Transcriptome Sequencing on Treatment of Kidney Deficiency and Blood Stasis-thin Endometrium in Rats with Bushen Huoxue Prescription Through Enema / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Bushen Huoxue enema in treating the rat model of kidney deficiency and blood stasis-thin endometrium （KDBS-TE） by transcriptome sequencing. MethodThe rat model of KDBS-TE was established by administration of tripterygium polyglycosides tablets combined with subcutaneous injection of adrenaline. The pathological changes of rat endometrium in each group were then observed. Three uterine tissue specimens from each of the blank group， model group， and Bushen Huoxue enema group were randomly selected for transcriptome sequencing. The differentially expressed circRNAs， lncRNAs， and miRNAs were screened， and the disease-related specific competitive endogenous RNA （ceRNA） regulatory network was constructed. Furthermore， the gene ontology （GO） functional annotation and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment were performed for the mRNAs in the network. ResultCompared with the blank group， the model group showed endometrial dysplasia， decreased endometrial thickness and endometrial/total uterine wall thickness ratio （P<0.01）， and differential expression of 18 circRNAs， 410 lncRNAs， and 7 miRNAs. Compared with the model group， the enema and estradiol valerate groups showed improved endometrial morphology and increased endometrial thickness and ratio of endometrial to total uterine wall thickness （P<0.05）. In addition， 21 circRNAs， 518 lncRNAs， and 17 miRNAs were differentially expressed in the enema group. The disease-related specific circRNA-miRNA-mRNA regulatory network composed of 629 nodes and 664 edges contained 2 circRNAs， 34 miRNAs， and 593 mRNAs. The lncRNA-miRNA-mRNA regulatory network composed of 180 nodes and 212 edges contained 5 lncRNAs， 10 miRNAs， and 164 mRNAs. The mNRAs were mainly enriched in Hippo signaling pathway， autophagy-animal， axon guidance， etc. ConclusionBushen Huoxue enema can treat KDBS-TE in rats by regulating specific circRNAs， lncRNAs， and miRNAs in the uterus and the ceRNA network.

Mechanism of Huashi Baidu Prescription in Treating Influenza Viral Pneumonia Based on Transcriptome / 中国实验方剂学杂志

ObjectiveTo clarify the therapeutic effect of Huashi Baidu prescription on pneumonia in mice caused by influenza A （H1N1） virus and explore its mechanism based on the transcriptome. MethodA mouse influenza viral pneumonia model was built by intranasal infection with influenza A virus， and mice were continuously administered the drug for five days， so as to investigate the general condition， lung index， viral load， pathological morphology of lung tissue， survival time， and prolongation rate of survival time of mice and clarify the therapeutic effect of Huashi Baidu prescription on influenza viral pneumonia. Transcriptome technology was used to detect the differentially expressed genes in the lung tissue of mice in the model group and the normal group， as well as the Huashi Baidu prescription group and the model group， and the potential core target of the Huashi Baidu prescription for the treatment of influenza viral pneumonia was screened. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to verify the effect of Huashi Baidu prescription on the mRNA expression level of core target genes. ResultCompared with the normal group， the lung index and viral load in the lung tissue of the model group were significantly increased （P<0.05， P<0.01）. Compared with the model group， the high-dose group of Huashi Baidu prescription significantly prolonged the survival time of mice infected with influenza A virus （P<0.05） and significantly reduced the lung index value of mice （P<0.05） and the viral load of lung tissue. The high-dose， medium-dose， and low-dose groups of Huashi Baidu prescription could significantly reduce lung tissue inflammation， blood stasis， swelling， and other pathological changes in mice （P<0.05， P<0.01）. Transcriptome analysis of lung tissue showed that core genes were mainly enriched in the nuclear transcription factor-κB （NF-κB） signaling pathway， interleukin-17 （IL-17） signaling pathway， cytokine-cytokine receptor interaction， and other pathways after the intervention of Huashi Baidu prescription. TRAF6， NFKBIA， CCL2， CCL7， and CXCL2 were the top five node genes with combined score values. Real-time PCR validation showed that Huashi Baidu prescription significantly downregulated the mRNA expression of key genes TRAF6 and NFKBIA in the NF-κB signaling pathway， as well as chemokines CCL2， CCL7， and CXCL2 （P<0.05， P<0.01）. ConclusionHuashi Baidu prescription has a therapeutic effect on influenza viral pneumonia， possibly by inhibiting the expression of key nodes TRAF6 and NFKBIA in the NF-κB signaling pathway and that of chemokines CCL2， CCL7， and CXCL2， reducing the recruitment of inflammatory cells and viral load， and exerting anti-influenza viral pneumonia effects.

Exploring the potential mechanism of artesunate in intervening with U87 cells and U251 cells with distinct therapeutic effects on the basis of transcriptome sequencing and network pharmacology / 药学学报

Artesunate possesses the potential of intervening with glioma, however, its pharmacological mechanisms remain unclarified. Firstly, the effects of artesunate on cell activity, proliferation and apoptosis of U87 and U251 human glioma cells were explored. It was found that artesunate exerted stronger inhibitory effects on the activity and proliferation of U87 cells than U251 cells. It could significantly promote apoptosis in U87 cells (P < 0.05), while only high dose of artesunate can promote that of U251 cells (P < 0.01), detected by Hoechst and TUNEL cell apoptosis staining. Further, the differential expression gene sets between artesunate-sensitive and non-sensitive cell line, as well the therapeutic effects-related genes of artesunate were obtained through transcriptome sequencing and differential data analysis by using the lysates of U87 and U251 cells before and after artesunate treatment, aiming to explore the molecular mechanism of distinct artesunate sensitivity to two types of cells. Then, key putative targets that related to therapeutic effects were screened by constructing the interaction network of differential genes of three above comparison groups, and calculating their topological characteristics. Pathway enrichment analysis showed that those key putative targets were significantly enriched in several signaling pathways that were closely associated with the main pathological changes of glioma, among which apoptosis-related activating transcription factor 4 (ATF4)-DNA damage induced transcript 3 (DDIT3)- polyadenosine diphosphate ribose polymerase 1 (PARP1) signaling axis was the most enriched in. Molecular docking indicated that artesunate had fine binding affinities with ATF4 and DDIT3. Above all, this study preliminarily revealed that ATF4-DDIT3-PARP1 signaling axis is the target pathway of artesunate intervening with U87 glioma cells, and PARP1 may be an important gene for U251 cells to develop resistance to artesunate. Our results not only provide fundamental experimental evidence for artesunate as a potential therapeutic drug in glioma treatment, but shed light into overcoming drug resistance in its clinical therapy.

Maackiain inhibits proliferation and promotes apoptosis of nasopharyngeal carcinoma cells by inhibiting the MAPK/Ras signaling pathway / 中国天然药物

Nasopharyngeal carcinoma (NPC) is the third most common malignancy with a high recurrence and metastasis rate in South China. Natural compounds extracted from traditional Chinese herbal medicines have been developed and utilized for the treatment of a variety of cancers with modest properties and slight side effects. Maackiain (MA) is a type of flavonoid that was first isolated from leguminous plants, and it has been reported to relieve various nervous system disorders and exert anti-allergic as well as anti-inflammatory effects. In this study, we demonstrated that MA inhibited proliferation, arrested cell cycle and induced apoptosis in nasopharyngeal carcinoma CNE1 and CNE2 cells in vitro and in vivo. The expression of the related proteins associated with these processes were consistent with the above effects. Moreover, transcriptome sequencing and subsequent Western blot experiments revealed that inhibition of the MAPK/Ras pathway may be responsible to the anti-tumor effect of MA on NPC cells. Therefore, the effects of MA and an activator of this pathway, tertiary butylhydroquinone (TBHQ), alone or combination, were investigated. The results showed TBHQ neutralized the inhibitory effects of MA. These data suggest that MA exerts its anti-tumor effect by inhibiting the MAPK/Ras signaling pathway and it has the potential to become a treatment for patients with NPC.

Transcriptomic Effects of LINC01614 on Lung Cancer A549 Cells and Relevance of Drug Resistance / 肿瘤防治研究

Objective To investigate the biological role of LINC01614 in non-small cell lung cancer A549 cells and its drug resistance-related mechanism. Methods The CRISPR/Cas9 technology was used to construct the A549 cell model with knockdown of LINC01614. Transcriptome sequencing was performed on A549 cells knocked down with LINC01614. We validated the transcriptomic differential genes MCAM and ABCC3 at the gene level and MCAM at the protein level, detected the IC50 changes of A549 cells after knockdown of LINC01614 under the effect of different concentrations of cisplatin, and detected the effect of knockdown of LINC01614 on the migration ability of A549 cells. Results Of the 2 713 DEGs after knockdown of LINC01614, a total of 1 626 genes were up-regulated and 1, 087 genes were down-regulated. GO analysis showed that DEGs were associated with intracellular signaling, cell adhesion, and so on. Meanwhile, the KEGG analysis showed that DEGs were associated with the Wnt signaling pathway, TGF-β signaling pathway, and Rap1 signaling pathway. Selection of drug resistance-associated gene ABCC3 from DEGs for validation with MCAM: qRT-PCR results showed that knockdown of LINC01614 significantly down-regulated the expression of MCAM (P<0.05) and upregulated the expression of ABCC3 on A549 cells (P<0.05). After knockdown of LINC01614, the protein expression of MCAM, was significantly decreased in A549 cells (P<0.05); the IC50 of A549 cells to cisplatin was significantly increased (P<0.05); and the scratch healing rate of A549 cells was also significantly decreased (P<0.05). Conclusion LINC01614 may be associated with the proliferation, invasion, and apoptotic pathways of A549 cells. In addition, LINC01614 may exert its migration ability through MCAM and chemoresistance to cisplatin through ABCC3.

Analysis of Tumor Immune Microenvironment Based on Bulk RNA-sequencing Data and Its Application / 肿瘤防治研究

Cancer immunotherapy is one of the most promising biological therapies. The dynamic changes of the immune microenvironment of heterogeneous tumors are critical factors in determining the interaction and therapeutic efficacy between tumor and immune microenvironment. Therefore, quantitative analysis of its constituent cells, related genes, and phenotypes in real time is of great importance. The methods used to analyze tumor immune microenvironment include immunohistochemistry, flow cytometry and so on. With the development of next-generation sequencing technology, transcriptome RNA sequencing data analysis has become one of the important methods to determine the composition of tumor immune microenvironment. This article focuses on the common methods of bulk RNA sequencing data analysis and related research progress.

Construction of CeRNA network of differential LncRNAs in THP-1 cells infected by Dengue virus type Ⅲ and its antibody dependent enhancement / 中国生物制品学杂志

@#ObjectiveTo establish models of Dengue virus type Ⅲ（DENV-3,DV-3）infection and antibody dependent enhancement(ADE)infection at the acute monocytic leukemia cells(THP-1),investigate the differential expression of long non-coding RNAs(LncRNAs),map the competitive endogenous RNA(CeRNA)regulatory network and predict the translation function of LncRNAs.MethodsThe culture supernatant was harvested 6 d after C6/36 cells were infected with DENV-3,the virus titer was determined by CCID50,and the type and full-length genome amplification were identified by PCR;The DENV-3 standard plasmid was amplified,identified by PCR,and the standard curve was drawn;THP-1 cells were divided into negative control group(THP-1),direct infection group(DV-3),ADE group and blank control group[1640(-)]. After 48 h of infection,the total RNA was extracted and the copy number of intracellular virus nucleic acid was measured;Through the whole transcriptome sequencing technology,the CeRNA regulatory network was constructed for the top five up-regulated and down-regulated LncRNAs in THP-1 vs DENV3,THP-1 vs ADE,DENV3 vs ADE groups,and the functions of their coding proteins were analyzed.ResultsC6/36 cells infected with DENV-3 for 3 d showed obvious cell fusion,vacuoles and abscission;The virus had a titer of about 1. 0 × 104. 64PFU/mL and was identified as DENV-3 by PCR specific primers,of which the complete gene sequence was obtained;The number of viral nucleic acid copies in ADE group was significantly higher than those in DV-3 group and blank control group;In THP-1 vs DENV-3,the expression of cytohesin interacting protein(CYTIP)was predicted to be up-regulated;In THP-1 vs ADE,the expression of kinesin family5A(KIF5A)was predicted to be down-regulated;In DENV-3 vs ADE,the expression of cluster differentiation antigen 9(CD9)and insulin like growth factor 2(IGF2)was predicted to be up-regulated. All of these differential LncRNAs had open reading frames(ORFs). Except Lnc-SH3BP1 and Lnc-RPL41,all of the other LncRNAs had internal ribosome binding site(IRES).ConclusionIn DENV-3 infection of THP-1 cells and ADE infection mediated by DENV-3,the expression of LncRNAs has changed significantly,and may regulate the process of infection through a variety of biological functions,which is helpful for a deeper understanding of the mechanism of ADE infection.

Transcriptome sequencing of transgelin-2 inhibiting high glucose induced microglia inflammation / 中华眼底病杂志

Objective:To analyze the change of differential genes and signaling pathways in high glucose induced BV2 cells, and to explore the mechanism of transgelin-2 (TAGLN2) regulating cellular inflammatory response and metabolic process.Methods:An experimental study. The cultured BV2 cells were divided into mannitol treatment (Man) group, glucose treatment (Glu) group, overexpression control Glu treatment (Con) group, overexpression TAGLN2 Glu treatment group, silence control Glu treatment (shCon Glu) group, and silence TAGLN2 Glu treatment (shTAGLN2 Glu) group. Cells in the Man group were cultured in modified Eagle high glucose medium (DMEM) containing 25 mmol/L mannitol and 25 mmol/L glucose, cells in other groups (Glu group, Con Glu group, TAGLN2 Glu group, shCon Glu group and shTAGLN2 Glu group) were cultured in DMEM medium containing 50 mmol/L glucose. After 24 hours of cells culture, transcriptome sequencing of cells in each group were performed using high-throughput sequencing technology, and significantly differentially expressed genes (DEG) were screened. |log 2 (fold change)|≥1 and P≤0.05 were adopted as criteria to screen for DEG. Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were performed. Real-time polymerase chain reaction (RT-PCR) was used to detect the relative expression level of DEG mRNA. The data between groups were compared by independent sample t-test. Results:When compared with Man group, a total of 517 differentially expressed genes were screened in Glu group, which including 277 up-regulated genes and 240 down-regulated genes. KEGG pathway enrichment analysis showed that the up-regulated genes were significantly enriched in immune system processes such as nuclear factor (NF)-κB signal pathway, Jak-signal transducers and activators of transcription (STAT) signal pathway, while down-regulated genes were significantly enriched in glycosaminoglycan degradation and glyceride metabolic pathway. Compared with Con Glu group, a total of 480 DEG were screened in TAGLN2 Glu group, among which 147 up-regulated and 333 down-regulated genes were detected. Up-regulated genes were significantly enriched in the metabolic processes of fatty acid, glyceride and pyruvate, while down-regulated genes were significantly enriched in immune system processes such as NF-κB signal pathway, Jak-STAT signal pathway and tumor necrosis factor (TNF) signal pathway. Compared with shCon Glu group, a total of 582 DEG were screened in shTAGLN2 Glu group, among which 423 up-regulated and 159 down-regulated genes were detected. Up-regulated DEG were significantly enriched in immune system processes such as TNF signal pathway and chemokine signal pathway, while down-regulated DEG were significantly enriched in pattern recognition receptor signal pathway. RT-PCR results showed that the relative expression levels of DEG mRNA Card11 ( t=13.530), Icos ( t=3.482), Chst3 ( t=6.949), Kynu ( t=5.399), interleukin (IL)-1β ( t=2.960), TNF-α ( t=5.800), IL-6 ( t=3.130), interferon-γ ( t=7.690) and IL-17 ( t=6.530) in the TAGLN2 Glu treatment group were decreased significantly compared with Con Glu group, and the difference was statistically significant. Conclusion:TAGLN2 can inhibit glucose induced microglia inflammation by NF-κB and Jak-STAT signaling pathways, Card11, Icos, Chst3 and Kynu play an important role in the anti-inflammatory process of TAGLN2.

Analysis of intercellular communication patterns in facioscapulohumeral muscular dystrophy based on single-cell nuclear transcriptome sequencing / 国际生物医学工程杂志

Objective:To analyze the patterns of intercellular communication in facioscapulohumeral muscular dystrophy (FSHD) by single-cell nuclear transcriptome sequencing.Methods:Bilateral asymmetrical lesions mouth orbicular muscle of two patients with FSHD and mouth orbicular muscle of two healthy patients were selected. Six samples were obtained, and were divided into control group, mild group and severe group. The normal orbicularis muscle sample was collected from 2 healthy individuals (the control group). The muscle samples in the mild group were from two patients with relatively normal muscle sides, and the samples in the severe group were from two patients with more severe muscle damage sides. Single-cell nuclear transcriptome sequencing was performed on all cells of the three groups. Reduced dimension clustering and cell definition were performed to identify differentially expressed genes and enrichment pathways. Intercellular communication patterns among major cell types and key signaling pathways were explored by cellular communication analysis.Results:Differential gene expression analysis of FSHD bilateral muscle samples identified 46 functionally differentially expressed genes associated with the disease in different cell types, related to apoptosis, oxidative stress, immune inflammation, and muscle function. Intercellular communication was generally increased in the severe group. Fibro-adipogenic progenitors (FAPs) and macrophages are important signaling sources in the abnormal muscle microenvironment of FSHD and are closely associated with disease progression. There are six unique signaling pathways in the mild group, including bone morphogenetic proteins (BMP), transforming growth factor-β (TGF-β), CXC motif chemokine ligand (CXCL), adhesion G protein-coupled receptor E5 (ADGRE5), interleukin-16 (IL-16), and wingless-type MMTV integration site family (WNT) signaling pathways. These signaling pathways are mainly involved in the interaction between macrophages, FAPs, and adipocytes and may be involved in the regulation of fat deposition and fibrosis changes in the diseased muscle.Conclusions:Single-cell nuclear transcriptome sequencing provides a relatively comprehensive pattern of intercellular communication between key cell types in FSHD, providing an appropriate reference for understanding the intercellular regulatory mechanisms of the FSHD muscle microenvironment.

Loss of ATM activity induces GADD45α-dependent apoptosis in cerebellar granular neurons / 中山大学学报(医学科学版)

objectiveTo explore the specific molecular mechanism of neuronal apoptosis induced by ATM inactivation. MethodsCGNs matured 7 days in vitro were cultured 8 h with 25 K， 5 K or 25 K medium containing ATM-specific inhibitors （Ku55933， 10 µmol/L； Ku60019， 15 µmol/L） for Hoechst stain and apoptosis analysis， or cultured for different lengths of time （2， 4， 8 h） to detect the protein expression levels of ATM， caspase-3 and cleaved caspase-3 by Western blotting. ATM and GADD45α specific siRNA was transfected into C6 cells and CGNs， and its interference efficiency was verified by q-PCR and Western blotting. CGNs matured for 5 days in vitro were transfected with ATM specific siRNA and pCMV-EGFP by calcium phosphate for 48 h， Hoechst staining and apoptosis analysis were performed. CGNs matured for 7 days in vitro were treated with 25 K medium containing ATM specific inhibitors for 8 h， transcriptome sequencing， differential expression gene identification and pathway enrichment analysis were performed. CGNs matured for 5 days in vitro were co-transfected with GADD45α specific siRNA and pCMV-EGFP by calcium phosphate for 48 h， then treated with 5 K or 25 K medium containing 15 µmol/L Ku6 for 8 h. Hoechst staining and apoptosis analysis were performed. ResultsCompared with the 25 K， CGNs nuclear pyknosis rate， cleaved Caspase-3 and ATM protein expression level were increased in the 5 K and ATM-specific inhibitor groups. The mRNA and protein expression levels of ATM and GADD45α were effectively reduced after transfection of ATM and GADD45α specific siRNA in C6 cells and CGNs. Compared with control， CGNs transfected with ATM specific siRNA showed a higher nuclear pyknosis rate. Totally 835 genes were identified to be up-regulated and 848 genes to be down-regulated in the Ku55933 treatment group； 454 genes were identified to be up-regulated and 314 genes to be down-regulated in the Ku6 treatment group； 274 genes were co-up regulated in the Ku5 and Ku60019 treatment groups， while 179 genes were co-down-regulated in the Ku5 and Ku6 treatment groups and the expression of ATM downstream target GADD45α was upregulated. The enrichment results showed that TNF signaling pathway， NF-κB signaling pathway and Apoptosis signaling pathway were significantly enriched. Compared with control， mRNA and protein expression levels of GADD45α were increased in inhibitor treatment and 5 K， while knocking down GADD45α resulted in a decrease in nuclear pyknosis rate in the Ku60019 and 5 K treatment group. ConclusionLoss of ATM activity induces GADD45α-dependent cerebellar granular neuronal apoptosis.

Mechanism of Huangjing Qianshi Decoction in treatment of prediabetic mice based on transcriptome sequencing / 中国中药杂志

Based on transcriptome sequencing technology, the mouse model of prediabetes treated with Huangjing Qianshi Decoction was sequenced to explore the possible mechanism of treating prediabetes. First of all, transcriptome sequencing was performed on the normal BKS-DB mouse group, the prediabetic model group, and the Huangjing Qianshi Decoction treatment group(treatment group) to obtain differentially expressed genes in the skeletal muscle samples of mice. The serum biochemical indexes were detected in each group to screen out the core genes of Huangjing Qianshi Decoction in prediabetes. Gene Ontology(GO) database and Kyoto Encyclopedia of Genes and Genomes(KEGG) database were used to conduct signaling pathway enrichment analysis of differentially expressed genes, and real-time quantitative polymerase chain reaction(RT-qPCR) was used to verify them. The results showed that the levels of fasting blood glucose(FBG), fasting insulin(FINS), insulin resistance index(HOMA-IR), total cholesterol(TC), triglycerides(TG), and low-density lipoprotein cholesterol(LDL-C) in the mouse model were significantly decreased after treatment with Huangjing Qianshi Decoction. In the results of differential gene screening, there were 1 666 differentially expressed genes in the model group as compared with the normal group, and there were 971 differentially expressed genes in the treatment group as compared with the model group. Among them, interleukin-6(IL-6) and NR3C2 genes, which were closely related to the regulation of insulin resis-tance function, were significantly up-regulated between the model group and the normal group, and vascular endothelial growth factor A(VEGFA) genes were significantly down-regulated between the model group and the normal group. However, the expression results of IL-6, NR3C2, and VEGFA genes were adverse between the treatment group and the model group. GO functional enrichment analysis found that the biological process annotation mainly focused on cell synthesis, cycle, and metabolism; cell component annotation mainly focused on organelles and internal components; and molecular function annotation mainly focused on binding molecular functions. KEGG pathway enrichment analysis found that it involved the protein tyrosine kinase 6(PTK6) pathway, CD28-dependent phosphoinositide 3-kinase/protein kinase B(PI3K/AKT) pathway, p53 pathway, etc. Therefore, Huangjing Qianshi Decoction can improve the state of prediabetes, and the mechanism may be related to cell cycle and apoptosis, PI3K/AKT pathway, p53 pathway, and other biological pathways regulated by IL-6, NR3C2, and VEGFA.

Protective Effects of Zhi Ciweipi Water Extracts on Dextran Sulfate Sodium Salt⁃Induced Ulcerative Colitis in Mice / 中国生物化学与分子生物学报

Ulcerative colitis (UC) is a disease characterized by chronic inflammation of the intestinal mucosa. However, its exact pathogenesis is not fully understood. Zhi Ciweipi (ZCWP), as a traditional Chinese medicine (TCM), has demonstrated suitable anti-inflammatory effects in the treatment of patients with bloody stool and hemorrhoids. However, its therapeutic effects on ulcerative colitis have yet to be investigated in depth. The aim of this study was to investigate the protective effect of the aqueous extract of ZCWP against dextran sulfate sodium salt (DSS)-induced ulcerative colitis in mice and its possible mechanism of action. Successful simulation of ulcerative colitis was achieved by providing mice with drinking water containing 3% DSS. The HE staining results revealed that the aqueous extract of ZCWP could significantly reduce DSS-induced colonic injuries. Transcriptome sequencing analysis identified 10 key inflammation-related genes (IL⁃6, IL⁃1β, CSF2, TNF, IL10, IFN⁃γ, CXCL1, CXCL2, CXCL9, CXCL10), all of which were significantly downregulated (P<0. 05) in response to treatment with the aqueous extract of ZCWP according to qRT-PCR and Western blotting analyses. The immunofluorescence results further indicated that the aqueous extract of ZCWP was able to reduce the DSS-induced increase in the proportion of M1-type macrophages in the colon. Using single-cell sequencing, we thoroughly investigated the signaling relationships between different cell types, which revealed particularly strong communication between M1-type macrophages and fibroblasts. Subsequent qRT-PCR and Western blot analyses verified that the aqueous extract of ZCWP significantly downregulated the expression of DSS-induced fibrosis-related genes in colonic tissues (P<0. 05). In conclusion, the results of this study suggest that the aqueous extract of prepared ZCWP exerts a protective effect against DSS-induced ulcerative colitis by inhibiting M1-type macrophage polarization, downregulating the expression of inflammatory factors, and preventing strong communication between M1-type macrophages and fibroblasts. These findings not only reveal the therapeutic mechanism by which ZCWP aqueous extract treats colitis, but also provide a new theoretical basis for its application in clinical practice.

Effect of Flavanomarein from Coreopsis tinctoria Ethanol Extract in Small Intestinal Organoids in Insulin-resistant Mice Based on Transcriptome Sequencing / 中国实验方剂学杂志

ObjectiveTo investigate the effects of flavanomarein on the transcriptome of small intestinal organoids in insulin-resistant mice. MethodFirstly， small intestinal organoids of C57BL/6J and db/db mice were established. Ki-67 and E-cadherin expression was determined by immunofluorescence. Small intestinal organoids were divided into the following three groups： C57BL/6J mouse small intestinal organoids as the normal control group， db/db mouse small intestinal organoids as the model group （IR group）， and db/db mouse small intestinal organoids treated with flavanomarein as the administration group （FM group）. Western blot was used to detect the expression of glucagon-like peptide-1（GLP-1） protein on the small intestinal organoids of the three groups. Finally， transcriptome sequencing was performed on samples from the three groups. ResultOn the 6th day of small intestine organoids culture， a cyclic structure was formed around the lumen， and a small intestine organoids culture model was preliminarily established. Immunofluorescence detection showed that ki-67 and E-cadherin were expressed in small intestinal organoids. Western blot results showed that the expression of GLP-1 protein was increased by flavanomarein. In the results of differential expressed gene （DEG） screening， there were 1 862 DEGs in the IR group as compared with the normal control group， and 2 282 DEGs in the FM group as compared with the IR group. Through protein-protein interaction（PPI） network analysis of the DEGs of the two groups， 10 Hub genes， including Nr1i3， Cyp2c44， Ugt2b1， Gsta1， Gstm2， Ptgs1， Gstm4， Cyp2c38， Cyp4a32， and Gpx3， were obtained. These genes were highly expressed in the normal control group， and their expression was reduced in the IR group. After the intervention of flavanomarein， the expression of the above genes was reversed. ConclusionFlavanomarein may play its role in improving insulin resistance by reversing the expression levels of 10 Hub genes， including Nr1i3， Cyp2c44， Ugt2b1， Gsta1， Gstm2， Ptgs1， Gstm4， Cyp2c38， Cyp4a32， and Gpx3.

Expression pattern of MMP-12 in long-term optic nerve regeneration induced by lens injury / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the expression changes of MMP-12 during the long-term axon regeneration induced by the lens injury after the optic nerve clamp trauma in sprague-dawley(SD)rats.METHODS: The optic nerve injury model and lens injury model of SD rats were established, and the 24 experimental animals were divided into control group; lens injury group; optic nerve injury group; lens injury combined with optic nerve injury group, with 6 rats in each group. Reference transcriptome sequencing was used to analyze the expression changes of differentially expressed genes in the injured optic nerve region, and relevant differentially expressed genes with high expression were screened. Quantitative real-time polymerase chain reaction(qRT-PCR)and enzyme-linked immunosorbent assay(ELISA)were used to quantify the expression changes of matrix metalloproteinase-12(MMP-12)in the injured optic nerve region.RESULTS: The Principal Component Analysis of transcriptome sequencing indicated that lens injury combined with optic nerve injury was the principal component of gene expression change. Analysis of gene expression differences showed that the expression of MMP-12 gene was up-regulated in the lens injury combined with optic nerve injury group. The mRNA expression level of MMP-12 in the lens injury combined optic nerve injury group was up-regulated compared with the control group, the optic nerve injury group and the lens injury group at 14d and 21d after successful modeling(P<0.05). At 7, 28d, there was no difference in expression among all groups. The protein expression level of MMP-12 in the lens injury combined with optic nerve injury group was up-regulated compared with the control group and optic nerve injury group at 7, 14 and 21d after successful modeling(P<0.05), and it was up-regulated in the lens injury group combined with optic nerve injury group compared with optic nerve injury group at 21d(P<0.05). At 28d, there was no difference in expression among all groups.CONCLUSION: The up-regulated expression of MMP-12 may be involved in the long-term regeneration of the optic nerve after lens injury.

Expression and Clinical Significance of ATP Citrate Lyase in Hepatocellular Carcinoma / 中国医学科学院学报

Objective To investigate the role of ATP citrate lyase(ACLY)in the development of hepatocellular carcinoma(HCC)and the impact of this enzyme on the immune microenvironment of HCC.Methods We utilized the University of Alabama at Birmingham Cancer Data Analysis Portal and the Gene Expression Profiling Interactive Analysis to identify the changes in ACLY expression and prognosis across different tumor types from The Cancer Genome Atlas.With HCC as the disease model,we analyzed the ACLY expression in HCC samples from the gene expression database.Furthermore,we collected the clinical specimens from HCC patients to verify the mRNA and protein levels of ACLY.In addition,we conducted transcriptome sequencing after knocking down the expression of ACLY to analyze the differentially expressed genes and investigated the impact of ACLY expression interference on cell proliferation and other functions.Finally,we explored the correlations of ACLY with immune cells and immune infiltration in the tumor microenvironment,new antigens,and immune checkpoint genes.Results ACLY expression was significantly up-regulated in solid tumors including HCC(all P<0.05),and high ACLY expression was associated with overall survival rate in HCC(P=0.005).Furthermore,high ACLY expression affected the presence of immune cells(e.g.,tumor-associated fibroblasts)and the expression of genes involved in lipid metabolism(all P<0.05).Conclusions ACLY is closely related to the occurrence and development of HCC and lipid metabolism abnormalities.Moreover,it has a specific impact on the immune microenvironment of HCC.

Deciphering the dynamic characteristics of non-neuronal cells in dorsal root ganglion of rat at different developmental stage based on single cell transcriptome data / 生物工程学报

Dorsal root ganglia (DRG) is an essential part of the peripheral nervous system and the hub of the peripheral sensory afferent. The dynamic changes of neuronal cells and their gene expression during the development of dorsal root ganglion have been studied through single-cell RNAseq analysis, while the dynamic changes of non-neuronal cells have not been systematically studied. Using single cell RNA sequencing technology, we conducted a research on the non-neuronal cells in the dorsal root ganglia of rats at different developmental stage. In this study, primary cell suspension was obtained from using the dorsal root ganglions (DRGs, L4-L5) of ten 7-day-old rats and three 3-month-old rats. The 10×Genomics platform was used for single cell dissociation and RNA sequencing. Twenty cell subsets were acquired through cluster dimension reduction analysis, and the marker genes of different types of cells in DRG were identified according to previous researches about DRG single cell transcriptome sequencing. In order to find out the non-neuronal cell subsets with significant differences at different development stage, the cells were classified into different cell types according to markers collected from previous researches. We performed pseudotime analysis of 4 types Schwann cells. It was found that subtype Ⅱ Schwann cells emerged firstly, and then were subtype Ⅲ Schwann cells and subtype Ⅳ Schwann cells, while subtype Ⅰ Schwann cells existed during the whole development procedure. Pseudotime analysis indicated the essential genes influencing cell fate of different subtypes of Schwann cell in DRG, such as Ntrk2 and Pmp2, which affected cell fate of Schwann cells during the development period. GO analysis of differential expressed genes showed that the up-regulated genes, such as Cst3 and Spp1, were closely related to biological process of tissue homeostasis and multi-multicellular organism process. The down regulated key genes, such as Col3a1 and Col4a1, had close relationship with the progress of extracellular structure organization and negative regulation of cell adhesion. This suggested that the expression of genes enhancing cell homestasis increased, while the expression of related genes regulating ECM-receptor interaction pathway decreased during the development. The discovery provided valuable information and brand-new perspectives for the study on the physical and developmental mechanism of Schwann cell as well as the non-neuronal cell changes in DRG at different developmental stage. The differential gene expression results provided crucial references for the mechanism of somatosensory maturation during development.

Whole-transcriptome sequencing analysis of placental differential miRNA expression profile in Down syndrome / 南方医科大学学报

OBJECTIVE@#To identify new biomarkers and molecular pathogenesis of Down syndrome (DS) by analyzing differentially expressed miRNAs in the placentas and their biological pathways.@*METHODS@#Whole transcriptome sequencing was used to identify the differentially expressed miRNAs in DS (n=3) and normal placental samples (n=3) diagnosed by prenatal diagnosis. The target genes were predicted using miRWalk, Targetscan and miRDB, and GO and KEGG pathway analyses were performed for gene enrichment studies.@*RESULTS@#We identified a total of 82 differentially expressed miRNAs in the placental tissues of DS, including 29 up-regulated miRNAs (fold change ≥2, P < 0.05) and 15 down-regulated miRNAs (fold change ≥2, P < 0.05), among which 10 miRNAs with relatively high expression abundance were selected for further analysis, including 4 up-regulated and 6 down-regulated miRNAs. These selected miRNAs shared the common target genes BTBD3 and AUTS2, both of which were associated with neurodevelopment. GO analysis showed that the target genes of the selected miRNAs were mainly enriched in protein binding, hydrolytic enzymes, metal ion binding protein combining, transferase activity, nucleotide, cytoplasmic constituents, nucleus composition, transcriptional regulation, RNA metabolism regulation, DNA-dependent RNA polymerase Ⅱ promoter transcriptional regulation, eye development, and sensory organ development. KEGG enrichment analysis showed that the target genes of these differentially expressed miRNAs were involved in the signaling pathways including tumor-related signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, Rap1 signaling pathway, cytoskeletal regulatory signaling pathway, purine metabolization-related signaling pathway and P53 signaling pathway.@*CONCLUSION@#The differentially expressed miRNAs may play important roles in placental damage and pregnancy pathology in DS and provide clues for the prevention and treatment of mental retardation-related diseases.

Application of single-cell RNA-sequencing in age-related macular degeneration / 国际眼科杂志(Guoji Yanke Zazhi)

@#Age-related macular degeneration(ARMD)is the most common eye disease that can cause irreversible loss of central vision in the elderly population. Since the complexity of the pathogenesis in ARMD, the underlying mechanism remains uncovered,and treatment limited. Conventional bulk transcriptome sequencing strategies can only provide the average gene profile in the dominant cells, while single-cell RNA-sequencing(scRNA-seq)is able to reveal the mRNA transcriptome at a single-cell level. The ScRNA-seq has been applied to discover novel cell subtypes, reveal intercellular heterogeneity, and unveil the process of cell differentiation.In this paper, we reviewed the technical principle of scRNA-seq and its application in retinal,choroid development and ARMD research, raised the defection of scRNA-seq and trended in emerging technologies,provided the new insights and perspectives for the in-depth study of retinal and choroid physiology, pathophysiology, and pathogenesis of ARMD diseases. It is hoped to provide theoretical foundation for the targeted therapy of ARMD.

Whole transcriptome analysis and critical gene regulatory network analysis during Schistosoma japonicum infection and praziquantel treatment in mice / 中国血吸虫病防治杂志

Objective To investigate long non-coding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA) interactions and identify the critical gene regulatory network during Schistosoma japonicum infections and praziquantel treatment using whole transcriptome sequencing. Methods A total of 110 male C57BL/6 mice were randomly divided into the control group, the infection group and the treatment group. Mice in the infection treatment and the control group were infected with S. japonicum cercariae via the abdomen, and liver specimens were sampled from 10 mice 3, 6, 8 weeks post-infection. Praziquantel treatment was given to mice in the treatment group 8 weeks post-infection, and liver specimens were sampled from 10 mice 2, 4, 6, 8, 10 weeks post-treatment. Total RNA was isolated from mouse liver specimens, and the transcriptome library was constructed for highthroughput whole transcriptome sequencing. The significant differentially expressed genes were subjected to functional annotations, Gene Ontology (GO) terms enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Correlation analysis of liver specimens was performed using R Corrplot and Himsc functions, and the lncRNAmiRNA-mRNA interaction network analysis was performed using R MixOmics and Himsc functions. Results There were 1 176 differentially expressed miRNAs, 5 270 differentially expressed mRNAs, and 2 682 differentially expressed lncRNAs between the infection group and the control group, 1 289 differentially expressed miRNAs, 7 differentially expressed mRNAs, and 69 differentially expressed lncRNAs between the treatment group and the infection group, and 1 210 differentially expressed miRNAs, 4 456 differentially expressed mRNAs, and 2 016 differentially expressed lncRNAs between the treatment group and the control group. Correlation analysis showed a higher correlation of gene expression between the treatment group and the control group. Principal component analysis showed obvious separate clustering between the infection group and the treatment group. The differentially expressed genes with significant relevance were significantly enriched in 24 GO terms, including arachidonic acid metabolic process, xenobiotic catabolic process, unsaturated fatty acid metabolic process, xenobiotic metabolic process, long-chain fatty acid metabolic process, and 8 KEGG metabolic pathways, including cholesterol metabolism, tyrosine metabolism, linoleic acid metabolism, retinol metabolism, and steroid hormone biometabolism. Conclusions There were 23 mRNAs including Cyp2b9 and 14 lncRNAs including Rmrpr in the core position of the gene regulatory network, which may play a critical role in S. japonicum infections and praziquantel treatment, and 9 miRNAs including miR-8105 may serve as potential molecular markers for diagnosis of S. japonicum infections.

Specific expression of FABP5 in type II alveolar epithelial cells in silicosis pulmonary fibrosis based on single-cell transcriptome sequencing / 环境与职业医学

Background Silicosis is caused by long-term inhalation of large amounts of free silica (SiO2) particles, and exploring its mechanism can provide new directions for the treatment of silicosis fibrosis. Objective To investigate the expression and role of fatty acid binding protein 5 (FABP5) in a silica-induced silicosis model. Methods In combination with the results of single-cell transcriptome sequencing, the expression pattern of FABP5 in mouse alveolar epithelial cells was explored by bioinformatic analysis, and the distribution pattern of fabp5 was detected by spatial transcriptomics. An in vivo model of silicosis was established by intratracheal injection with SiO2 into mice and four groups were set up: normal saline (NS) 7 d group, NS 56 d group, SiO2 7 d group, and SiO2 56 d group. An in vitro model of silicosis was established in SiO2-treated mouse lung epithelial cell line (MLE-12). At the whole animal level, the marker of epithelial cells (E-Cad) and the protein level of FABP5 were detected by tissue immunofluorescence assay; in vitro, the changes of fabp5 mRNA expression and protein level in MLE-12. Results The results of single-cell transcriptome sequencing and spatial transcriptome sequencing showed that the mRNA expression of fabp5 was upregulated in type II alveolar epithelial cells in the focal area of silicosis in mice, accompanied by elevated tissue immunofluorescent protein levels, and there was co-localization of E-CAD. Meanwhile, SiO2 stimulation induced a 1.58-fold increase in fabp5 mRNA expression and a 2-fold increase in protein levels in MLE-12 cells, with significant differences (P<0.05). Conclusion The protein level of FABP5 is increased in alveolar epithelial cells in a pulmonary fibrosis model, suggesting that FABP5 may be involved in the pathological process of epithelial cells in pulmonary fibrosis.