Transcriptome analysis of adherens junction pathway-related genes after peripheral nerve injury

The neural regeneration process is driven by a wide range of molecules and pathways. Adherens junctions are critical cellular junctions for the integrity of peripheral nerves. However, few studies have systematically characterized the transcript changes in the adherens junction pathway following injury. In this study, a rat model of sciatic nerve crush injury was established by forceps. Deep sequencing data were analyzed using comprehensive transcriptome analysis at 0, 1, 4, 7, and 14 days after injury. Results showed that most individual molecules in the adherens junctions were either upregulated or downregulated after nerve injury. The m RNA expression of ARPC1 B, ARPC3, TUBA8, TUBA1 C, CTNNA2, ACTN3, MET, HGF, NME1 and ARF6, which are involved in the adherens junction pathway and in remodeling of adherens junctions, was analyzed using quantitative real-time polymerase chain reaction. Most of these genes were upregulated in the sciatic nerve stump following peripheral nerve injury, except for CTNNA2, which was downregulated. Our findings reveal the dynamic changes of key molecules in adherens junctions and in remodeling of adherens junctions. These key genes provide a reference for the selection of clinical therapeutic targets for peripheral nerve injury.

Transcriptomic analysis reveals essential microRNAs after peripheral nerve injury

Studies have shown that microRNAs(miRNAs) mediate posttranscriptional regulation of target genes and participate in various physiological and pathological processes, including peripheral nerve injury. However, it is hard to select key miRNAs with essential biological functions among a large number of differentially expressed miRNAs. Previously, we collected injured sciatic nerve stumps at multiple time points after nerve crush injury, examined gene changes at different stages(acute, sub-acute, and post-acute), and obtained mRNA expression profiles. Here, we jointly analyzed mRNAs and miRNAs, and investigated upstream miRNAs of differentially expressed mRNAs using Ingenuity Pathway Analysis bioinformatic software. A total of 31, 42, 30, and 23 upstream miRNAs were identified at 1, 4, 7, and 14 days after rat sciatic nerve injury, respectively. Temporal expression patterns and biological involvement of commonly involved upstream miRNAs(miR-21, let-7, miR-223, miR-10 b, miR-132, miR-15 b, miR-127, miR-29 a, miR-29 b, and miR-9) were then determined at multiple time points. Expression levels of miR-21, miR-132, miR-29 a, and miR-29 b were robustly increased after sciatic nerve injury. Biological processes involving these miRNAs include multicellular organismal response to stress, positive regulation of the epidermal growth factor receptor signaling pathway, negative regulation of epithelial cell differentiation, and regulation of myocardial tissue growth. Moreover, we constructed mechanistic networks of let-7, miR-21, and miR-223, the most significantly involved upstream miRNAs. Our findings reveal that multiple upstream miRNAs(i.e., let-7, miR-21, and miR-223) were associated with gene expression changes in rat sciatic nerve stumps after nerve injury, and these miRNAs play an important role in peripheral nerve regeneration. This study was approved by the Experimental Animal Ethics Committee of Jiangsu Province of China(approval No. 20190303-18) on March 3, 2019.

Biological characteristics of dynamic expression of nerve regeneration related growth factors in dorsal root ganglia after peripheral nerve injury

The regenerative capacity of peripheral nerves is limited after nerve injury.A number of growth factors modulate many cellular behaviors,such as proliferation and migration,and may contribute to nerve repair and regeneration.Our previous study observed the dynamic changes of genes in L4–6 dorsal root ganglion after rat sciatic nerve crush using transcriptome sequencing.Our current study focused on upstream growth factors and found that a total of 19 upstream growth factors were dysregulated in dorsal root ganglions at 3,9 hours,1,4,or 7 days after nerve crush,compared with the 0 hour control.Thirty-six rat models of sciatic nerve crush injury were prepared as described previously.Then,they were divided into six groups to measure the expression changes of representative genes at 0,3,9 hours,1,4 or 7 days post crush.Our current study measured the expression levels of representative upstream growth factors,including nerve growth factor,brain-derived neurotrophic factor,fibroblast growth factor 2 and amphiregulin genes,and explored critical signaling pathways and biological process through bioinformatic analysis.Our data revealed that many of these dysregulated upstream growth factors,including nerve growth factor,brain-derived neurotrophic factor,fibroblast growth factor 2 and amphiregulin,participated in tissue remodeling and axon growth-related biological processes Therefore,the experiment described the expression pattern of upstream growth factors in the dorsal root ganglia after peripheral nerve injury.Bioinformatic analysis revealed growth factors that may promote repair and regeneration of damaged peripheral nerves.All animal surgery procedures were performed in accordance with Institutional Animal Care Guidelines of Nantong University and ethically approved by the Administration Committee of Experimental Animals,China(approval No.20170302-017)on March 2,2017.

Quantitative proteomic determination of diethylstilbestrol action on prostate cancer

Diethylstilbestrol （DES） has a direct cellular mechanism inhibition on prostate cancer. Its action is independent from the oestrogen receptors and is preserved after a first-line hormonal therapy. We aimed to identify proteins involved in the direct cellular inhibition effects of DES on prostate cancer. We used a clonogenic assay to establish the median lethal concentration of DES on 22RV1 cells. 22RV1 cells were exposed to standard and DES-enriched medium. After extraction, protein expression levels were obtained by two-dimensional differential in-gel electrophoresis （2D-DIGE） and isotope labelling tags for relative and absolute quantification （iTRAQ）. Proteins of interest were analysed by quantitative RT-PCR and western blotting. The differentially regulated proteins （P〈0.01） were interrogated against a global molecular network based on the ingenuity knowledge base. The 2D-DIGE analyses revealed DES-induced expression changes for 14 proteins （〉 1.3 fold; P〈0.05）. The iTRAQ analyses allowed the identification of 895 proteins. Among these proteins, 65 had a modified expression due to DES exposure （i.e., 23 overexpressed and 42 underexpressed）. Most of these proteins were implicated in apoptosis and redox processes and had a predicted mitochondrial expression. Additionally, ingenuity pathway analysis placed the OAT and HSBP1 genes at the centre of a highly significant network. RT-PCR confirmed the overexpression of OAT （P=0.006） and HSPB1 （P=0.046）.

Prediction of Triptolide Targets in Colorectal Cancer Using Network Pharmacology and Molecular Docking

[Objectives]To investigate the potential mechanisms of action of triptolide,an active component in the traditional Chinese medicine Tripterygium wilfordii Hook F,in colorectal cancer(CRC).[Methods]Public databases were first searched for genes and proteins known to be associated with CRC,as well as those predicted to be targets of triptolide,and then Ingenuity Pathway Analysis(IPA)was applied to identify enriched gene pathways and networks.Networks and pathways that overlapped between CRC-associated proteins and triptolide target proteins were then used to predict candidate protein targets of triptolide in CRC.[Results]The following proteins were found to be expressed in both CRC-associated networks and triptolide target networks:JUN,FOS,CASP3,BCL2,IFNG,and VEGFA.Docking studies suggested that triptolide can fit in the binding pocket of the four top candidate triptolide target proteins(CASP3,BCL2,VEGFA and IFNG).The overlapping pathways were activation of neuroinflammation signaling,glucocorticoid receptor signaling,T helper(Th)cell differentiation,Th1/Th2 activation,and colorectal cancer metastasis signaling.[Conclusions]These results show that network pharmacology can be used to generate hypotheses about how triptolide exerts therapeutic effects in CRC.Network pharmacology may be a useful method for characterizing multi-target drugs in complex diseases.

Prediction of Apigenin Targets in Lung Cancer Using Network Pharmacology and Molecular Docking

[Objectives]To elucidate the mechanism of action of apigenin against lung cancer.[Methods]First,drug-target pathways and networks were constructed to predict potential protein targets of apigenin and their main interactions with the drug.Then,the ingenuity pathway analysis(IPA)was carried out to identify enriched gene pathways and networks,and the candidate protein targets of apigenin were predicted using networks and pathways that overlapped between proteins associated with lung cancer and proteins targeted by apigenin.[Results]Docking studies with apigenin indicated that BCL-2,CASP9,CDK2,CYCLIND1,PI3K,NF-κB,Rb1,p53,and AKT are the top candidate targets of apigenin,suggesting that the drug acts against lung cancer by regulating proteins involved in molecular mechanisms of cancer,as well as small cell lung cancer,pancreatic adenocarcinoma,glucocorticoid receptor,and p53 signaling.It was also found that apigenin affects networks mainly involved in the cell cycle,cell-to-cell signaling and interactions,hematological system development and function,cell death and survival,and organismal injury and abnormalities.[Conclusions]This study shows that network pharmacology is useful in generating hypotheses about how apigenin exerts therapeutic effects in lung cancer,as well as in discovering new multitarget drugs against other complex diseases.

Mechanism of herbal pairs with the properties of Qi-tonifying,blood activation,blood-stasis breaking in treating coronary heart disease

OBJECTIVE:To investigating the molecular mechanism of herbal pairs in three types of Chinese medicinals:Qi-tonifying,blood activation,blood-stasis breaking in treatment of coronary heart disease(CHD).METHODS:The components of six herbs were searched in Chinese medicine dictionary and their target proteins were found in PubChem.CHD genes were obtained from PubMed gene database.Ingenuity Pathways Analysis was used to build the pharmacological network of three herbal pairs and CHD molecular network.The canonical pathways between each herbal pair network and CHD network was compared to decipher the molecular mechanism on three herbal pairs in treating CHD.RESULTS:The network analysis showed that there were the common signal pathways of three herbal pairs in treating CHD including hypoxia signaling in the cardiovascular system,Hypoxia-inducible factor 1-alpha signaling,glucocorticoid receptor signaling,G-Protein coupled receptor signaling and pregnane X receptor/retinoid X receptor(PXR/RXR)activation.Further to analyze cardiovascular signaling,cytokine signaling and cytokine signaling,the effective molecules for three herbal pairs in treating CHD included HIF1α and estrogen receptor 1,Qi-tonifying herbal pair included albumin and matrix metallopeptidase 2,and blood-activation herbal pair included estrogen receptor 2 and peroxisome proliferator-activated receptor-γ.CONCLUSION:Each herbal pair can affect some respective CHD-related functions and pathways,meanwhile three herbal pairs exert some mutual effects on CHD-related functions and pathways.Mutual effects of three herbal pairs may be the key components of their total molecular mechanisms and respective effects of each herbal pair may be the characteristic components of their respective molecular mechanism.