Preliminary Research of Main Components of Dll4/ Notch-VEGF Signaling Pathway Under High-Glucose Stimulation in vitro.

Purpose: To establish a high-glucose (HG) stressed cell model and study the expression of main components of the Dll4/Notch-VEGF signaling pathway under high-glucose stimulation. Methods: A model of HG-conditioned cells (human umbilical vein endothelial cells, HUVECs) was first established, and then the expression of Dll4, Notch1, Notch4 and VEGF in HG-stressed cells with or without Notch pathway blockage was analyzed by RT-PCR and Western blot. To observe cell migration, we also evaluated the Transwell assay. Results: HUVECs stimulated with 30mmol/L HG was selected as a cell model. RT-PCR and Western blot results showed that HG stimulation induced the expression of Dll4, Notch1 and VEGF and downregulated Notch4. The expressions were reversed after Notch pathway blockage; meanwhile, the blockage of Notch pathway inhibited cell migration under HG condition. Conclusion: The function of Notch4 in responses to HG stimulation deserves further researching. Combination therapy by blocking Dll4/Notch and VEGF pathways may provide us with a new way for anti-neovascularization.

The intestinal microbiota influences the microenvironment of metastatic colon cancer by targeting miRNAs.

This study aimed to investigate the molecular mechanisms through which the intestinal microbiota and microRNAs (miRNAs) participate in colon cancer metastasis. Intestinal flora data, and the GSE29621 (messenger RNA/long non-coding RNA [mRNA/lncRNA]) and GSE29622 (miRNA) datasets, were downloaded from The Cancer Gene Atlas and Gene Expression Omnibus databases, respectively. Immune-related cells in M1 vs. M0 samples were analyzed using the Wilcoxon test. Furthermore, an lncRNA-miRNA-mRNA (competing endogenous RNA [ceRNA]) network was constructed, and survival analysis of RNAs in the network was performed. A total of 16 miRNA-genus co-expression pairs containing eight microbial genera and 15 miRNAs were screened; notably, Porphyromonas and Bifidobacterium spp. were found to be associated with most miRNAs, and has-miR-3943 was targeted by most microbial genera. Furthermore, five immune cell types, including activated natural killer cells, M1 macrophages, resting mast cells, activated mast cells and neutrophils, were differentially accumulated between the M1 and M0 groups. Enrichment analysis suggested that mRNAs related to colon cancer metastasis were mainly involved in pathways related to bacterial and immune responses. Survival analysis revealed that TMEM176A and PALM3 in the ceRNA network were significantly associated with the prognosis of patients with colon cancer. In conclusion, this study revealed a potential mechanism by which the intestinal microbiota influences the colon cancer microenvironment by targeting miRNAs.

Knockdown of ZFAS1 improved the cardiac function of myocardial infarction rats via regulating Wnt/ß-catenin signaling pathway.

Myocardial infarction (MI) is a big health threat in the world, and it is characterized by high morbidity and mortality. However, current treatments are not effective enough, and novel therapeutic strategies need to be explored. ZFAS1 has been proved to be involved in the regulation of MI, but the specific mechanism remains unclear. MI rats were constructed through left anterior descending artery ligation, and hypoxia cell model was also established. The proliferation, invasion, and migration of cells were detected via CCK8, traswell, and wound healing methods. Immunohistochemistry staining, western blotting, and qRT-PCR were used to detect the levels of molecules. Knockdown of ZFAS1 significantly increased the proliferation, migration, and invasion of cardiac fibroblasts. Knockdown of ZFAS1 remarkably improved cardiac function via decreasing infarction ratio and increasing vWF expression, left ventricular ejection fraction, and left ventricular fractional shortening compared with group MI. Knockdown of ZFAS1 also suppressed Wnt/ß-catenin pathway in vivo. The inhibition of Wnt/ß-catenin remarkably reversed the influence of shZFAS1 on cardiac function and cardiac fibroblasts viability. Therefore, Knockdown of ZFAS1 could improve the cardiac function of myocardial infarction rats via regulating Wnt/ß-catenin signaling pathway. The present study might provide new thoughts for the prevention and treatment of MI damage.

A strategy for preparing non-fluorescent graphene oxide quantum dots as fluorescence quenchers in quantitative real-time PCR.

In recent years, graphene oxide quantum dots (GOQDs) have emerged as novel nanomaterials for optical sensing, bioimaging, clinical testing, and environmental testing. However, GOQDs demonstrate unique photoluminescence properties, with GOQDs having quantum limitations and edge effects that often affect the accuracy of the test results in the sensory field. Herein, GOQDs with a large content of hydroxyl groups and low fluorescence intensity were first prepared via an improved Fenton reaction in this study, which introduces a large amount of epoxy groups to break the C-C bonds. The synthesized GOQDs show no significant variation in the fluorescence intensity upon ultraviolet and visible light excitations. We further utilized the GOQDs as fluorescence quenchers for different fluorescent dyes in real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and verified that the addition of GOQDs (5.3 µg ml-1) into a qRT-PCR system could reduce the background fluorescence intensity of the reaction by fluorescence resonance energy transfer (FRET) during its initial stage and its non-specific amplification, and improve its specificity. In addition, the qRT-PCR method could detect two different lengths of DNA sequences with a high specificity in the 104 to 1010 copies per µl range. It is of paramount importance to carry out further investigations to establish an efficient, sensitive, and specific RT-PCR method based on the use of GOQD nanomaterials as fluorescence quenchers.

Exosome-transmitted circ_MMP2 promotes hepatocellular carcinoma metastasis by upregulating MMP2.

Exosomes released by tumor cells have been recently identified as important determinants of tumor progression. They often carry circular RNAs that are differentially expressed in tumors and may regulate tumorigenesis and metastasis. Here, we showed that supernatant of 97H hepatocellular carcinoma (HCC) cell line could promote metastasis in L02 human liver cells and HCC cell lines. Moreover, we determined that circ_MMP2 (has_circ_0039411) could be delivered by 97H- or LM3 cell-derived exosomes to L02 and HepG2 cell cultures. High expression of circ_MMP2 led to the upregulation of its host gene matrix metallopeptidase 2 (MMP2) via the sponging of miR-136-5p. Rescue assays demonstrated that miR-136-5p and MMP2 were two essential participants in HCC metastasis. Finally, high level of circ_MMP2 or MMP2, as well as low level of miR-136-5p, was correlated with low overall survival of HCC patients. Our study highlights a novel molecular pathway in HCC cell-derived exosomes.

Cell-Type Specific Distribution of T-Type Calcium Currents in Lamina II Neurons of the Rat Spinal Cord.

Spinal lamina II (substantia gelatinosa, SG) neurons integrate nociceptive information from the primary afferents and are classified according to electrophysiological (tonic firing, delayed firing, single spike, initial burst, phasic firing, gap firing and reluctant firing) or morphological (islet, central, vertical, radial and unclassified) criteria. T-type calcium (Cav3) channels play an essential role in the central mechanism of pathological pain, but the electrophysiological properties and the cell-type specific distribution of T-type channels in SG neurons have not been fully elucidated. To investigate the electrophysiological and morphological features of T-type channel-expressing or -lacking neurons, voltage- and current-clamp recordings were performed on either transverse or parasagittal spinal cord slices. Recording made in transverse spinal cord slices showed that an inward current (I T) was observed in 44.5% of the SG neurons that was fully blocked by Ni2+ and TTA-A2. The amplitude of I T depended on the magnitude and the duration of hyperpolarization pre-pulse. The voltage for eliciting and maximizing I T were -70 mV and -35 mV, respectively. In addition, we found that most of the I T-expressing neurons are tonic firing neurons and exhibit more negative action potential (AP) threshold and smaller difference of AP threshold and resting membrane potential (RMP) than those neurons lacking I T. Consistently, a specific T-type calcium channel blocker TTA-P2 increased the AP threshold and enlarged the difference between AP threshold and membrane potential (Ihold = 0). Meanwhile, the morphological analysis indicated that most of the I T-expressing neurons are islet neurons. In conclusion, we identify a cell-type specific distribution and the function of T-type channels in SG neurons. These findings might provide new insights into the mechanisms underlying the contribution of T-type channels in sensory transmission.