Transcriptome analysis of immune-inflammatory regulation in Tremella fuciformis-derived polysaccharide reeducated B16 cells: A subcutaneous model.

BACKGROUND: Circulating cancer cells have characteristics of tumor self-targeting. Modified circulating tumor cells may serve as tumor-targeted cellular drugs. Tremella fuciformis-derived polysaccharide (TFP) is related to immune regulation and tumor inhibition, so could B16 cells reeducated by TFP be an effective anti-tumor drug? OBJECTIVES: To evaluate the intrinsic therapeutic potential of B16 cells exposed to TFP and clarify the therapeutic molecules or pathways altered by this process. MATERIAL AND METHODS: RNA-seq technology was used to study the effect of TFP-reeducated B16 cells on the immune and inflammatory system by placing the allograft subcutaneously in C57BL/6 mice. RESULTS: Tremella fuciformis-derived polysaccharide-reeducated B16 cells recruited leukocytes, neutrophils, dendritic cells (DCs), and mast cells into the subcutaneous region and promoted the infiltration of several cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 1ß (IL-1ß), and interleukin 1 (IL-1). Tumor necrosis factor alpha also activated Th17 lymphocytes to secrete interleukin 17 (IL-17) and interferon gamma (IFN-γ). The co-expression of IFN-γ and IL-17 was favorable for tumor immunity to shrink tumors. In short, TFP-reeducated B16 cells activated the innate and adaptive immune responses, especially Th17 cell differentiation and IFN-γ production, as well as the TNF-α signaling pathway, which re-regulated the inflammatory and immune systems. CONCLUSION: B16 cells subcutaneously exposed to TFP in mice induced an immune and inflammatory response to inhibit tumors. The study of the function of TFP-reeducated B16 cells to improve cancer immunotherapy may be of particular research interest. This approach could be an alternative and more efficient strategy to deliver cytokines and open up new possibilities for long-lasting, multi-level tumor control.

From tradition to innovation: conventional and deep learning frameworks in genome annotation.

Following the milestone success of the Human Genome Project, the 'Encyclopedia of DNA Elements (ENCODE)' initiative was launched in 2003 to unearth information about the numerous functional elements within the genome. This endeavor coincided with the emergence of numerous novel technologies, accompanied by the provision of vast amounts of whole-genome sequences, high-throughput data such as ChIP-Seq and RNA-Seq. Extracting biologically meaningful information from this massive dataset has become a critical aspect of many recent studies, particularly in annotating and predicting the functions of unknown genes. The core idea behind genome annotation is to identify genes and various functional elements within the genome sequence and infer their biological functions. Traditional wet-lab experimental methods still rely on extensive efforts for functional verification. However, early bioinformatics algorithms and software primarily employed shallow learning techniques; thus, the ability to characterize data and features learning was limited. With the widespread adoption of RNA-Seq technology, scientists from the biological community began to harness the potential of machine learning and deep learning approaches for gene structure prediction and functional annotation. In this context, we reviewed both conventional methods and contemporary deep learning frameworks, and highlighted novel perspectives on the challenges arising during annotation underscoring the dynamic nature of this evolving scientific landscape.

Effect of electroacupuncture on cyclic adenosine monophosphate-protein kinase A-vanillic acid receptor subtype 1 of the transient receptor potential/PLK-protein kinase C-vanillic acid receptor subtype 1 of the transient receptor potential pathway based on RNA-seq analysis in prostate tissue in rats with chronic prostatitis/chronic pelvic pain syndrome.

Objective: To investigate the analgesic mechanism of electroacupuncture (EA) in rats with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Methods: Thirty male SD rats were randomly divided into sham group, model group and EA group, with ten rats in each group. The CP/CPPS model was prepared by injecting 50 µL of complete Freund's adjuvant (CFA) into the ventral lobes of the prostate tissue, and the sham group was injected with the same dose of saline. After 14 days of modeling, EA was applied to Guanyuan (CV4), Zhongji (CV3), Sanyinjiao (SP6) and Huiyang (BL35) in the EA group. After four courses, H&E staining was performed to observe the prostate tissue morphology, transcriptome sequencing (RNA-Seq) was performed for each group, and the selected signaling pathways were verified by qRT-PCR. Results: The RNA-Seq analysis results suggested that the analgesic effect of EA on CP/CPPS may be achieved by regulating prostate gene expression, which may be related to multiple biological processes and signaling pathways. qRT-PCR results showed that the vanillic acid receptor subtype 1 of the transient receptor potential (TRPV1), phospholipase C (PLC), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA) were all upregulated in the model group compared to the sham group (p < 0.01). Compared with the model group, TRPV1, PLC, PKC, cAMP, and PKA were all downregulated in the EA group (p < 0.05, p < 0.01). Conclusion: The analgesic mechanism of EA on CP/CPPS may be achieved through modulation of cAMP-PKA-TRPV1/PLC-PKC-TRPV1 signaling pathway.

Phosphate-Solubilizing Pseudomonas sp. Strain WS32 Rhizosphere Colonization-Induced Expression Changes in Wheat Roots.

Rhizosphere colonization is a pre-requisite for the favorable application of plant growth-promoting rhizobacteria (PGPR). Exchange and mutual recognition of signaling molecules occur frequently between plants and microbes. Here, the luciferase luxAB gene was electrotransformed into the phosphate-solubilizing strain Pseudomonas sp. WS32, a type of plant growth-promoting rhizobacterium with specific affinity for wheat. A labeled WS32 strain (WS32-L) was applied to determine the temporal and spatial traits of colonization within the wheat rhizosphere using rhizoboxes experimentation under natural condition. The effects of colonization on wheat root development and seedling growth were evaluated, and RNA sequencing (RNA-seq) was performed to explore the transcriptional changes that occur in wheat roots under WS32 colonization. The results showed that WS32-L could survive in the wheat rhizosphere for long periods and could expand into new zones following wheat root extension. Significant increases in seedling fresh and dry weight, root fresh and dry weight, root surface area, number of root tips, and phosphorus accumulation in the wheat leaves occurred in response to WS32 rhizosphere colonization. RNA-seq analysis showed that a total of 1485 genes in wheat roots were differentially expressed between the inoculated conditions and the uninoculated conditions. Most of the transcriptional changes occurred for genes annotated to the following functional categories: "phosphorus and other nutrient transport," "hormone metabolism and organic acid secretion," "flavonoid signal recognition," "membrane transport," and "transcription factor regulation." These results are therefore valuable to future studies focused on the molecular mechanisms underlying the growth-promoting activities of PGPR on their host plants.

Effect of 4-chloro-2-methylphenoxy acetic acid on tomato gene expression and rhizosphere bacterial communities under inoculation with phosphate-solubilizing bacteria.

The herbicide 4-chloro-2-methylphenoxy acetic acid (MCPA) is widely used to control the spread of broad-leaved weeds in agricultural soils, though it remains unclear how tomato plants cope with the phytotoxic effects of MCPA at the molecular level. In this study, RNA-seq and Illumina MiSeq were used to sequence bacterial communities in tomato rhizosphere soils treated with MCPA and the phosphate-solubilizing bacterial strain N3. The results showed that MCPA induced abnormal growth of lateral roots in tomato seedlings and reduced uptake of the nutrients N, P, and K as well as the hormone (ABA and GA3) levels. Inoculation with strain N3 increased nutrient uptake by roots and increased levels of the hormones ABA, ZEA, and JA in tomato seedlings and also increased the abundance of the phyla Proteobacteria and Gemmatimonadetes in soil under MCPA treatment. GO functional groups in which differentially expressed genes (DEGs) are involved included DNA binding transcription factor activity, transcriptional regulator activity, enzyme inhibitor activity, and cell wall biogenesis. The highest numbers of DEGs are annotated to ribosome, photosynthesis, and carbon metabolism categories. Our findings provide valuable information for the application of strain N3, which is beneficial for reducing the toxic effect of MCPA on vegetable plants.

Differential miRNA expression in the peripheral blood of patients with Keshan disease and its mechanism / 中华地方病学杂志

Objective:Through differential miRNA expression profiles and bioinformatics in the peripheral blood of patients with Keshan disease (KD) and healthy control, to explore the possible pathogenesis of KD.Methods:Ten patients with chronic KD (KD group) were selected in the severe disease area of KD in Wulian County, and 10 healthy subjects (control group) were selected in non-KD area of Dongchangfu District, Shandong Province. Blood sample of elbow vein was collected and plasma was separated. RNA-seq technology was used to construct the differential expression profiles of miRNA in KD and control groups. Target mRNAs were screened using Starbase, miRTarBase, miRDB and TargetScan. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted to investigate the possible pathogenesis of KD.Results:Compared the control group and KD group, 132 differentially expressed miRNAs were screened out, including 90 upregulated and 42 downregulated miRNAs. Through Starbase, miRTarBase, miRDB and TargetScan, 53 miRNAs were obtained, 737 targeted mRNAs were obtained. GO analysis showed that the differential genes were mainly involved in the biological processes of Ras protein signal transduction, transmembrane transport, cell cycle regulation, cell adhesion, etc. KEGG pathway analysis showed that the differential genes were mainly involved in viral infection, endocytosis, adhesion spot and actin regulation.Conclusion:In this study, RNA-seq technology is used to obtain differential miRNA expression profiles of KD patients and healthy control, and target pathogenic genes and signaling pathways that may be related to KD are screened out.

Analysis of the Mechanism of Yam Protein in the Prevention and Treatment of Diabetes-induced Erectile Dysfunction Based on RNA-Seq Technology / 中国药房

OBJECTIVE：To study the potential mechanism of yam protein （DOT） in the prevention and treatment of diabetes-induced erectile dysfunction （DIED）. METHODS ：DIED model was induced by high-glucose and high-fat diet and intraperitoneal injection of streptozotocin （40 mg/kg）. The experiment was set up in the normal control group （normal saline ）， model group （normal saline ），DOT low-dose ，medium-dose and high-dose groups （0.3，0.6，0.9 mg/kg），sildenafil group （positive control ，4.4 mg/kg），with 9 rats in each group. In the stage of successful establishment of diabetes model and initiation of inducing DIED ，rats in each group were given relevant solution intragastrically ，once a day ，for consecutive 11 weeks. Body weight，fasting plasma glucose （FPG），the times and rate of penile erection ，fasting insulin （FINS），insulin resistance index （IR），the contents of endothelial nitric oxide synthase （eNOS）and cyclic guanosine monophosphate （cGMP）in penile cavernous tissue were determined so as to evaluate the intervention effects of DOT on DIED model rats. High-glucose damaged mice cavernous endothelial cells （MCECs）model was induced by 30 mmol/L glucose for 48 h，and then give DOT 125，250，500 μg/mL. The cell viability was detected so as to evaluate the effects of DOT on high-glucose damaged MCECs model. RNA-Seq mail：xingxin0902@163.com technology was adopted to screen the differentially expressed genes between normal MCECs and high-glucose damaged MCECs，high-glucose damaged MCECs and MCECs treated with 250 μg/mL DOT. Gene ontology（GO）function enrichment analysis and KEGG pathway enrichme nt analysis were performed for differentially expressed genes. The common differentially expressed genes between 2 groups were analyzed ，and mRNA expressions of six key genes were validated. RESULTS ：Different doses of DOT could reverse the reduction of body weight ，the increase of FINS and IR ，the reduction of the times and rate of penile erection ，the decrease of eNOS and cGMP contents in penile cavernous tissue of DIED model rats ；above indexes of DIED model rats were reversed significantly after treated with high-dose of DOT（P＜0.05 or P＜0.01）. 125，250，500 μg/L DOT could significantly improve the activity of high-glucose damaged MCECs （P＜0.05 or P＜0.01）. RNA-Seq technology showed that compared with normal MCECs ，a total of 48 differentially expressed genes were found in high-glucose damaged MCECs. Compared with high-glucose damaged MCECs ，a total of 779 differentially expressed genes were found in MCECs treated with DOT. The differentially expressed genes of 2 groups were mainly cellular process in biological process annotation ，cellular part in cell component annotation and binding molecular function in molecular function annotation ，which were mainly enriched in extracellular matrix receptor interaction pathway ，mismatch repair pathway ， phosphatidylinositol 3 kinase-protein kinase B （PI3K-Akt）signal pathway and so on. Among differentially expressed genes of 2 groups，13 common differentially expressed genes such as Aldh1a1，Abcc5，Tac1 were found. DOT could significantly reverse the expression of the above common differentially expressed genes in high-glucose damaged MCECs. After validation ，DOT could significantly reverse the mRNA expression of TGF-β3，Txnip，Aldh1a1，Loxl1，Mt1 and Mt2 in high-glucose damaged MCECs. CONCLUSIONS：DOT could improve the symptom of DIED model rats ，the mechanism of which may be related to biological pathway of inhibiting fibrosis and reducing oxidative stress ，so as to improve the endothelial function of cavernous body.

Biological functions of nirS in Pseudomonas aeruginosa ATCC 9027 under aerobic conditions.

Through our previous study, we found an up-regulation in the expression of nitrite reductase (nirS) in the isothiazolone-resistant strain of Pseudomonas aeruginosa. However, the definitive molecular role of nirS in ascribing the resistance remained elusive. In the present study, the nirS gene was deleted from the chromosome of P. aeruginosa ATCC 9027 and the resulting phenotypic changes of ΔnirS were studied alongside the wild-type (WT) strain under aerobic conditions. The results demonstrated a decline in the formations of biofilms but not planktonic growth by ΔnirS as compared to WT, especially in the presence of benzisothiazolinone (BIT). Meanwhile, the deletion of nirS impaired swimming motility of P. aeruginosa under the stress of BIT. To assess the influence of nirS on the transcriptome of P. aeruginosa, RNA-seq experiments comparing the ΔnirS with WT were also performed. A total of 694 genes were found to be differentially expressed in ΔnirS, of which 192 were up-regulated, while 502 were down-regulated. In addition, these differently expressed genes were noted to significantly enrich the carbon metabolism along with glyoxylate and dicarboxylate metabolisms. Meanwhile, results from RT-PCR suggested the contribution of mexEF-oprN to the development of BIT resistance by ΔnirS. Further, c-di-GMP was less in ΔnirS than in WT, as revealed by HPLC. Taken together, our results confirm that nirS of P. aeruginosa ATCC 9027 plays a role in BIT resistance along with biofilm formation and further affects several metabolic patterns under aerobic conditions.

Differentially expressed genes of HepG2 cells treated with gecko polypeptide mixture.

Gecko (Gekko japonicus) extracts have been used in traditional Chinese medicine for many years. It has been proven that the gecko polypeptide mixture (GPM) extracted from gecko can inhibit the growth of multiple types of tumor cells. In order to investigate the possible anti-tumor molecular mechanisms of GPM, we used RNA-seq technology to identify the differentially expressed genes (DEGs) of human hepatocellular carcinoma (HCC) HepG2 cells treated with or without GPM. MTT assay was used to detect the viability of HepG2 cells. DAPI fluorescence staining was performed to observe morphological changes in the nuclei of HepG2 cells. Western blot analysis was applied to observe the expressions of apoptosis-related and endoplasmic reticulum stress (ERS)-related proteins in HepG2 cells. Flow cytometry assay was performed to detect the apoptosis and reactive oxygen species (ROS) in HepG2 cells. Our results showed that GPM inhibited HepG2 cells proliferation and induced the apoptosis of HepG2 cells. RNA-seq analysis suggested that the ER-nucleus signaling pathway involved in the anti-cancer molecular mechanism of GPM. Therefore, GPM may induce apoptosis in HepG2 cells via the ERs pathway.

Integrated study on comparative transcriptome and skeletal muscle function in aged rats.

The present study aimed to reveal aging-related changes in the skeletal muscle of SD rats by comparing transcriptome analysis, integrated with muscle physiological parameters. Ten rats aged 25 months were set as the old group (OG) and ten rats aged 6 months were set as the young group (YG). After 6 weeks of feeding, the body mass, grip strength, and gastrocnemius muscle mass were determined, and the differentially expressed genes were analyzed by transcriptome sequencing, followed by GO enrichment analysis and KEGG analysis. The results showed that the muscle index and the relative grip strength were lower in OG rats than YG rats. The expressions of AMPK, UCP3, IGF-1, several ion channel associated genes and collagen family genes were down-regulated in OG rats. MGMT, one of the strength determining genes and CHRNa1, a subunit of the acetylcholine receptor were up-regulated in OG rats. The present results supply the global transcriptomic information involved in aging related skeletal muscle dysfunction in rats. The reduced expressions of AMPK, IGF-1, and CASK can explain the losses of muscle mass and function in the aged rats. In addition, the up-regulation of MGMT and CHRNa1 also contribute to muscle wasting and weakness during aging.

Differential expression of genes in HepG2 cells caused by UC001kfo RNAi as shown by RNA-seq.

The differential expression of genes in HepG2 cells caused by UC001kfo RNAi was investigated using RNA-seq. HepG2 cells were infected by Lenti-shUC001kfo lentivirus particles. The expression of UC001kfo mRNA in the HepG2-shUC001kfo cell line was detected by real-time PCR. RNA-seq technology was used to identify the difference in the expression of genes regulated by lncRNA UC001kfo in the HepG2 cell line. Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different mRNAs. The results showed that mRNAs were differentially expressed between the HepG2-shUC001kfo cell line and the HepG2 cell line. The UC001kfo mRNA was significantly down-regulated in the stable cell line HepG2-shUC001kfo (P<0.001). Additionally, we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics, cell adhesion, invasion and migration. The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lncRNA UC001kfo. LncRNA UC001kfo may play a role in regulating cancer cell invasion and metastasis. It was suggested that mRNAs were differentially expressed in the HepG2 cell line after the down-regulation of lncRNA-UC001kfo. Some took part in the extracellular matrix, cell adhesion, motility, growth, and localization. The genes encoding of differentially expressed mRNAs may participate in cell invasion and metastasis.

Transcriptome Analysis of a Female-sterile Mutant (fsm) in Chinese Cabbage (Brassica campestris ssp. pekinensis).

Female-sterile mutants are ideal materials for studying pistil development in plants. Here, we identified a female-sterile mutant fsm in Chinese cabbage. This mutant, which exhibited stable inheritance, was derived from Chinese cabbage DH line 'FT' using a combination of isolated microspore culture and ethyl methanesulfonate mutagenesis. Compared with the wild-type line 'FT,' the fsm plants exhibited pistil abortion, and floral organs were also relatively smaller. Genetic analysis indicated that the phenotype of fsm is controlled by a single recessive nuclear gene. Morphological observations revealed that the presence of abnormal ovules in fsm likely influenced normal fertilization process, ultimately leading to female sterility. Comparative transcriptome analysis on the flower buds of 'FT' and fsm using RNA-Seq revealed a total of 1,872 differentially expressed genes (DEGs). Of these, a number of genes involved in pistil development were identified, such as PRETTY FEW SEEDS 2 (PFS2), temperature-induced lipocalin (TIL), AGAMOUS-LIKE (AGL), and HECATE (HEC). Furthermore, GO and KEGG pathway enrichment analyses of the DEGs suggested that a variety of biological processes and metabolic pathways are significantly enriched during pistil development. In addition, the expression patterns of 16 DEGs, including four pistil development-related genes and 12 floral organ development-related genes, were analyzed using qRT-PCR. A total of 31,272 single nucleotide polymorphisms were specifically detected in fsm. These results contribute to shed light on the regulatory mechanisms underlying pistil development in Chinese cabbage.

Differential Expression of Genes in HepG2 Cells Caused by UC001kfo RNAi as Shown by RNA-seq / 华中科技大学学报（医学）（英德文版）

The differential expression of genes in HepG2 cells caused by UC001kfo RNAi was investigated using RNA-seq.HepG2 cells were infected by Lenti-shUC001kfo lentivirus particles.The expression of UC001kfo mRNA in the HepG2-shUC001kfo cell line was detected by real-time PCR.RNA-seq technology was used to identify the difference in the expression of genes regulated by lncRNA UC001kfo in the HepG2 cell line.Gene ontology and signaling pathway analysis were performed to reveal the biological functions of the genes encoding of significantly different mRNAs.The results showed that mRNAs were differentially expressed between the HepG2-shUC001kfo cell line and the HepG2 cell line.The UC001kfo mRNA was significantly down-regulated in the stable cell line HepG2-shUC001kfo (P＜0.001).Additionally,we found 19 signaling pathways or functional classifications encompassing 30 genes that played a role in cancer characteristics,cell adhesion,invasion and migration.The results also showed that the expression of many genes associated with cancer cell invasion and metastasis was decreased with the down-regulation of the lncRNA UC001kfo.LncRNA UC001kfo may play a role in regulating cancer cell invasion and metastasis.It was suggested that mRNAs were differentially expressed in the HepG2 cell line after the down-regulation of lncRNA-UC001kfo.Some took part in the extracellular matrix,cell adhesion,motility,growth,and localization.The genes encoding of differentially expressed mRNAs may participate in cell invasion and metastasis.

Transcriptomic analysis of human breast cancer cells reveals differentially expressed genes and related cellular functions and pathways in response to gold nanorods.

Breast cancer is the leading cause of cancer deaths in women. Recent advances in nanomedicine have shown that gold nanorods (AuNRs), as multifunctional drug delivery and photothermal therapeutic agents, have potential for use in cancer therapy. However, the effect of AuNRs on the transcriptome of breast cancer cells is unknown. In the present study, cells of the triple-negative human breast cancer cell line MDA-MB-231, which has high metastatic activity, were treated with AuNRs for transcriptomic analysis using RNA-seq technology. In total, 3126 genes were found to be up-regulated and 3558 genes were found to be down-regulated in AuNR-treated MDA-MB-231 cells. These differentially expressed genes presumably take part in multiple biological pathways, including glycolysis and regulation of the actin cytoskeleton, and impact a variety of cellular functions, including chemoattractant activity. The distinct gene expression profile of MDA-MB-231 cells treated with AuNRs provides a foundation for further screening and validation of important genes involved in the interaction between AuNRs and MDA-MB-231 cells.