Augmentation of miR-202 in varicose veins modulates phenotypic transition of vascular smooth muscle cells by targeting proliferator–activated receptor-γ coactivator-1α

In varicose veins, vascular smooth muscle cells (VSMCs) often show abnormal proliferative and migratory rates and phenotypic transition. This study aimed to investigate whether microRNA (miR)-202 and its potential target, peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α), were involved in VSMC phenotypic transition. miR-202 expression was analyzed in varicose veins and in VSMCs conditioned with platelet-derived growth factor. The effect of miR-202 on cell proliferation and migration was assessed. Furthermore, contractile marker SM-22α, synthetic markers vimentin and collagen I, and PGC-1α were analyzed by Western blot analysis. The modulation of PGC-1α expression by miR-202 was also evaluated. In varicose veins and proliferative VSMCs, miR-202 expression was upregulated, with decreased SM-22α expression and increased vimentin and collagen I expression. Transfection with a miR-202 mimic induced VSMC proliferation and migration, whereas a miR-202 inhibitor reduced cell proliferation and migration. miR-202 mimic constrained luciferase activity in HEK293 cells that were cotransfected with the PGC-1α 3′-untranslated region (3′-UTR) but not those with mutated 3′-UTR. miR-202 suppressed PGC-1α protein expression, with no influence on its messenger RNA expression. PGC-1α mediated VSMC phenotypic transition and was correlated with reactive oxygen species production. In conclusion, miR-202 affects VSMC phenotypic transition by targeting PGC-1α expression, providing a novel target for varicose vein therapy.     

Methyl B12 protects PC12 cells against cytotoxicity induced by Aβ25−35

Alzheimer's disease (AD) is the most common aging-associated dementia. The population of AD patients is increasing as the world age grows. Currently, there is no cure for AD. Given that methyl vitamin B12 (methylcobalamin) deficiency is related to AD and Aβ-induced oxidative damage and that methylcobalamin can scavenge reactive oxygen species (ROS) by direct or indirect ways, we studied the effect of methylcobalamin on the cytotoxicity of Aβ. PC12 cells were chronically exposed (24 hours) to Aβ_25-35 (25 μM) to establish an AD cell model. The cells were pretreated with or without methylcobalamin (1-100 μM) to investigate the role of methylcobalamin. Cell viability and apoptosis were tested, followed by testing of mitochondrial damage, oxidative stress, and mitochondrial calcium concentration. We observed that methylcobalamin improved the cell viability by decreasing the ratio of apoptosis cells in this AD cell model. Further experiments suggested that methylcobalamin functioned as an antioxidant to scavenge ROS, reducing the endoplasmic reticulum-mitochondria calcium flux through IP3R, preventing mitochondria dysfunction, ultimately protecting cells against apoptosis and cell death. Taken together, our results presented, for the first time, that methyl vitamin B12 can protect cells from Aβ-induced cytotoxicity and the mechanism was mainly relevant to the antioxidative function of methyl B12.     

Mechanically stimulated osteocytes reduce the bone-metastatic potential of breast cancer cells in vitro by signaling through endothelial cells

Bone metastases occur in 65% to 75% of patients with advanced breast cancer and significantly worsen their survival and quality of life. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced the transendothelial migration of breast cancer cells. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. In this study, we investigated the specific effects of flow-stimulated osteocytes on the interaction between endothelial cells and breast cancer cells in vitro. We observed that CM from flow-stimulated osteocytes reduced endothelial permeability by 15% and breast cancer cell adhesion onto endothelial monolayers by 18%. The difference in adhesion was abolished with anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing antibodies. Furthermore, CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered the gene expression in bone-metastatic breast cancer cells, as shown by RNA sequencing. Specifically, breast cancer cell expression of matrix metallopeptidase 9 (MMP-9) was downregulated by 62%, and frizzled-4 (FZD4) by 61%, when the osteocytes were stimulated with flow. The invasion of these breast cancer cells across Matrigel was also reduced by 47%, and this difference was abolished by MMP-9 inhibitors. In conclusion, we demonstrated that flow-stimulated osteocytes downregulate the bone-metastatic potential of breast cancer cells by signaling through endothelial cells. This provides insights into the capability of bone mechanical regulation in preventing bone metastases; and may assist in prescribing exercise or bone-loading regimens to patients with breast cancers.     

Asymmetric dimethylarginine targets MAPK pathway to regulate insulin resistance in liver by activating inflammation factors

Insulin resistance is associated with impaired glucose uptake and altered protein kinase B (Akt) signaling. Previous studies have suggested asymmetric dimethylarginine (ADMA) and inflammation are two distinguish factors that correlate with insulin resistance (IR). How ADMA and inflammation factors interact and synchronize in the regulation of IR in liver remain to be elucidated. In this study, we systematically investigated whether ADMA is involved in IR using primary hepatocytes, if yes, by via which molecular mechanism. Our results demonstrated that ADMA inhibits insulin sensitivity in a concentration-dependent manner by activating inflammation factors tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6 in primary hepatocytes. Further analysis revealed that mitogen-activated protein kinase (MAPK) signaling pathway act downstream of ADMA and inflammation factors, and inhibition of MAPK pathway rescued the IR. Furthermore, metformin effects has been found which could reverse ADMA-induced IR by suppressing MAPK signaling pathway. To our knowledge, we, for the first time, unveiled the complicated regulatory network and interactions among ADMA, inflammation, and MAPK signaling pathway, which advanced current research on the development and regulation of IR in liver. This study also certainly provided novel insights on comprehensive diagonistics roles of ADMA as a potential biomarker.     

Rab6A GTPase contributes to phenotypic modulation in pulmonary artery smooth muscle cells under hypoxia

Previous studies have demonstrated that hypoxia can induce phenotypic modulation of pulmonary smooth muscle cells; however, the mechanisms remain unclear. The present study aimed to investigate the effect of the GTPase Rab6A-mediated phenotypic modulation and other activities of rat pulmonary artery smooth muscle cells (RPASMCs). We revealed that Rab6A was induced by hypoxia (1% O₂) and was involved in a hypoxia-induced phenotypic switch and endoplasmic reticulum stress (ERS) in RPASMCs. After 48 hours of hypoxia, the expression of the phenotype marker protein smooth muscle actin was downregulated and vimentin (VIM) expression was upregulated. Rab6A was upregulated after 48 hours of hypoxia, and the level of glucose-regulated protein, 78 kDa (GRP78) after 12 hours of hypoxic stimulation was also increased. After transfection with a Rab6A short interfering RNA under hypoxic conditions, the expression levels of GRP78 and VIM in RPASMCs were downregulated. Overall, hypoxia-induced RPASMCs to undergo ERS followed by phenotypic transformation. Rab6A is involved in this hypoxia-induced phenotypic modulation and ERS in RPASMCs.     

MiR-134-5p attenuates neuropathic pain progression through targeting Twist1

Neuropathic pain is a kind of chronic pain because of dysfunctions of somatosensory nerve system. Recently, many studies have demonstrated that microRNAs (miRs) play crucial roles in neuropathic pain development. This study was designed to investigate the effects of miR-134-5p on the process of neuropathic pain progression in a rat model established by chronic sciatic nerve injury (CCI). First, we observed that miR-134-5p was significantly decreased in CCI rat models. Overexpression of miR-134-5p strongly alleviated neuropathic pain behaviors including mechanical and thermal hyperalgesia. Meanwhile, inflammatory cytokine expression, such as IL-6, IL-1β and TNF-α in CCI rats were greatly repressed by upregulation of miR-134-5p. Twist1 has been widely regarded as a poor prognosis biomarker in diverse diseases. Here, by using bioinformatic analysis, 3′-untranslated region (UTR) of Twist1 was predicted to be a downstream target of miR-134-5p in our study. Here, we found that overexpression of miR-134-5p was able to suppress Twist1 dramatically. Furthermore, it was exhibited that Twist1 was increased in CCI rats time-dependently and Twist1 was inhibited in vivo. Subsequently, downregulation of Twist1 in CCI rats could depress neuropathic pain progression via inhibiting neuroinflammation. In conclusion, our current study indicated that miR-134-5p may inhibit neuropathic pain development through targeting Twist1. Our findings suggested that miR-134-5p might provide a novel therapeutic target for neuropathic pain.     

Suppressing ROS-TFE3-dependent autophagy enhances ivermectin-induced apoptosis in human melanoma cells

Melanoma is an aggressive skin malignancy with a high mortality rate; however, successful treatment remains a clinical challenge. Ivermectin, a broad-spectrum antiparasitic drug, has recently been characterized as a potential anticancer agent due to its observed antitumor effects. However, the molecular mechanisms of ivermectin remain poorly understood. In the current study, we tested the involvement of autophagy in the ivermectin mechanism of action in human melanoma cells. We exposed SK-MEL-28 cells to different concentrations of ivermectin (2.5, 5, and 10 μM) for 24 hours. Here, ivermectin-induced apoptosis, as evidenced by the upregulation of cleaved poly (ADP-ribose) polymerase, BAX expression, and caspase-3 activity and downregulation of BCL-2 expression. In line with the apoptosis response, ivermectin triggered autophagy. Pharmacological or genetic inhibition of autophagy further sensitized SK-MEL-28 cells to ivermectin-induced apoptosis. Mechanistically, ivermectin-induced TFE3^(Ser321) dephosphorylation, activated TFE3 nuclear translocation and increased TFE3 reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes, and subsequently, initiated autophagy in SK-MEL-28 cells. Moreover, N-acetyl-cysteine, an reactive oxygen species (ROS) scavenger, abrogated the effects of ivermectin on TFE3-dependent autophagy. Taken together, we demonstrated that ivermectin increases TFE3-dependent autophagy through ROS signaling pathways in human melanoma cells and that inhibiting autophagy enhances ivermectin-induced apoptosis in human melanoma cells.