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Background Pulmonary fibrosis currently lacks screening and diagnostic methods in the early stages and effective treatments in the later stages, so there is an urgent need to explore the mechanisms and develop targeted treatments. Objective To screen the expression of differentially expressed circular RNA (circRNA) hsa_circUCK2 under pathological conditions and to explore its effect on pulmonary fibrosis. Methods In the cell-based experiments, hsa_circUCK2 was knocked down in HPF-a cells using small interfering RNA (siRNA), and HPF-a cells were stimulated by TGF-β1. Four groups were set up: si-NC group, si-circUCK2 group, si-NC+TGF-β1-treated group, and si-circUCK2+TGF-β1-treated group. Western blot assay was used to detect the expression of fibronectin (FN1) in HPF-a cells of the four groups, scratch assay was used to detect the migration ability of HPF-a cells, and CCK-8 assay was used to detect the proliferation ability of HPF-a cells in the two groups with TGF-β1 stimulation, the si-NC+TGF-β1-treated group and the si-circUCK2+TGF-β1-treated group. In the animal experiments, forty-eighty healthy SPF-grade male C57BL/6 mice were randomly divided into four groups: saline+si-con group, saline+si-circ_0000115 group, SiO2+si-con group, and SiO2+si-circ_0000115 group. Mouse lung circRNA mmu_circ_0000115 (mouse homolog of hsa_circUCK2) was knocked down by tracheal drip injection of siRNA, and a mouse lung fibrosis model was constructed by tracheal drip injection of SiO2 suspension (0.2 g·kg−1, 50 mg·mL−1) after 48 h. Real-time fluorescence quantitative PCR was used to detect the knockout efficiency in each organ of the mouse, Western blot was applied to detect the expression of type I collagen α2 (COL1A2) in the lung tissues, and Sirius red was used to detect collagen synthesis in the lung tissues. Results In the cell-based experiments, after the knockdown of hsa_circUCK2, the Western blot results showed that the expression level of the FN1 protein in TGF-β1-stimulated HPF-a cells was significantly down-regulated (P <0.05); the CCK-8 assay and cell scratch assay showed that the down-regulation of hsa_circUCK2 gene significantly inhibited the proliferation and migration of HPF-a cells (P<0.01). In the animal experiments, the real-time fluorescence quantitative PCR results showed that among the detected organs, mmu_circ_0000115 was significantly knocked down only in the lung tissues (P<0.0001); the Western blot results showed that knocking down mmu_circ_0000115 significantly reduced the COL1A2 protein expression level when compared with the SiO2+si-con group (P<0.0001); the Sirius red results showed that knocking down mmu_circ_0000115 significantly reduced collagen production and deposition in lung tissues of mice in the model group. Conclusion Knockdown of hsa_circUCK2 inhibits fibroblast activation and reduces collagen deposition in lung fibrosis model mice. It is suggested that the hsa_circUCK2 is involved in the process of pulmonary fibrosis and may be a potential therapeutic target for pulmonary fibrosis.
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Objective:To detect key genes of local glucocorticoid therapy in oral lichen planus(OLP)through transcriptome sequencing.Methods:The study prospectively enrolled 28 symptomatic patients who visitied Department of Oral Mucosa,Peking University Hospital of Stomatology from November 2019 to March 2023.Topical inunction of 0.1 g/L of dexamethasone was applied for 1 min,3 times daily for 4 weeks.The patients'signs and pain symptoms were recorded and they were classified as effective group and ineffective group according to the treatment outcome.Their mucosa samples were collected before treatment.After isolating total RNA,transcriptome sequencing was performed.The gene expression data obtained by sequencing were analyzed differently using the DESeq2 package in R software,and the Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis was performed on the basis of the hypergeometric distribution algorithm to describe the biological function of differentially expressed genes(DEGs),accordingly detecting sensitivity related molecular affecting therapeutic effect of dexamethasone.Results:After 4 weeks treatment by topical dexamethasone,13 cases of the 28 OLP pa-tients responding well with the sign score reducing from 7.0(4.5,9.0)to 5.0(3.0,6.3),pain score decreasing from 5.0(2.0,5.5)to 2.0(0.0,3.5),oral health impact profile lessening from 5.0(3.5,9.0)to 1.0(0.0,5.0)significantly(P＜0.01)were classified as effective group and 15 cases with poor response to the drug were sorted as ineffective group.There were no significant differences of demographic and baseline levels of clinical features,especially disease severity between these two groups.A total of 499 DEGs including 274 upregulated and 225 downregulated genes were identified between ef-fective group and ineffective group.KEGG enrichment analysis showed that upregulated genes in effective group compared with ineffective group including CLDN8,CTNNA3,MYL2 and MYLPF were associated with leukocyte transendothelial migration,while downregulated genes were significantly enriched in tumor necrosis factor(TNF),interleukin-17(IL-17),nuclear factor kappa B(NF-κB)signaling pathways,and cortisol synthesis and secretory.Conclusion:High expressions of CLDN8,CTNNA3,MYL2 and MYLPF genes in patients with oral lichen planus have a good clinical response to topical dexamethasone,while patients with high expression genes of inflammation pathway such as TNF,IL-17,NF-κB and corti-sol synthesis and secretion received poor effect.
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Aim To investigate the effect of benzyl iso-thiocyanate (BITC) on the proliferation of mouse U14 cervical cancer cells and to explore the mechanism of cytotoxicity based on transcriptomic data analysis. Methods The effect of BITC on U14 cell activity was detected by MTT, nuclear morphological changes were observed by Hochest 33258 and fluorescent inverted microscope, cell cycle and apoptosis were determined by flow cytometry, and the transcriptome database of U14 cells before and after BITC (20 μmol · L
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Objective @#To study the molecular mechanism of fat mass and obesity⁃associated protein (FTO) regulating hepatocellular carcinoma (HCC) . @*Methods@#HepG2 cells of knock⁃down FTO were constructed , HepG2 cells of knock⁃down FTO and HepG2 cells were collected , and high⁃throughput sequencing was performed using Illumina Hiseq platform to screen the gene expression differences between the two groups . Through GO and KEGG enrichment analysis of these differential genes , FTO regulatory pathways were studied and downstream target genes of FTO were screened . The role of FTO downstream target gene in HCC was revealed by bioinformatic analysis and cell experiments .@*Results@#Transcriptome sequencing showed that 386 genes were differentially expressed between HepG2 cells of knock⁃down FTO and HepG2 cells , and they were involved in biological processes such as response to interferon⁃gamma . The expression of IFIT2 , one of the most responsive interferon⁃stimulating genes , was up⁃regulated after FTO knockdown . Potential m6 A methylation occurred at multiple sites of IFIT2 . The survival of HCC patients with high expression of IFIT2 was significantly prolonged , and knock⁃down of IFIT2 promoted the growth and migration of HepG2 cells . @*Conclusion @#FTO may regulate IFIT2 by mediating m6 A , and further promote the occurrence and development of HCC .
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AIM:To explore the molecular mechanism of Tiaopi Chengqi decoction (TpCqD) improving hyperthermia and high-protein food-induced hyperphagia mice based on transcriptomics. METHODS:C57 mice were randomly divided into a control group, model group, low-dose TpCqD group, high-dose TpCqD group, and domperidone group. The general condition of the experimental mice was observed and the average food intake was counted, and the rate of gastric emptying and intestinal propulsion was determined for each group of mice. H&E staining was used to observe pathological changes in gastric tissue. PAS staining was used to observe glycogen changes in gastric tissue. Pepsin activity was determined by colorimetry. pH value of gastric contents was measured by acid-base titration. Transcriptome sequencing was used to analyze the differential genes in gastric tissue, a volcano map and a cluster heat map were made for the differential genes, and KEGG was used to analyze the signal pathway enrichment of the differential genes. RT-qPCR verified the differential genes obtained by screening. RESULTS:After treatment with TpCqD, the body weight and average food intake of mice with food accumulation increased (P<0.05), and the intestinal propulsion rate and gastric emptying speed of mice with food accumulation accelerated (P<0.05). TpCqD could protect gastric tissue structure and glycogen degradation, increase pepsin activity (P<0.05), and reduce gastric content pH (P<0.05). Transcriptome results showed that TpCqD could regulate the expression of Acox2 and cilp2, regulate fat digestion and absorption, protein digestion and absorption, and pancreatic secretion signals. RT-qPCR showed that compare with model group, TpCqD up-regulated Acox2 (P<0.05) and down-regulated the mRNA level of cilp2 (P<0.05). CONCLUSION:TpCqD ameliorated digestive dysfunction in mice with high-calorie and high-protein diets leading to food accumulation involving the regulation of the fat and sugar metabolism genes Acox2 and cilp2, and pancreatic secretory signaling.
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AIM:To identify transcriptional differences between the ocular surface ectoderm(OSE)and surface ectoderm(SE)using RNA-seq, and elucidate the OSE transcriptome landscape and the regulatory networks involved in its development.METHODS:OSE and SE cells were differentiated from human embryonic stem(hES)cells. Differentially expressed genes(DEGs)between OSE and SE were analyzed using RNA-seq. Based on the DEGs, we performed gene ontology(GO)analysis, Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis, and protein-protein interaction(PPI)network analysis. Transcription factors(TFs)and hub genes were screened. Subsequently, TF-gene and TF-miRNA regulatory networks were constructed using the NetworkAnalyst platform.RESULTS:A total of 4 182 DEGs were detected between OSE and SE cells, with 2 771 up-regulated and 1 411 down-regulated genes in OSE cells. GO-BP analysis revealed that up-regulated genes in OSE were enriched in the regulation of ion transmembrane transport, axon development, and modulation of chemical synaptic transmission. Down-regulated genes were primarily involved in nuclear division, chromosome segregation, and regulation of cell cycle phase transition. KEGG analysis indicated that up-regulated genes in OSE cells were enriched in signaling pathways such as cocaine addiction, axon guidance, and amphetamine addiction, while down-regulated genes were enriched in proteoglycans in cancer, ECM-receptor interaction, protein digestion and absorption, and cytokine-cytokine receptor interaction. Additionally, compared with SE, 204 TFs(including FOS, EGR1, POU5F1, SOX2, and PAX6)were up-regulated, and 80 TFs(including HAND2, HOXB6, HOXB5, HOXA5, and HOXB8)were down-regulated in OSE cells. Furthermore, we identified 6 up-regulated and 9 down-regulated hub genes in OSE cells, and constructed TF-gene and TF-miRNA regulatory networks based on these hub genes.CONCLUSIONS:The transcriptome characteristics of OSE and SE cells were elucidated through RNA-seq analysis. These findings may provide a novel insight for studies on the development and in vitro directed induction of OSE and corneal epithelial cells.
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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.
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ObjectiveTo develop traditional Chinese medicine （TCM） formulae for the treatment of nonsevere coronavirus disease 2019 （COVID-19） and to explore its anti-inflammatory mechanism. MethodsThe dysregulated signaling pathways were determined in macrophages from bronchoalveolar lavage fluid of COVID-19 patients and in lung epithelial cells infected with SARS-CoV-2 in vitro based on transcriptome analysis. A total of 102 TCM formulae for the clinical treatment of nonsevere COVID-19 were collected through literature. The pathway-reversing rates of these formulae in macrophages and lung epithelial cells were evaluated based on signature signaling pathways， and the basic formula was determined in conjunction with TCM theory. The commonly used Chinese materia medica for nonsevere COVID-19 were summarized from the 102 TCM formulae as abovementioned. And together with the screening results from the Pharmacopoeia of the People's Republic of China， a “Chinese materia medica pool” was esta-blished for the development of TCM formulae for COVID-19. The regulatory effects of each herb on signaling pathways were obtained based on targeted transcriptome analysis. Oriented at reversing dysregulated signaling pathways of COVID-19， the calculation was carried out， and the artificial intelligent methods for compositing formulae， that are exhaustive method and parallel computing， were used to obtain candidate compound formulas. Finally， with reference to professional experience， an innovative formula for the treatment of nonsevere COVID-19 was developed. The ethanol extract of the formula was evaluated for its anti-inflammatory effects by detecting the mRNA expression of interleukin 1b （Il1b）， C-X-C motif chemokine ligand 2 （Cxcl2）， C-X-C motif chemokine ligand 10 （Cxcl10）， C-C motif chemokine ligand 2 （Ccl2）， nitric oxide synthase 2 （Nos2）， and prostaglandin-endoperoxide synthase 2 （Ptgs2） using reverse transcription-quantitative polymerase chain reaction （RT-qPCR） in RAW264.7 cells treated with lipopolysaccharide （LPS）. ResultsIn macrophages and lung epithelial cells， 34 dysregulated signaling pathways associated with COVID-19 were identified respectively. The effects of the 102 formulae for clinical treatment of nonsevere COVID-19 were evaluated based on the dysregulated signaling pathways and targeted transcriptome， and the result showed that Yinqiao Powder and Pingwei Powder （银翘散合平胃散， YQPWP） ranked first， reversing 91.18% of the dysregulated signaling pathways in macrophages and 100% of the dysregulated signaling pathways in lung epithelial cells. Additionally， YQPWP had the function of scattering wind and clearing heat， resolving toxins and removing dampness in accordance with the pathogenesis of wind-heat with dampness in COVID-19. It was selected as the basic formula， and was further modified and optimized to develop an innovative fomula Qiaobang Zhupi Yin （翘蒡术皮饮， QBZPY） based on expert experience and artificial intelligence in composing formulae. QBZPY can reverse all the dysregulated signaling pathways associated with COVID-19 in macrophages and lung epithelial cells， with the reversing rates of 100%. The chief medicinal of QBZPY， including Lianqiao （Fructus Forsythiae）， Xixiancao （Herba Siegesbeckiae） and Niubangzi （Fructus Arctii）， can down-regulate multiple signaling pathways related with virus infection， immune response， and epithelial damage. RT-qPCR results indicated that compared with the model group， the QBZPY group down-regulated the mRNA expression of Il1b， tumor necrosis factor （Tnf）， Cxcl2， Cxcl10， Ccl2， Nos2 and Ptgs2 induced by LPS in RAW264.7 cells （P＜0.05 or P＜0.01）. ConclusionBased on targeted transcriptome analysis， expert experience in TCM and artificial intelligence， QBZPY has been developed for the treatment of nonsevere COVID-19. The ethanol extract of QBZPY has been found to inhibit mRNA expression of several pro-inflammatory genes in a cellular inflammation model.
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【Objective】 To explore the effects of finasteride on the gene expression in patients with benign prostatic hyperplasia (BPH) through transcriptome analysis. 【Methods】 Postoperative prostate tissues from patients who underwent prostatectomy at Peking University Third Hospital during Oct.2020 and Oct.2021 were collected.The patients were divided into medication group and non-medication group based on whether they had taken finasteride for a long time before surgery, with 8 patients in either groups.Transcriptome sequencing analysis was performed and the results were validated with qPCR and immunohistochemistry analysis. 【Results】 Compared with the non-medication group, 857 up-regulated and 806 down-regulated genes were screened in the medication group.Pathway enrichment analysis showed that finasteride induced down-regulation of vascular endothelial growth factor D (VEGFD) expression in the focal adhesion pathway.Inter group network analysis suggested that the calcium signaling pathway was key in the entire process.GSEA enrichment analysis further revealed the up regulation of CD38 gene expression in the calcium signaling pathway.The qPCR and immunohistochemistry analysis supported the transcriptome results mentioned above, and found that androgen receptor (AR) expression was also increased. 【Conclusion】 Finasteride reduces prostate microvascular formation by downregulating the expression of VEGFD in the focal adhesion pathway, thereby reducing the risk of bleeding during prostate hyperplasia surgery. Long-term use of finasteride leads to the up regulation of CD38 expression in the calcium signaling pathway, which may lead to the development of finasteride resistance.
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Objective @#To screen the core genes of diabetic kidney disease ( DKD) based on bioinformatics , ex- plore the therapeutic targets of DKD , and discuss its possible regulatory mechanism .@*Methods @#The expression da- ta matrix of glomerular transcriptome in patients with DKD in GEO database (GSE30528 , GSE47183) was extrac- ted , and the differentially expressed genes ( DEGs) were screened by bioinformatics methods to identify the core differential genes , and then gene expression and enrichment analysis (GSEA) were conducted to predict effective targets . @*Results @#By screening and identifying the mRNA expression matrix of DKD , five core genes were screened out. Among them , C1orf21 and NPHS1 were significantly down regulated , and CD48 , COL1A2 , and TGFBI were up regulated . NPHS1 and CD48 were significantly related to immune differences , intercellular communication , and cell surface interaction . Through receiver operating characteristic curve ( ROC) analysis and GSEA analysis and drug target prediction , it might be of great significance for the treatment of DKD .@*Conclusion @#The core genes screened in this study have significant correlation with DKD , which may be used as effective markers for the treat- ment of diabetes . And then , this study provides a theoretical basis for the treatment and identification of DKD .
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Artemisia argyi (A. argyi), a plant with a longstanding history as a raw material for traditional medicine and functional diets in Asia, has been used traditionally to bathe and soak feet for its disinfectant and itch-relieving properties. Despite its widespread use, scientific evidence validating the antifungal efficacy of A. argyi water extract (AAWE) against dermatophytes, particularly Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum, remains limited. This study aimed to substantiate the scientific basis of the folkloric use of A. argyi by evaluating the antifungal effects and the underlying molecular mechanisms of its active subfraction against dermatophytes. The results indicated that AAWE exhibited excellent antifungal effects against the three aforementioned dermatophyte species. The subfraction AAWE6, isolated using D101 macroporous resin, emerged as the most potent subfraction. The minimum inhibitory concentrations (MICs) of AAWE6 against T. rubrum, M. gypseum, and T. mentagrophytes were 312.5, 312.5, and 625 μg·mL-1, respectively. Transmission electron microscopy (TEM) results and assays of enzymes linked to cell wall integrity and cell membrane function indicated that AAWE6 could penetrate the external protective barrier of T. rubrum, creating breaches ("small holes"), and disrupt the internal mitochondrial structure ("granary"). Furthermore, transcriptome data, quantitative real-time PCR (RT-qPCR), and biochemical assays corroborated the severe disruption of mitochondrial function, evidenced by inhibited tricarboxylic acid (TCA) cycle and energy metabolism. Additionally, chemical characterization and molecular docking analyses identified flavonoids, primarily eupatilin (131.16 ± 4.52 mg·g-1) and jaceosidin (4.17 ± 0.18 mg·g-1), as the active components of AAWE6. In conclusion, the subfraction AAWE6 from A. argyi exerts antifungal effects against dermatophytes by disrupting mitochondrial morphology and function. This research validates the traditional use of A. argyi and provides scientific support for its anti-dermatophytic applications, as recognized in the Chinese patent (No. ZL202111161301.9).
Subject(s)
Antifungal Agents/chemistry , Arthrodermataceae , Artemisia/chemistry , Molecular Docking Simulation , Mitochondria , Microbial Sensitivity Tests摘要
ObjectiveTo provide more basic information of comparative medicine for the study of biological changes and pathogenesis of COVID-19 by systematical sorting and analyzing the transcriptome data.MethodsFollowing a retrieval strategy, using COVID-19 and SARS-CoV-2 as key words, transcriptome datasets related to COVID-19 from January 2020 to May 2023 were collected from GEO, ArrayExpress and GEN Transcriptome databases. The composition, distribution, and research application of COVID-19 transcriptome data resources were analyzed. Data distribution was visually displayed and correlation analysis was performed. The research applications and limitations of existing COVID-19 transcriptome data were analyzed from the perspectives of clinical medicine and comparative medicine, focusing on clinical-related molecular mechanisms, biomarkers and related immune responses, treatment intervention strategies, etc. The research value and application prospects were discussed.Results A total of 975 sets of COVID-19 transcriptome data were included. Among three databases, datasets from humans accounted for the highest proportion, namely 71.9%, 77.9%, and 90%, respectively. Species other than humans, such as mice, were the main sources of data, with the respiratory and nervous systems having the highest distribution of data. Twenty-seven datasets were associated with clinical significance. Analysis revealed that respiratory tract injury and other related molecular mechanisms were obtained through transcriptome data mining. Biomarkers such as cfDNA could be used as therapeutic targets. The severity of COVID-19 infection was associated with cell changes and disorders represented by M1 macrophages. Comparative medical analysis showed that mice, hamsters, and other animals were susceptible to SARS-CoV-2. Rhesus monkeys and cynomolgus monkeys exhibited infection characteristics highly similar to human. Apart from respiratory symptoms, hamsters also exhibited digestive system symptoms. SARS-CoV-2 can replicate in the respiratory organs of various susceptible animals, the intestines of ferrets and the ears of minks, resulting in interstitial pneumonia, diffuse lung injury and other pathological changes of varying degrees. Based on the differences in immune responses, hamsters can be used for neutralizing antibody reaction research.Conclusion Currently there is a wealth of COVID-19 transcriptome data, but there is a lack of comparative transcriptome research. Transcriptomics can be combined with comparative medicine to further explore the differences in comparative medicine of COVID-19.
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Objective:Study on the correlation between the active components of Salviea Miltiorrhizae Radix et Rhizoma screened by high-throughput sequencing and the regulation of lncRNA-mRNA in human lung adenocarcinoma A549 cells.Methods:A549 cells were cultured, and the IC 50 dose of cryptotanshinone and tanshinone ⅡA was confirmed according to the cell proliferation experiment. A549 cells were randomly divided into blank control group, cryptotanshinone group, and tanshinone IIA group using a random number table method. After 24 hours of intervention, the cell cycle was detected by flow cytometry. High-throughput sequencing technique was used to detect the expressions of lncRNA and mRNA in A549 cells in intervention group and non-intervention group. By analyzing the expression profiles of differential genes related to cryptotanshinone and tanshinone ⅡA, the obtained differential genes were analyzed by GO and KEGG. Results:The cell cycle results showed that the proportion of G0/G1 phase cells in cryptotanshinone and Tanshinone ⅡA was increased ( P<0.01), the proportion of S phase cells was decreased ( P<0.01), and the proportion of G2/M phase cells in cryptotanshinone was decreased ( P<0.01). The results of high-throughput screening showed that cryptotanshinone could up-regulate 4 698 lncRNA, down-regulate 1 557 lncRNA, up-regulate 4 810 mRNA and down-regulate 5 644 mRNA. Tanshinone ⅡA could up-regulate 1 348 lncRNA, down-regulate 1 299 lncRNA, up-regulate 4646 mRNA and down-regulate 4 741 mRNA. The function and pathway enrichment analysis of differential lncRNA and mRNA showed that the differentially expressed genes of cryptotanshinone and tanshinone ⅡA were mainly related to cell cycle, autophagy, AMPK signaling pathway, FoxO signaling pathway and EGFR signaling pathway. GAS5 may be one of the targets for the inhibitory effects of cryptotanshinone and tanshinone ⅡA. Conclusion:Cryptotanshinone and tanshinone ⅡA have certain inhibitory effects on A549 cells, and there are differentially expressed genes of lncRNA-mRNA, which are closely related to cell cycle and signal pathway.
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Objective To investigate the effect of cisplatin treatment on the transcriptional level of human liver cancer cells by conducting transcriptome sequencing analysis after treating human liver cancer cell lines with differ-ent concentrations of cisplatin(CDDP).Methods Liver cancer cell lines HepG2 and Huh7 were incubated with cisplatin at different final concentrations of 0,20,50,100 and 200 μmol/L.After 12 hours,cell viability,immuno-fluorescence and RNA-sequencing(RNA-seq)were performed.Differential gene expression analysis(DEG),KEGG pathway analysis,and protein-protein interaction network analysis were conducted.Results Cisplatin de-creased cell viability and increased DNA damage in HepG2,Huh7 cells.Among the genes regulated after cisplatin treatment at different concentrations,59 genes were commonly up-regulated in both HepG2 and Huh7 cells,while 81 genes were commonly down-regulated.The commonly upregulated genes were mainly enriched in cancer initiation and progression pathways.The 81 commonly down-regulated genes were mainly enriched in Rap1 signaling pathway,Ras signaling pathway,signaling pathways regulating pluripotency of stem cells,axon guidance,and cell adhesion-related pathways.Survival analysis of key nodes in the protein-protein interaction network of commonly up-regulated and downregulated genes revealed a significant correlation between high expression of Jun proto-oncogene,AP-1 transcription factor subunit(JUN)and prolonged patient survival and a significant correlation between low ex-pression of growth arrest and DNA damage inducible alpha(GADD45A)and prolonged patient survival.Conclu-sions The study revealed common transcriptional changes in liver cancer cells under cisplatin treatment.Differential expression of JUN and GADD45A is a potential core mechanism to explain drug resistance.This conclusion provides some important prognostic indicators for clinical treatment.
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Objective To explore the role and possible molecular mechanism of Isocitrate dehydrogenase 1(IDH1)gene in proliferation and migration of intrahepatic cholangiocarcinoma(iCCA)cell HuCCT1.Methods HuCCT1 cells with IDH1 gene knockout(HuCCT1IDH1-/-)were constructed by CRISPR/Cas9 gene editing technology.To investigate the capacities of proliferation,migration and invasion of HuCCT1WT(HuCCT1 cells with wild-type IDH1 gene)and HuCCT1IDH1-/-cells,assays of CCK-8,clone formation,scratch and transwell were performed.Western blotting was used to detect the expression levels of epithelial-mesenchymal transition(EMT)associated proteins E-cadherin,N-cadherin,Vimentin,MMP-9,Wnt3a and β-catenin in two groups of cells.The transcriptome sequencing data of HuCCT1WT and HuCCT1IDH1-/-cells were analyzed by bioinformatics methods,Western blotting was used to verify the expression of signaling pathway-related proteins.Results Compared with HuCCT1WT cells,HuCCT1IDH1-/-cells showed the number of proliferation and clone formation significantly reduced(P<0.05),the proportion of cells blocked in G2/M phase was significantly increased(P<0.01),the rate of scratch healing was significantly decreased(P<0.01),and the number of migrated cells(P<0.001)and invaded cells(P<0.05)was significantly reduced.qRT-PCR assay showed that the expression levels of IDH1,Vimentin,MMP-9 and genes related to the regulation of G2/M cycle proliferation,Cyclin A2,Cyclin B1 and CDK1 mRNA were down-regulated in HuCCT1IDH1-/-cells(P<0.05),and the expression of CDH1 mRNA encoding E-cadherin was up-regulated(P<0.01);Western blotting assay showed that the expression level of E-cadherin in HuCCT1IDH1-/-cells was significantly increased(P<0.05),and the expression level of N-cadherin,Vimentin and MMP-9 protein was significantly decreased(P<0.05)than that in HuCCT1WT cells.Data of transcriptome sequencing revealed 1476 differentially expressed genes(DEGs)between two groups of HuCCT1 cells.Go enrichment analysis showed the DEGs were significantly enriched in cell biological processes associated with inflammatory response,cell signaling and cell metabolism.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis suggested that the DEGs may be involved in some signaling pathways such as Wnt,MAPK,Rap1,Hippo and TNF,which are closely related to the regulation of proliferation and invasion of tumor cells.Western blotting verification results showed that compared with HuCCT1WT cells,the relative expression of Wnt3a and β-catenin proteins of HuCCT1IDH1-/-cells was significantly decreased(P<0.05).Conclusions IDH1 gene may participate in the control of biological functions of HuCCT1 cells,including cell proliferation,migration,invasion and epithelial mesenchymal transition.The mechanism may be related to the activation of the Wnt/β-catenin signaling pathway.
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BACKGROUND:A previous study by our group found that protein phosphatase 2Cm(PP2Cm)null mice developed significantly fewer symptoms of renal failure relative to wild-type mice,and thus it was speculated that PP2Cm may play an important protective role in the development of renal fibrosis,however,the molecular mechanisms remain undefined. OBJECTIVE:To investigate the effect of the PP2Cm gene on the transcriptome of human renal tubular epithelial cells. METHODS:Cultured human renal tubular epithelial cells were transfected with the PP2Cm gene into human renal tubular epithelial cells using plasmids.The expression of PP2Cm in the cells was detected by fluorescence quantitative PCR assay and western blot assay,and subsequently,cell RNA was separately extracted for transcriptome sequencing to look for differentially expressed genes between transfected and control groups.The resulting differential genes were further subjected to GO analysis and KEGG analysis using bioinformatics methods. RESULTS AND CONCLUSION:There were 796 differentially expressed genes,553 of which were downregulated genes and 243 upregulated genes,in human renal tubular epithelial cells transfected with the PP2Cm gene compared with untransfected blank cells by sequencing analysis.GO analysis results showed that the upregulated genes were significantly enriched in cellular biosynthetic processes,protein translation,intrinsic apoptotic signaling pathways,and so on.The downregulated expressed genes were significantly enriched in endothelial cell proliferation,cell adhesion and other signaling pathways.KEGG analysis results showed that the significantly up-regulated genes were enriched in metabolism-related signaling pathways such as amino acid metabolism and biosynthesis.The downregulated expressed genes were significantly enriched in signaling pathways such as pantothenate and coenzyme A biosynthesis.Our results show that PP2Cm overexpression can affect a number of signaling pathways related to a range of biological processes in renal tubular epithelial cells,which may be important in metabolism-related signaling pathways such as amino acid metabolism and biosynthesis.
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BACKGROUND:Fluorosis is a disorder of enamel development caused by long-term intake of large amounts of fluoride during enamel development. OBJECTIVE:To further explore the molecular mechanism of dental fluorosis formation by screening the differentially expressed genes associated with calcium homeostasis in ameloblasts by transcriptome sequencing technology. METHODS:LS8 cells were treated with 0,0.4,0.8,1.6,3.2 and 6.4 mmol/L sodium fluoride(NaF)for 24,48 and 72 hours to observe the effects of different concentrations of NaF on the morphology,cell activity and intracellular Ca2+ concentration of LS8 cells.The differentially expressed genes were screened by transcriptome sequencing and validated. RESULTS AND CONCLUSION:After 24 hours of treatment,the cells treated with 0,0.4,and 0.8 mmol/L NaF were in good growth condition,with increased cell number and clear cell outline.When the NaF concentration was≥1.6 mmol/L,the cells were gradually shrunken and became smaller and the number of cells decreased with the increase of NaF concentration.After 48 and 72 hours of treatment,the number of cells increased in the 0,0.4 mmol/L NaF groups,while gradually decreased in the 0.8,1.6,3.2 mmol/L NaF groups,with rounded and smaller cell morphology.The cells in the 6.4 mmol/L NaF group were shrunken,rounded and suspended in the medium,with almost no adherent cells.When treated with the same concentration of NaF,LS8 cells were in optimal growth after 24 hours of treatment.Results from cell counting kit-8 assay showed that when treated with the same concentration of NaF,the cell activity decreased with the increase of treatment time;when the treatment time was the same,the cell activity decreased with the increase of NaF concentration.After 24 hours of treatment,the intracellular Ca2+ concentration increased with the increase of NaF concentration.Transcriptome sequencing analysis identified genes involved in the regulation of cellular calcium homeostasis:Hsp90b1,Canx,Calr,and Hspa5 that were significantly upregulated(P＜0.05)and Cacna1a that was significantly downregulated(P＜0.05).To conclude,the inhibitory effect of NaF on LS8 cell proliferation may be related to the abnormal increase in intracellular Ca2+ concentration,and the mechanism may be caused by the upregulation of the expression of protein processing and synthesis pathways Hsp90b1,Canx,Calr,and Hspa5 and the downregulation of the expression of calcium signaling pathway Cacna1a.
摘要
BACKGROUND:Ferroptosis-mediated ischemia-reperfusion injury plays a crucial role in the occurrence and progression in pressure ulcers,and there may be pressure ulcer-associated ferroptosis biomarkers,but the mechanism has not been elucidated. OBJECTIVE:To investigate the molecular mechanisms underlying pressure ulcers using bioinformatic analysis,with a focus on identifying differentially expressed genes associated with ferroptosis during the process of pressure ulcer formation,thereby providing novel insights into the clinical treatment of pressure ulcers. METHODS:The single-cell transcriptome sequencing dataset and ferroptosis-related genes were obtained and preprocessed from the Gene Expression Omnibus(GEO)and FerrDb databases.We performed clustering and proportion analyses,metabolic activity and pseudotime analysis,cell communication analysis,ferroptosis gene set cell population identification,and enrichment analysis to determine differentially expressed genes related to ferroptosis.Animal experiments were then conducted for further validation,with 20 Sprague-Dawley rats randomly assigned into a control group and a model group(n=10 per group).The control group received no treatment,while the model group underwent a cycle of ischemia-reperfusion to establish pressure ulcer models.Changes in differentially expressed genes and proteins in the wound tissues of pressure ulcer rats were detected using fluorescent quantitative PCR and western blot,respectively. RESULTS AND CONCLUSION:The single-cell transcriptome sequencing data were clustered into six cell types,with a higher proportion of type 2 and type 3 keratinocytes observed in the pressure ulcer group.There was evident metabolic heterogeneity and evolutionary trajectory among cell populations.Type 2 and type 3 keratinocytes exhibited stronger cell communication,while type 2 keratinocytes demonstrating optimal ligand-receptor interactions.Type 2 keratinocytes demonstrated higher scores for ferroptosis,accompanied by significant upregulation or downregulation of specific genes.A total of 27 Gene Ontology enrichments,20 Kyoto Encyclopedia of Genes and Genomes enrichments,and 24 ferroptosis-related differentially expressed genes,including glutathione peroxidase 4(GPX4)and acyl-CoA synthetase long chain family member 4(ACSL4),were identified.Animal experiments further confirmed the downregulation of GPX4,the ferroptosis-inhibiting protein,and the upregulation of ACSL4,the ferroptosis-promoting protein,in the model group.Overall,these findings indicate the presence of ferroptosis in pressure ulcer tissue.GPX4 and ACSL4 are important genes regulating ferroptosis in pressure ulcer tissues.
摘要
Background & objectives: During the COVID-19 pandemic, the death rate was reportedly 5-8 fold lower in India which is densely populated as compared to less populated western countries. The aim of this study was to investigate whether dietary habits were associated with the variations in COVID-19 severity and deaths between western and Indian population at the nutrigenomics level. Methods: In this study nutrigenomics approach was applied. Blood transcriptome of severe COVID-19 patients from three western countries (showing high fatality) and two datasets from Indian patients were used. Gene set enrichment analyses were performed for pathways, metabolites, nutrients, etc., and compared for western and Indian samples to identify the food- and nutrient-related factors, which may be associated with COVID-19 severity. Data on the daily consumption of twelve key food componentsacross four countries were collected and a correlation between nutrigenomics analyses and per capita daily dietary intake was investigated. Results: Distinct dietary habits of Indians were observed, which may be associated with low death rate from COVID-19. Increased consumption of red meat, dairy products and processed foods by western populations may increase the severity and death rate by activating cytokine storm-related pathways, intussusceptive angiogenesis, hypercapnia and enhancing blood glucose levels due to high contents of sphingolipids, palmitic acid and byproducts such as CO2 and lipopolysaccharide (LPS). Palmitic acid also induces ACE2 expression and increases the infection rate. Coffee and alcohol that are highly consumed in western countries may increase the severity and death rates from COVID-19 by deregulating blood iron, zinc and triglyceride levels. The components of Indian diets maintain high iron and zinc concentrations in blood and rich fibre in their foods may prevent CO2 and LPS-mediated COVID-19 severity. Regular consumption of tea by Indians maintains high high-density lipoprotein (HDL) and low triglyceride in blood as catechins in tea act as natural atorvastatin. Importantly, regular consumption of turmeric in daily food by Indians maintains strong immunity and curcumin in turmeric may prevent pathways and mechanisms associated with SARS-CoV-2 infection and COVID-19 severity and lowered the death rate. Interpretation & conclusions: Our results suggest that Indian food components suppress cytokine storm and various other severity related pathways of COVID-19 and may have a role in lowering severity and death rates from COVID-19 in India as compared to western populations. However, large multi-centered case?control studies are required to support our current findings.
摘要
Karnal bunt of wheat is an important quarantine disease that interrupts India’s wheat trade in the international market. The whole transcriptome of germinating and dormant teliospores of Tilletia indica was performed using the RNA Seq approach to identify germination-related genes. Approximately 63 million reads were generated using the RNA sequencing by the Illumina NextSeq500 platform. The high-quality reads were deposited in NCBI SRA database (accession: PRJNA522347). The unigenes from the pooled teliospores were 16,575 having unigenes length of 28,998,753 bases. The high-quality reads of germinating teliospores mapped on to 21,505 predicted CDSs. 9,680 CDSs were common between dormant and germinating teliospores of T. indica. 11,825 CDSs were found to be in germinating teliospores while only 91 were unique in dormant spores of pathogen. The pathway analysis showed the highest number of pathways was found in germinating spores than dormant spores. The highest numbers of CDSs were found to be associated with translation (431 in number), transport and catabolism (340), signal transduction (326), and carbohydrate metabolism (283). The differential expression analysis (DESeq) of germinating and dormant teliospores showed that 686 CDS were up-regulated and 114 CDS were down-regulated in the germinating teliospores. Significant germination-related genes in the spores were validated using qPCR analysis. Ten genes viz. Ti3931, Ti6828, Ti7098, Ti7462, Ti7522, Ti 9289, Ti 8670, Ti 7959, Ti 7809,and Ti10095 were highly up-regulated in germinated teliospores which may have role in germination of spores.Further, these differentially expressed genes provide insights into the molecular events. This first study of transcriptome will be helpful to devise better management strategies to manage Karnal bunt disease.