis. Autophagy is a cytoprotective process recognized as one of the major pathways for degradation and recycling of cellular constituents. Autophagy as a protective or maladaptive response was still confused in ALI during sepsis. Acute lung injury was performed by cecal ligation and puncture (CLP). Autophagic inducer rapacymin and inhibitor 3-MA and autophagosomal-lysosome fusion inhibitor bafilomucin (Baf) A1 and chloroquine (CQ) were administrated by intraperitoneal injection at 1h after CLP operation. Microtubule-associated protein light chain 3 II (LC3II), Beclin 1, Rab7, and lysosome-associated membrane protein type 2 (LAMP2) were detected by western blotting. Seven-day survival rate of septic mice was observed. Histologic scores, lung wet-to-dry (W/D) weight ratio, oxygenation index (PaO2/FiO2), total cells and polymorphonuclear neutrophils (PMN) in bronchial alveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity and cytokine tumor necrosis factor (TNF)-α, high-mobility group box (HMGB)1, interleukin (IL)-6, IL-10, and monocyte chemotactic protein (MCP)1 were measured after sham or ALI operation. ALI induced the increasing expression of autophagy-related protein LC3II, Beclin 1, Rab7, and LAMP2 in CLP operation. Autophagic inducer rapacymin significantly induced the expression of LC3II, Beclin 1, LAMP2, and Rab7 in mice model of CLP, and inhibitor 3-MA reduced expression of LC3II, Beclin 1, LAMP2, and Rab7 expressions in CLP + RAP mice compared to CLP group. Compared with ALI group, Baf and CQ obviously elevated the level of LC3II and Beclin 1, and reduced the LAMP2 and Rab7 expressions in CLP + Baf group and ALI + CQ group. Compared with CLP group, autophagic inducer rapacymin improved the survival rate, histologic scores, lung wet/dry weight ratio, PaO2/FiO2, total cells, and PMNS in BALF and MPO activity and cytokines TNF-α, HMGB1, IL-6, IL-10, and MCP1 in CLP + RAP group, but there were exacerbated above indicators in CLP + 3-MA group, CLP + Baf group, and CLP + CQ group. Autophagy activation participated in the pathophysiologic process of sepsis, and alleviated the cytokine excessive release and lung injury in sepsis.

RAB31-Mediated Endosomal Trafficking Is Defective in RUNX1 Haplodeficiency

RAB GTPases are key players in vesicle trafficking, granule targeting of proteins, granule biogenesis and secretion. RAB31 (Ras-related protein 31) has been implicated in the regulation of vesicular trafficking between the Golgi/TGN and endosomes, and from early endosome (EE) to late endosome (LE). Studies in neuronal cells have implicated RAB31 in the transport of epidermal growth factor receptor (EGFR) from EE to LE. RUNX1, a major hematopoietic transcription factor, plays a critical role in megakaryocyte (MK) maturation and platelet production. Patients with RUNX1 haplodeficiency have thrombocytopenia, abnormal platelet function, and impaired granule contents and secretion. Our studies in a patient with a heterozygous RUNX1 mutation (c.969-323G>T ) (Sun et al, Blood 103: 948-54, 2004) associated with thrombocytopenia, platelet dysfunction, granule deficiency and impaired platelet responses revealed decreased platelet expression of RAB31. The role of RAB31 in MK and platelets is unknown. We addressed the hypothesis that RAB31 is a transcriptional target of RUNX1. We studied the role of RAB31 on vesicle transport in MK cells in human erythroleukemia (HEL) cells transformed into megakaryocytic cells by PMA (phorbol 12-myristate 13-acetate). RAB31 mRNA was decreased on platelet expression profiling of the patient (fold change: 0.28, p<0.0076, Sun et al J Thromb Haemost 5: 146-154). With real-time PCR platelet platelet RAB31 mRNA was decreased compared to 5 healthy controls by 60-80% in our patient (P1) and in two additional unrelated patients (siblings, P2 and P3) with RUNX1 mutation (c.508+1G>A). Platelet RAB31 protein was decreased compared to that in 5 healthy controls in patients P2 and P3. RAB31 promoter region (-2023/-1bp from the ATG) revealed 4 RUNX1 consensus sites: site I (-813/-808), site II (-972/-967), site III (-1500/-1495) and site IV (-2007/-2002). Chromatin immunoprecipitation (ChIP) revealed RUNX1 binding to RAB31 sites II and IV but not to other sites. Electrophoretic mobility shift assays using HEL cell proteins showed RUNX1 binding to sites II and IV. In luciferase reporter assays, mutation of individual sites II and IV decreased promoter activity indicating that they are functional sites. RAB31 promoter activity and protein expression were inhibited by RUNX1 siRNA and enhanced by RUNX1 overexpression. These indicate that RAB31 is a direct RUNX1 target, providing a mechanism for decreased RAB31 in patient platelets. We investigated putative roles of RUNX1 and its target RAB31 in endosomal dynamics in PMA treated HEL cells. We used immunofluorescent staining for markers of early endosomes (EE, EEA1) and late endosomes/multivesicular bodies (LE/MVB, CD63) in RUNX1- or RAB31-depleted cells by siRNA transfection. Either RUNX1 or RAB31 siRNA yielded a striking enlargement of early endosomes, as indicated by the EE marker EEA1. This finding suggests a role for RUNX1/RAB31 in EE maturation, either by mediating vesicle fission, or maturation to late endosomes by fusion with other endosomal vesicles. This effect of RUNX1 knockdown on EE enlargement was partially reversed by reconstitution of RAB31 by plasmid co-transfection, indicating that RAB31 is a significant but non-exclusive contributor to this RUNX1 function.Conclusions: These studies provide the first evidence that RAB31 is a direct transcriptional target of RUNX1 and a mechanism for RAB31 downregulation in RUNX1 haplodeficient patients. Downregulation of RAB31 or RUNX1 results in impaired endosomal maturation/trafficking, and this may contribute to the defective handling of α-granule proteins recognized in patients with RUNX1 mutations. DisclosuresLambert:Sysmex: Consultancy; Rigel: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees; Educational Concepts in Medicine: Consultancy; CSL: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Summus: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Shionogi: Consultancy.