Serum exosomal miR-210 as a potential biomarker for clear cell renal cell carcinoma

Exosomal microRNAs (miRNAs) are suggested to reflect molecular changes occurring in their cells of origin and are potential indicators in the early detection of cancers. This study aimed to determine whether certain exosomal miRNAs from tumor tissue can be used as noninvasive biomarkers for clear cell renal cell carcinoma (ccRCC). Based on ccRCC miRNA expression profiles and the literature, we selected six miRNAs (miR-210, miR-224, miR-452, miR-155, miR-21, and miR-34a) and analyzed their expression in tissues, sera, and serum exosomes through quantitative real-time polymerase chain reaction in hypoxia-induced (with CoCl₂) renal cell lines. miR-210, miR-224, miR-452, miR-155, and miR-21 were upregulated in tumor tissues compared with normal tissues. Serum miR-210 and miR-155 levels were higher in patients with ccRCC than in healthy controls (HCs). Furthermore, only exosomal miR-210 was significantly upregulated in patients with ccRCC than in HCs. Moreover, receiver operating characteristic (ROC) curve analysis revealed an area under the ROC curve of 0.8779 (95% confidence interval, 0.7987-0.9571) and a sensitivity and specificity of 82.5% and 80.0%, respectively. Moreover, exosomal miR-210 was upregulated at an advanced stage, and Fuhrman grade and metastasis decreased significantly one month after surgery. Acute hypoxia exposure activates miR-210 and release of exosomes with upregulated miR-210 in both normal and tumor RCC cell lines and interferes with vacuole membrane protein 1 mRNA expression, especially in the metastatic ccRCC cell line. In conclusion, Serum exosomal miR-210 originating from tumor tissue has potential as a novel noninvasive biomarker for the detection and prognosis of ccRCC.     

Isofraxidin attenuates IL-1β-induced inflammatory response in human nucleus pulposus cells

Inflammation has been demonstrated to be the key factor for intervertebral disc degeneration (IVD), which remains a major public health problem. Isofraxidin is a coumarin compound that possesses strong anti-inflammatory activity. However, the role of isofraxidin in IVD remains unclear. The aim of this study was to evaluate the effects of isofraxidin on inflammatory response in human nucleus pulposus cells (NPCs) exposed to interleukin-1β (IL-1β). The results proved that isofraxidin attenuated the IL-1β-induced significant increases in inflammatory mediators and cytokines including nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), and IL-6. Besides, isofraxidin also inhibited the induction effect of IL-1β on matrix metalloproteinases (MMP)-3 and MMP-13. Moreover, the NF-κB activation caused by IL-1β was significantly inhibited by isofraxidin treatment. These findings suggested that isofraxidin alleviates IL-1β-induced inflammation in NPCs. Our work provided an idea that isofraxidin might act as a novel preventive role in IVD.     

Gamma-secretase inhibitor suppressed Notch1 intracellular domain combination with p65 and resulted in the inhibition of the NF-κB signaling pathway induced by IL-1β and TNF-α in nucleus pulposus cells

In this experiment, the cross-talk betweenNotch and the NF-κB signaling pathway was examined to reveal the mechanism of slowing down the type II collagen (ColII) and aggrecan degeneration affected by inflammatory cytokines. The expression levels of ColII and aggrecan in the intervertebral disc were observed through immunohistochemistry and hematoxylin-eosin staining+alcian blue staining, respectively. The expression levels of ColII, aggrecan, Runx2, and NF-κB in the nuclei of human nucleus pulposus cells (hNPCs) in each group, as well as the phosphorylation and acetylation levels of p65, were examined through Western blot analysis. The 293T cells were transfected with a plasmid containing the overexpressed relative domain of Notch1 intracellular domain (NICD1), and immunoprecipitation (IP) was performed to observe the combination of NICD1 and p65. HNPCs were transfected with a lentiviral-contained overexpression lacking the ANK region of NICD1, and IP was performed to observe the combination of NICD1 and p65. The expression of ColII and aggrecan in the intervertebral disc culture increased when γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-Sphenylglycine t-butyl ester (DAPT) was added to the disc culture medium. Western blot revealed that DAPT inhibited p65 phosphorylation and acetylation, and the p65 and p50 levels in the nucleus decreased. NICD1 was found to be combined with p65 in contrast to the reverse consequences after ANK domain deletion in hNPCs. In nucleus pulposus cells, the combination of p65 and the ANK domain of NICD1 is a critical procedure for the degeneration related to the NF-κB signaling pathway activation induced by IL-1β and TNF-α.     

3,3′,4,4′,5-Pentachlorobiphenyl influences mitochondrial apoptosis pathway in granulosa cells

3,3′,4,4′,5-Polychlorinated biphenyl (PCB126) is a persistent organic environmental pollutant which can affect various biological activities of organisms, such as immunity, neurological function, and reproduction. In our study, we aimed to investigate the effects of PCB126 on granulosa cells (GCs). GCs were collected from ovaries in PMSG-treated mice, after 24 hours culture. GCs were then incubated with 10 pg/mL, 100 pg/mL, and 10 ng/mL of PCB126 for another 24 hours. Following these steps, exposed GCs were collected for further experimentation. Our data showed that the number of GCs in the 10 ng/mL PCB126 decreased. Meanwhile, pyknotic nuclei and condensed chromatin increased, while the apoptotic cells in the 10 ng/mL PCB126 group were significantly increased. Furthermore, the expression of the apoptotic executive protein caspase-3 increased after PCB126 treatment. The expression of Bax, Bcl-2, and Bim related to the mitochondrial apoptosis pathway were also influenced to different degrees. Thus, our data suggested that PCB126 affect the GCs apoptosis, and mitochondrial apoptosis pathway was involved in this process.     

Erbin plays a critical role in human umbilical vein endothelial cell migration and tubular structure formation via the Smad1/5 pathway

Angiogenesis is an important process in atherosclerosis. ErbB2 was proved to have an important role in vascular development, but it is still unclear whether Erbin expresses in vessels as well as its location and function in the vessels. In the current study, we investigated the location and function of Erbin in human umbilical veins. The human umbilical veins were prepared, and immunofluorescent analysis was performed to determine the expression of Erbin. Human umbilical vein endothelial cells (HUVECs) were cultured and the lentivirus (LV) containing Erbin RNAi was also prepared. After transfection with the lentivirus, CCK-8 assay and Annexin V-PI assay were used for cell proliferation and apoptosis, respectively. Cell migration was studied using the scratch wound healing assay and the transwell assay. The capillary-like tube formation assay was performed to illustrate the effect of Erbin on HUVEC tube formation. Expression of signaling pathway molecules was assessed with Western blot. The immunofluorescent analysis suggested that Erbin expressed in human umbilical veins and the majority of the Erbin is strongly colocalized in endothelial cells. Although knockdown of Erbin did not affect HUVEC proliferation and apoptosis, it significantly suppressed HUVEC migration and tubular structure formation. Erbin knockdown showed no effect on the ERK1/2 and Smad2/3 signaling pathways but significantly promoted Smad1/5 phosphorylation and nuclear translocation. Ablation of the Smad1/5 pathway decreased the effects of Erbin on endothelial cells. Erbin is mainly localized in endothelial cells in human umbilical veins and plays a critical role in endothelial cell migration and tubular formation via the Smad1/5 pathway.     

Functional variants of hepatocyte growth factor identified in patients with adolescent idiopathic scoliosis

The genetic etiology of adolescent idiopathic scoliosis (AIS) remains obscure. Whole-genome sequencing was performed in four members of one family. Then, we performed a rigorous computational analysis to determine the deleterious effects of the identified variants. Furthermore, the structural differences between the native hepatocyte growth factor (HGF) protein and a protein encoded by an HGF variant containing one mutation (p.T596M) were analyzed using molecular dynamic stimulation. A novel heterozygous mutation (p.T596M) within the HGF gene was identified and found to cosegregate with scoliosis phenotypes in three affected family members. Subsequent modeling and structure-based analyses supported the theory that this mutation is functionally deleterious. Functional analyses demonstrated that the HGF p.T596 M mutation changed the ability of the HGF protein to be secreted and impaired migration and invasion in HEK293T cells. Furthermore, an HGF knockdown zebrafish model exhibited a curly tailed phenotype. Mutation in HGF is associated with an autosomal dominant pattern of inheritance of AIS. This finding increases our understanding of the genetic heterogeneity of AIS.     

Microarray analysis reveals the changes of circular RNA expression and molecular mechanism in acute lung injury mouse model

Acute lung injury (ALI) is a severe disease with sudden onset, rapid progression, poor treatment response, and high mortality. An increasing number of studies had found that circular RNAs (circRNAs) has significant functions in various diseases, while the role of circRNAs in ALI is not yet clear. The purpose of this study was to find circRNAs related to ALI and their mechanism of action. Expression profiles of lung circRNAs and messenger RNAs (mRNAs) were analyzed by microarray in the ALI mice models and healthy controlled mice. Differentially expressed RNAs were identified, function and pathways were analyzed by bioinformatics analysis. Moreover, the results of the microarray were verified by real-time PCR. We identified 2262 differentially expressed mRNAs and 581 circRNAs between ALI mice and control. Validation of candidate circRNAs by real-time PCR indicates that the majority of circRNAs identified by microarray are reliable and worthy of further study. ALI induced circRNAs primarily function in the metabolic regulatory process. Moreover, differentially expressed circRNAs were mainly involved in signaling pathways of mitogen-activated protein kinases, focal adhesion, FoxO, neurotrophin, and Wnt. In addition, a competitive endogenous RNA network was constructed to further interpret the molecular mechanism of ALI. This study observed significantly changed circRNAs profiles in LPS-induced mouse model and revealed a potential role of circRNAs in ALI.