Firmicutes/Bacteroidetes ratio changed with corylin intervention, and the microbial diversity and community richness of the AOM/DSS mice were improved by corylin. The comparative analysis of gut microbiota revealed that Bacteroidetes, Patescibacteria, Candidatus Saccharimonas, Erysipelatoclostridium, and Enterorhabdus were significantly increased but Firmicutes, Turicibacter, Romboutsia, and Blautia decreased after corylin treatment. Altogether, corylin administration showed cancer-ameliorating effects by reducing the risk of colitis-associated colon cancer via regulation of inflammation, carcinogenesis, and compositional change of gut microbiota. Therefore, corylin could be a novel, potential health-protective, natural agent against CAC.Inflammatory bowel disease (IBD) involves chronic inflammation, loss of epithelial integrity, and gastrointestinal microbiota dysbiosis, resulting in the development of a colon cancer known as colitis-associated colorectal cancer (CAC). In this study, we evaluated the effects of corylin in a mouse model of dextran sodium sulfate (DSS)-induced colitis. The results showed corylin could improved the survival rate and colon length, maintained body weight, and ameliorated the inflammatory response in the colon. Then, we further identified the possible antitumor effects after 30-day treatment of corylin on an azoxymethane (AOM)/DSS-induced CAC mouse model. Biomarkers associated with inflammation, the colon tissue barrier, macrophage polarization , and microbiota dysbiosis were monitored in the AOM/DSS group versus corylin groups. Corylin downregulated pro-inflammatory cytokines  mRNA expression and inflammatory signaling-associated markers . In addition, a colon barrier experiment revealed that epithelial cell proliferation of the mucus layer  was downregulated and tight junction proteins (claudin-1 and ZO-1) were upregulated. Furthermore, the  Colorectal cancer (CRC) is the most commonly diagnosed malignancy and the leading cause of cancer mortality worldwide . ChronicGut permeability plays a pivotal role in the maintenance of intestinal homeostasis, metabolism, and immune tolerance. A single epithelial layer of the intestinal barrier consists of multiple components: a robust junctional complex consisting of tight junctions (TJs), which are comprised of multiple strands of adhesive transmembrane molecules , connected via intracellular adaptor proteins  to the actin cytoskeleton ,7 to forDisruption of the gut microbiota or dysbiosis can increase lipopolysaccharide (LPS) production by Gram-negative bacteria, which leads to impaired gut barrier function and causes endotoxemia ,11,12. LPsoralea corylifolia L. [Corylin is a compound isolated from the fruit and seed of folia L.  and possfolia L.  and ostefolia L. . In addifolia L. . Furtherfolia L. , are thaUpon DSS challenge, mice exhibited a lower survival rate B, increaFurthermore, we confirmed that DSS-induced colitis mice colons showed reduced barrier function. The expression levels of claudin-1, ZO-1, and occludin were significantly decreased in the DSS-alone treatment group. Compared with the DSS-alone treatment group, the expression levels of claudin-1, occludin, and ZO-1 were significantly increased in the corylin treatment groups. Furthermore, corylin, at a dose of 100 mg/kg (H), significantly increased the levels of claudin-1 and occludin, which were even higher than for the effects produced by corylin at a dose of 25 mg/kg (L) compared with colitis mice. We propose that the effects of corylin on DSS-induced colitis mice are related to the regulation of the colonic mucosal barrier function, where the expression levels of claudin-1, ZO-1, and occludin play important roles in maintaining the intestinal mucosal barrier function H,I.Growing evidence indicates that the TLR4/NF-κB signaling pathway plays an important role in the pathogenesis of IBD. Thus, targeted suppression of the TLR4 signaling pathway has become a therapeutic strategy for IBD in recent years. To determine the effect of corylin on TLR4 expression, an immunohistochemical analysis and a Western blot analysis were carried out. The immunohistochemical assay showed that DSS markedly increased the overexpression of TLR4, MyD88, p-p38, and AP-1 compared with the normal group. However, the treatment with corylin could decrease signaling on the TLR4 signal pathway A,B.Ifnγ, Tnf-α, Il-6, Il-1β, Nlrp3, Asc, Pannexin, and Pro-caspase 1 mRNA expression and protein levels in DSS-induced colitis mice. Administration of 100 mg/kg (high dose) corylin significantly reduced the inflammatory response and had a more beneficial effect than 25 mg/kg corylin in DSS-induced colitis mice , which promote proliferation and tumor formation. To understand whether cancer stem cells and intestinal epithelial cells are involved in DSS-induced colitis, LGR5, CD44, Ki67, PCNA, BrdU, and Cyclin D1 were examined in the colon A. To furtis mice B. Corylin = 5)  every day for 30 days. The fourth group of mice was untreated  and served as the control group (n = 5) A. There n = 5) B–F. Addin = 5) H,I. The n = 5) B–I. Meann = 5) E–I.The expression levels of claudin-1, ZO-1, and occludin were significantly decreased in the AOM/DSS treatment group. Compared with the AOM/DSS treatment group, the levels of claudin-1, occludin, and ZO-1 were significantly increased in the corylin treatment groups. Furthermore, as shown in Progressively activated TLR4 encourages the proliferation of colon cancer cells and extricates cancer cells from death. As shown in Ifnγ, Tnf-α, Il-6, Il-1β, Nlrp3, Asc, Pannexin, and Pro-caspase 1 , CD11b, CCR7 (M1 markers), CD163, and CD206 (M2 markers) macrophage markers in colon tissue. According to Lgr5, Olfm4, and Cyclin D1 was increased in the AOM/DSS group, and corylin reversed that effect in the colon of mice  was employed to characterize the overall pattern of the gut microbiota community in AOM/DSS- and corylin-treated mice. In order to demonstrate the bacterial diversity, richness, abundance, and structural differences in each group, alpha diversity indices were evaluated. The alpha diversity parameters were determined using the Simpson reciprocal, the Shannon index, and ENSPIE A–C. In tNext, we investigated the hierarchical relationship of all the gut microbiota. In the interactive html result, the circles show the different classification levels from the phylum to the genus (inside to outside) in the community, and the area of the sector shows the respective proportion of different OTU annotation results F,G. CoryActinobacteria, Patescibacteria, Bacteroidetes, Bacteroidetes/Firmicutes ratio, Candidatus Saccharimonas, Erysipelatoclostridium, Enterorhabdus, Coriobacteriaceae UCG-002, and Enterorhabdus mucosicola  was calculated. We created a bar plot to present the results. All sequences were classified and identified from phylum to species level . The 16Sum lowii H–K,P–R. Psoralea corylifolia L. (Fabaceae) [P. corylifolia L., corylin is the main flavonoid and shows several therapeutic effects, such as anti-inflammatory, anti-oxidation, and anti-cancer effects for hepatocellular carcinoma [Corylin is a natural flavonoid compound that is extracted and purified from abaceae) . The herabaceae) , and as abaceae) ,26. Amonarcinoma , as wellarcinoma . Furtherarcinoma . This prThe pathogenesis of inflammatory bowel disease (IBD) may be characterized by a loss of intestinal epithelial integrity and the dysregulation of several proteins, including transmembrane proteins, such as zona occludens (ZO)-1, occludin, claudins, and tight junctional molecules ,29,30, iThese results showed that orally administered corylin markedly inhibited AOM/DSS-induced CAC in mice, as evidenced by the tumor number, size, ACF, colon polyps and histopathological examination. In this study, AOM/DSS treatment of normal mice increased pro-inflammatory cytokines and inflammasome gene expression, with the observation of shorter colon lengths, and epithelial cell proliferation, potentially associated with this. However, administration of corylin to AOM/DSS-induced CAC mice showed the opposite pattern regarding the production of those cytokines, which was probably associated with the recovered colon length and reduced number of stem-like cells and epithelial cell proliferation, suggesting the potential anti-inflammatory properties of corylin in the AOM/DSS model. Furthermore, corylin ameliorates AOM/DSS-induced colonic carcinogenesis in mice, probably due to the breakdown of the imbalanced M1/M2 macrophage ratio; administration of corylin ameliorates this symptom by restoring the homeostasis of the macrophage polarization.In this study, it appears that corylin’s anti-carcinogenic properties are most likely due to its effects on multiple molecular targets, such as macrophage polarization and epithelial cell proliferation. Macrophages are the most common non-neoplastic cells in human tumors, including colorectal cancer ,39. TradBacteroidetes and Firmicutes ratio was relatively somewhat maintained in AOM/DSS mice. In an American patient cohort, an increased abundance of Firmicutes was observed compared with non-adenoma subjects in adenoma biopsies [Firmicutes/Bacteroidetes (F/B) ratio is widely accepted as having an important influence in maintaining normal intestinal homeostasis. An increased or decreased F/B ratio is regarded as dysbiosis, whereby the former is usually observed with obesity and has been associated with inflammatory bowel disease (IBD) [Firmicutes is a direct result of AOM/DSS stimuli or an indirect effect of the decrease in tumorigenesis from corylin’s contribution. Firmicutes, which, as part of the gut microbiome, has been shown to be involved in energy resorption [In our study, we have characterized how tumorigenesis was suppressed in the corylin treatment groups, where the relative signaling of the TLR4 cascade and gut barrier permeability were also repaired and the abundance of the phylum biopsies . The Firse (IBD) . Our ressorption , has higsorption . Microbisorption . We obseBacteroidetes, Patescibacteria, and Actinobacteria was detected in the antibiotic-induced diarrhea model [Fusobacteria, Bacteroidetes, and Patescibacteria [Patescibacteria was enriched throughout the luminal content in the small intestine when compared with that of the colon of preterm lambs [Patescibacteria was most abundantly involved in corylin treatment in AOM/DSS mice. Patescibacteria lack a tricarboxylic acid (TCA) cycle and most elements of the electron transfer chain, suggesting that they are strict anaerobic fermenters [Patescibacteria may illustrate a genomic reduction process towards an ectosymbiotic lifestyle [Patescibacteria signature, which could show a negative trend in AOM/DSS mice.In a previous study, using a magnetically controlled sampling capsule endoscope (MSCE), a dramatic decrease in the abundance of ea model . In contbacteria . The phyrm lambs . These rrmenters . Based oifestyle . The triTuricibacter, Romboutsia, Blautia, and Acetatifactor than the normal controls. Moreover, a decrease in the relative abundance of Candidatus saccharimonas, Erysipelatoclostridium, Enterorhabdus, and Coriobacteriaceae UCG-002 was observed. Blautia are anaerobic bacteria with probiotic characteristics that occur widely in the feces and intestines of mammals. Evidence indicates the importance of the survival and evolution of Blautia in the gut and other microenvironments [Blautia was the only gut microbe significantly and inversely associated with visceral fat area (VFA) regardless of sex [Blautia and Ruminococcus in colorectal neoplasm patients were higher than in healthy controls [Blautia were significantly increased in a colorectal cancer group [Erysipelatoclostridium was identified in the early detection of colorectal cancer across populations [Erysipelatoclostridium [Erysipelatoclostridium was enriched in AOM/DSS mice treated with corylin. Therefore, we suspect that Erysipelatoclostridium is a beneficial bacterium in the gut. However, the specific role of these gut microbiota needs to be further investigated. Taken together, the present study suggests that corylin may protect the intestinal barrier from AOM/DSS damage by altering the Blautia/Erysipelatoclostridium ratio, as well as modifying the levels of other specific bacterial species.Changes in the microbiota have been observed in previous studies; decreased bacterial diversity and increased bacterial instability were verified in patients with IBD compared with healthy individuals ,56. In oronments . Blautias of sex . The abucontrols . Blautiaer group . On the ulations . Vitaminstridium . As showIn summary, we found that corylin administration during the late stage is effective at reducing tumorigenesis signaling. This was associated with a decrease in select M1/M2 macrophage polarization in the colon tissue linked to CAC. Overall, these data support the development of therapeutic strategies to target macrophages in CAC, and the results provide a theoretical basis for understanding the relationship between the intestinal barrier function and gut microbiota modulated by corylin, although further investigations, based on metabolomics, should be conducted in future studies.Eight-week-old male wild-type C57BL/6J mice were purchased from the National Laboratory Animal Center (Taiwan) and housed with free access to sterile chow diets and water under a standard temperature (23~25 °C) and humidity-controlled (40–60%) environment. These studies were approved by the Animal Care and Use Committee at the Chang Gung University (CGU106-189).n = 5) of eight-week-old male wild-type C57BL/6J mice were fed dextran sulfate sodium  dissolved in sterile distilled water (4%) for up to 12 days and fed corylin  at 25 mg/kg/day (L) and 100 mg/kg/day (H) orally from day 3 for 10 days [Four groups ( 10 days . The DSS 10 days .n = 5) of eight-week-old male wild-type C57BL/6J mice were first induced to form AOM/DSS models and then fed 25 mg/kg/day (L) and 100 mg/kg/day (H) corylin from day 70 to day 100 (30 days). Body weight and survival were recorded daily during experiments. Animals were euthanized with CO2 inhalation followed by cervical dislocation at the end of the experiments. Colon length was measured, and colon tissues were fixed in PBS-buffered 10% formalin for histopathology or processed for molecular biology studies.The AOM/DSS model was created by injecting 10 mg/kg body weight of AOM once, with four further cycles of 2% DSS treatment, as described previously . Four grColonic tissues were fixed in 10% formalin and then embedded in paraffin wax after dehydration with xylene and alcohol. The tissues were used to cut 5-micrometer sections, which were then stained with hematoxylin and eosin according to the standard protocols. The slides were then incubated with ZO-1 , claudin-1 , occludin , TLR4 , MyD88 , p-p38 , F4/80 , CD11c , CCR7 , CD163 , CD206 , Ki67 , LGR5 , BrdU , PCNA , CD44 , cyclin D1 , and AP-1  primary antibodies overnight night at 4 °C. Secondary antibody was conjugated with HRP-conjugated anti-rabbit , anti-mouse , and anti-rat  secondary antibodies, followed by incubation in DAB peroxidase solution  and subsequent counterstaining with hematoxylin . Sections were incubated with the secondary antibody   for immunofluorescence and DAPI  for nuclear staining. Images were obtained using Olympus cellSens .o C. Secondary antibody was conjugated with HRP-conjugated anti-rabbit , anti-mouse , and anti-rat  secondary antibody. Quantifications were performed using Image J.Colon tissues that had been stored at −80 °C were lysed in Thermo-Fisher Tissue Protein Extraction Reagent  containing a protease inhibitor cocktail . Protein concentrations were determined using the Bio-Rad Protein Assay . Total extracted proteins were then incubated with tris-buffered saline containing 0.1% Tween-20 with 5% non-fat dried milk for 1 h, the blots were incubated with ZO-1 , claudin-1 , occludin , TLR4 , MyD88 , p-p38 , p38 , CD11c , CCR7 , CD163 , CD206 , β-actin , AP-1 , IFNγ , TNF-α , IL-6 , IL-1β , NLRP3 , ASC , Pannexin , and Caspase 1  antibodies overnight at 4Total RNA was extracted from colon tissue using Trizol reagent . Complementary DNA (cDNA) was synthesized using the High-Capacity cDNA Reverse Transcription Kit . The cDNA was analyzed by quantitative real-time (qRT) PCR using specific primers , 0.5 μM of forward and reverse primers, and approximately 1 ng template DNA. The main tool of the workflow was QIIME, which contained multiple analysis process programs [Amplification of the 16S rRNA V3–V4 (341F-805R) hypervariable region was carried out using the 16S V3 (5′-CCTACGGGNGGCWGCAG-3′) and 16s_illumina_V4R (5′-GACTACHVGGGTATCTAATCC-3′) with added Illumina adapter overhang nucleotide sequences. The 16S/18S rRNA genes were amplified using specific primers with barcodes. All PCR reactions were carried out in 25-microliter reactions with 0.5 μL of KAPAprograms .The 16S rRNA sequences were compared for similarity with the reference species of bacteria using the NCBI nucleotide database and the PhiX Control Library. To begin, sequences were clustered into operational taxonomic units (OTUs) using mothur v.1.39.5 with 97% identity. Chimera sequences were identified using UCHIME v4.2 (with reference data: GOLD database) and an OTU table was made . We analThe serum level of C-reactive protein (CRP) was measured in mouse serum obtained at day 12 using a mouse CRP ELISA kit . ELISA assays were performed in accordance with the manufacturer’s instructions. The optical densities of the samples were determined using a microplate reader set at 450 nm.The serum level of leucine-rich alpha2-glycoprotein (LRG) was measured in mouse serum obtained at day 12 using a mouse LRG Assay Kit . ELISA assays were performed in accordance with the manufacturer’s instructions. The optical densities of the samples were determined using a microplate reader set at 450 nm.To evaluate the severity of colitis, DAI scores were monitored daily. The DAI score was determined based on the methods of Friedman et al.  The DAI t-tests with two-tailed distributions were performed to examine statistical differences between two experimental groups using Graph Pad Prism software 7.0 . p-values < 0.05 are regarded as statistically significant.Data are expressed as the mean ± SEM. Unpaired, non-parametric Student’s


The development of cancer is a multistep and complex process involving interactions between tumor cells and the tumor microenvironment (TME). C-X-C chemokine ligand 13 (CXCL13) and its receptor, CXCR5, make crucial contributions to this process by triggering intracellular signaling cascades in malignant cells and modulating the sophisticated TME in an autocrine or paracrine fashion. The CXCL13/CXCR5 axis has a dominant role in B cell recruitment and tertiary lymphoid structure formation, which activate immune responses against some tumors. In most cancer types, the CXCL13/CXCR5 axis mediates pro-neoplastic immune reactions by recruiting suppressive immune cells into tumor tissues. Tobacco smoke and haze (smohaze) and the carcinogen benzo(a)pyrene induce the secretion of CXCL13 by lung epithelial cells, which contributes to environmental lung carcinogenesis. Interestingly, the knockout of CXCL13 inhibits benzo(a)pyrene-induced lung cancer and azoxymethane/dextran sodium sulfate-induced colorectal cancer in mice. Thus, a better understanding of the context-dependent functions of the CXCL13/CXCR5 axis in tumor tissue and the TME is required to design an efficient immune-based therapy. In this review, we summarize the molecular events and TME alterations caused by CXCL13/CXCR5 and briefly discuss the potentials of agents targeting this axis in different malignant tumors. Chemokines are a family of chemotactic cytokines with small molecular weights 8–14 kDa) [4 kDa [1]Chemokines and their receptors also modulate lymphocyte populations in the TME, thus inducing resistance to immune checkpoint inhibitors that exhibit remarkable efficacies on a proportion of patients with many cancer types ,10. IncoCXCL13 gene localizes on chromosome 4q21 and encodes CXCL13 protein, which has 109 amino acids, a molecular mass of 12,664 Da, and a crystal structure as below (+ T cells .,46.45,46CXCL13 is aberrantly expressed and acts as the main orchestrator in TLSs ,52,53,54hiCXCR5−CD4+ T cells . An. AnCXCL1tor (AR) . In addi pathway . In a muand Kras ,118. HowMounting evidence demonstrates a high concentration of CXCL13 and/or high expression of CXCR5 in tumor tissues or tumor cell lines. The CXCL13/CXCR5 axis in both hematological malignancies and solid tumors mediates multiple intracellular signal cascade reactions and yields various phenotypes responding to the signaling pathways. In addition, the CXCL13/CXCR5 axis also potentiates the crosstalk between tumor cells and lymphocytes or non-lymphocytes, shaping a complex TME. The roles of the CXCL13/CXCR5 axis participating in the malignant tumors are context-dependent, including pro-tumor and anti-tumor activities . On one + CD23+ B cells in the peripheral blood, secondary lymphoid tissues, and bone marrow. Most CLL tumor cells are inert and arrested in the G0/G1 of the cell cycle, and there is only a small proliferative compartment; however, the progressive accumulation of malignant cells will ultimately lead to symptomatic disease [Eμ-Tcl1 model of CLL, the CXCR5-expressing malignant B cells are trafficked to GC light zones of B cell follicles in SLOs, where leukemic B cells contact FDCs [Chronic lymphocytic leukemia (CLL) is the most frequently diagnosed subtype of leukemia in adults and is a lymphoproliferative disorder that is characterized by the expansion of monoclonal, mature CD5 disease ,143. CLL disease ,143. In act FDCs . Leukemiact FDCs ,144, whiact FDCs . This cract FDCs . In addiact FDCs . The malact FDCs . Taken t+ and/or CXCR5+ malignant lymphocytes (B cells and Tfh cells) promote the accumulation and proliferation of lymphoma cells, but so do the CXCL13+ FDC and circulating CXCR5+ CD4+ T cells [H. pylori-induced gastric mucosa-associated lymphoid tissue (MALT) lymphomas [Lymphoid neoplasms, characterized by the malignant clonal proliferation of lymphocytes, such as mature B cells, T cells, and natural killer (NK) cells, are classified as Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL), according to morphologic and immunologic characteristics ,146. Mos T cells ,85. CXCL T cells ,165 and ymphomas ,69. Due ymphomas , AITL [1ymphomas ,163, MCLymphomas , PIOL [1ymphomas , and ENKymphomas .+ CD68+ macrophages to the TME, where macrophages produce secreted phosphoprotein 1 (SPP1) to promote cell migration and tumor progression. Interestingly, deficiency in CXCL13 or CXCR5 significantly inhibits BaP-induced lung cancer in mice, indicating the critical role of this axis in environmental lung carcinogenesis [Chronic inflammation provides a favorable context for lung carcinogenesis . CXCL13 ogenesis .+ CD8+ T cells, mediates the immune cells recruited to TLSs. As a result, the TLS is infiltrated by CXCR5+ CD4+ T cells, CD4+ Bcl6+ Tfh cells, and B cells [+ CXCR5+ CD4 T cells in the GCs, which promotes CD8+ T cells to produce IFN-γ, induces the proliferation of B cells, and potentiates B cells to produce IgM and IgG, forming an anti-tumor immunity microenvironment [Anti-tumor activity of the CXCL13/CXCR5 axis has also been reported, which is shown to be able to promote the formation of TLSs and is linked to improved survival of lung cancer patients ,54,171.  B cells . Anotherironment . Furtherironment . AlthougPten deficiency upregulates the release of CXCL13, leading to tumorigenesis and metastasis of PC through the CXCL13/CXCR5 axis in an autocrine manner [Prostate cancer (PC) represents a leading cause of cancer-related mortality among men due to its metastasis . Chemokie manner . Based oe manner ,101,140.H−/−mice lacking mature B cells, the emergence and expansion of castration resistant prostate cancer (CRPC) after androgen ablation relies on B cells, rather than T cells, and the CRPC microenvironment is subsequently infiltrated by B cells [vβ3-integrin clustering [CXCL13 also has an essential role in configuring a complex TME. Although patients initially benefit from the treatment of androgen ablation, the responses are transient with a duration of 12–18 months when they progress to castration-resistant (CR) PC ,175,176. B cells . The inf B cells . The int B cells ,178 and  B cells , as a re B cells ,115. Apaustering .+ B cells and T cells homing to tumors, thereby eliciting an anti-tumor immune response [+ Tfh cells express CXCL13, which has been speculated to initiate GC formation and enhance TLS development by homing immune cells to peritumoral or tumor sites [CXCL13 was found to be overexpressed in breast cancer and in the peripheral blood of patients with breast cancer ,181. Theresponse ,183. Infor sites ,184. In or sites .+ innate lymphoid cell group 3 (ILC3) to the TME, where ILC3 stimulates stromal cells to secrete CXCL13 [However, a large number of studies have demonstrated that the CXCL13/CXCR5 axis is closely associated with breast cancer growth and lymph node metastasis ,136,185.e CXCL13 . CXCL13,e CXCL13 . After te CXCL13 . Moreovee CXCL13 . A recene CXCL13 , though G12DHif1αKOKras model of pancreatic cancer exhibits an abundant expression of CCL19, CCL20, CCL21, CXCL12, and CXCL13, all of which mediate Breg migration and infiltration into tumor tissue to promote pancreatic tumorigenesis [G12DKras-harboring pancreatic ductal adenocarcinoma (PDAC), CXCL13 expression by stromal fibroblasts enhances the infiltration of IL-35 producing B cells into the TME, which potentiates the expansion of pancreatic cancer cells [LSL-+/G12DKras and Pdx1-cre with IL-1β overexpression, the combination of IL-1β stimulation with Kras mutation mediates the upregulation of CXCL13 expression and the expansion of Breg and PD-L1+ B cells, which play an immune-suppressive role in inhibiting CD8+ T cell activity, in addition to facilitating pancreatic tumorigenesis [In pancreatic cancer, CXCL13 signaling participates in the establishment of the pro-oncogenic microenvironment by recruiting tumor-associated B cells ,117,118.er cells . Recentl+) granulocytic myeloid cells promotes Foxp3 expression, Stat3 phosphorylation, and Treg proliferation, as a result of immune suppression and tumorigenesis [In colorectal or colon cancer, the CXCL13/CXCR5 axis mediates the pathogenesis, development, and distal metastasis of tumor cells through upregulating the expression and secretion of MMP13, as well as activating the PI3K/Akt pathway ,187,188.igenesis . CXCL13 igenesis . These rThe CXCL13/CXCR5 axis makes prominent contributions to oral squamous cell carcinoma (OSCC) invasion of bone/osteolysis ,84,189. + MDSC migration to gastric cancer, leading to immune escape and tumor progression [+ CD8+ T cells mediate immune evasion with increased Treg cells and exhausted cytotoxic T cells [+ CD8+ T cells in tumor tissue is associated with poor clinical outcomes for the patients, and elimination of these cells could be helpful for gastric cancer immunotherapy. CXCL13 has been reported to be increased in the serum of patients with hepatocellular carcinoma (HCC) [CXCL13 abounds in the serum and tumor tissue of patients with gastric cancer and serves as a prognostic marker for patients under postoperative adjuvant chemotherapy ,192,193.gression . CXCL13+ T cells . High inma (HCC) ,195. CXCma (HCC) . The CXCThe CXCL13/CXCR5 axis makes pivotal contributions to the initiation and progression of tumors, and therefore may serve as an attractive therapeutic target for related malignant neoplasms ,104,112.+ T cell activity [+ T cells [CXCL13-producing non-cancerous cells in the TME, such as myofibroblasts, myeloid cells, B cells, and T cells, may also serve as potential targets for cancer therapy . A previactivity . Therapeactivity . In antiactivity ,49. Emer T cells ,191. An  T cells ,195,197.+ T cells are linked to the proinflammatory features of macrophages and show enhanced cytotoxicity following anti-PD-L1 therapy in triple-negative breast cancer [The CXCL13/CXCR5 axis plays multifaceted roles in intracellular signaling transduction pathways and interactions among tumor cells, stromal cells, and lymphocytes. The CXCL13/CXCR5 axis not only modulates molecular events inside malignant cells to promote tumor initiation and progression, but also recruits multiple populations of lymphocytes to exert pro-tumor or anti-tumor immunity reactions in the TME. An intriguing phenomenon is that those anti-neoplastic lymphocytes attracted by CXCL13 signaling, such as B cells and Tfh, participate in the organization of the TLS. However, the circumstances, which contain immune-suppressive cells recruited by the CXCL13/CXCR5 axis, with or without TLSs, have not been fully illuminated. In addition, CXCL13-expressing CD8t cancer . Thus, b


SNA is one of the essential EMT transcriptional factors capable of suppressing epithelial maker while upregulating mesenchymal markers. However, the mechanisms for SNA to transactivate mesenchymal markers was not well elucidated. Recently, we demonstrated that SNA collaborates with EGR1 and SP1 to directly upregulate MMP9 and ZEB1. Remarkably, a SNA-binding motif (TCACA) upstream of EGR/SP1 overlapping region on promoters was identified. Herein, we examined whether four other mesenchymal markers, lymphoid enhancer-binding factor (LEF), fibronectin (FN), cyclooxygenase 2 (COX2), and collagen type alpha I (COL1A1) are upregulated by SNA in a similar fashion. Expectedly, SNA is essential for expression of these mesenchymal genes. By deletion mapping and site directed mutagenesis coupled with dual luciferase promoter assay, SNA-binding motif and EGR1/SP1 overlapping region are required for TPA-induced transcription of LEF, FN, COX2 and COL1A1. Consistently, TPA induced binding of SNA and EGR1/SP1 on relevant promoter regions of these mesenchymal genes using ChIP and EMSA. Thus far, we found six of the mesenchymal genes are transcriptionally upregulated by SNA in the same fashion. Moreover, comprehensive screening revealed similar sequence architectures on promoter regions of other SNA-upregulated mesenchymal markers, suggesting that a general model for SNA-upregulated mesenchymal genes can be established. The poor prognosis of hepatocellular carcinoma (HCC) is due to frequent metastasis occurring via complicated processes, including epithelial mesenchymal transition (EMT), migration, and invasion of primary tumor . Among tThe cultured conditions for HCC340 and HepG2 cells were the same as described in our previous report . HCC340 Tetradecanoyl phorbol acetate (TPA) was purchased from Sigma-Aldrich . The monoclonal Snail (C15D3) antibody; and the antibodies against EGR1, SP1, MMP9, ZEB1, LEF1, Fibronectin, COX2, COL1A1, Histone H3, and GAPDH were purchased from Cell Signaling  and Santa Cruz Biotechnology , respectively. The snail expression plasmid, p-Snail, driven by CMV promoter within the p-cDNA3 vector, was a gift from Dr. Cheng K.K., Tzu Chi University.The promoter regions in the full-length promoter plasmids of LEF, FN, COX2, and COL1A1 were amplified from the genomic sequence of each gene. The PCR products were ligated into pGL3 vector . The promoter plasmids of deletion constructs were derived from each full-length promoter by double digestion with various restriction enzymes, followed by filling in the restriction site overhangs by Klenow enzyme. Subsequently, the digested DNA fragments were ligated into pGL3 vector.The full-length promoter plasmids of LEF, FN, COX2, and COL1A1 were used as templates for site-directed mutagenesis using a GeneEditor in vitro site-directed mutagenesis system  to obtain various mutant promoters according to the manufacturer’s protocol. The bases changed in the site-directed mutagenesis for the proposed Snail binding motif (TCACA), and the EGR1/SP1 overlapping binding region were the same as those described in our previous report ,18.Transwell and wound healing migration assays were performed as described in previous report ,24.Dual-Luciferase promoter assay was performed according to the protocol of manufacture .ChIP assay was performed as described in our previous Reports ,18. SuppRT-PCR was performed as described in our previous report ; the priEMSA was performed as described in our previous report . NuclearWestern Blot was performed as described in our previous Reports ,18.shRNA Technology was performed as described in our previous Reports . The seqt-test in Excel. All the quantitative studies were performed at least in triplicate, as appropriate. Statistical significance between groups were indicated by * p < 0.05 and ** p < 0.001.Data were analyzed using Student’s Initially, by RT-PCR, we found TPA elevated mRNA of SNA by 3.7-fold at 1 h and declined until 6 h, whereas it significantly increased mRNA of FN, LEF, and COX2 by 1.5–2-fold within 1–2 h, which was further increased to 2.5–4.8-fold until 6 h in HepG2, one of the conventional HCC cell lines a. Also, We further examined whether TPA can induce promoter activation of FN, LEF, COX2, and COL1A1 in a SNA-dependent manner. To this end, full-length promoter plasmid of FN (FNpro1150), LEF (LEFpro1595), COX2 (COX2pro979), and COL1A1 (COL1A1pro1090) were constructed by inserting the promoter fragment containing 1150 bp and 1595 bp, 979 bp and 1090 bp, respectively, upstream of the translation start site of each promoter into PGL3 vector. As shown in Further, we sought to identify the TPA-responsive element on promoters of FN and LEF, COX2, and COL1A1 and examined whether it contained the proposed SNA binding region “TCACA” and putative EGR1/SP1 overlapping motif, as described previously . AccordiTo prove whether the candidate SNA binding motifs are required for transcription of the aforementioned mesenchymal genes, site-directed mutagenesis coupled with promoter analysis were performed. For FN, promoter mutants with the altered sequence (TCACA→TTGTA) at indicated SNA motifs (PM 1–3) were produced for analysis in both HepG2 . In addiThus far, it appears that SNA coupled with EGR1/SP1 activate transcription of the aforementioned mesenchymal genes, whether the relevant transcriptional factors can bind to the indicated putative regions was examined within cells using ChIP assay. As shown in We further confirmed the binding of SNA on the aforementioned promoters by EMSA. Nuclear extract of HCC340 treated with TPA at different time points were incubated with biotin-labeled probe containing the SNA binding motif on FN, LEF, COX2, and COL1A1 promoter . As shown in In the past decades, it appeared that the mechanisms by which SNA upregulates the expression of mesenchymal genes were more complicated than it downregulates epithelial markers. As demonstrated in this and our previous  reports,Previously, the role of SNA in upregulating transcription of FN, LEF, COX2, and COL1A1 has been reported in different contexts. SNA can mediate TGFβ-triggered LEF expression for EMT in MDCKII cells . OverexpThus far, we have demonstrated six of the mesenchymal genes, FN, LEF, MMP9, ZEB1, COX2, and COL1A1, are transcriptionally upregulated by SNA in the same manner, i.e., by direct binding of SNA coupled with EGR1/SP1 on their putative regions. To predict whether this could be a general model, we screened the mesenchymal genes that can be upregulated by SNA from PubMed and examined whether they contain the aforementioned SNA binding motif (TCACA) coupled with a downstream EGR1/SP1 overlapping region in the gene bank. In addition to what we have investigated, five of the well-known mesenchymal genes such as vimentin, vitronectin, α-SMA, N-cadherin, and TWIST1 were identified . All of In conclusion, we proposed a general model for SNA to upregulate transcription of mesenchymal genes via collaboration with EGR1/SP1 in HCC. Remarkably, a specific putative SNA binding motif “TCACA” close to the EGR1/SP1 overlapping region, required for activating their promoters, was identified. In the future, this model will be validated after more of the candidate mesenchymal genes are investigated in HCC and other cancers.


Rhynchophorus ferrugineus (Olivier), have become a severe problem for palm species. In present work, fat body transcriptome of adult female red palm weevil was analyzed, focusing on the identification of reproduction control genes. Transcriptome study was completed by means of next-generation sequencing (NGS) using Illumina Hiseq 2000 sequencing system. A total of 105,938,182 raw reads, 102,645,544 clean reads, and 9,238,098,960 clean nucleotides with a guanine–cytosine content of 40.31%, were produced. The processed transcriptome data resulted in 43,789 unique transcripts . It was found that 20% of total unique transcripts shared up to 80%–100% sequence identity with homologous species, mainly the mountain pine beetle Dendroctonus ponderosae (59.9%) and red flour beetle Tribolium castaneum (26.9%). Nearly 25 annotated genes were predicted to be involved in red palm weevil reproduction, including five vitellogenin (Vg) transcripts. Among the five Vg gene transcripts, one was highly expressed compared with the other four , and the five Vg gene transcripts were designated as RfVg, RfVg-equivalent1, RfVg-equivalent2, RfVg-equivalent3, and RfVg-equivalent4, respectively. The high expression level of RfVg verified by RT-polymerase chain reaction analysis suggested that RfVg is the primary functional Vg gene in red palm weevil. A high similarity of RfVg with other Coleopterans was also reflected in a phylogenetic tree, where RfVg was placed within the clade of the order Coleoptera. Awareness of the major genes that play critical roles in reproduction and proliferation of red palm weevil is valuable to understand their reproduction mechanism at a molecular level. In addition, for future molecular studies, the NGS dataset obtained will be useful and will promote the exploration of biotech-based control strategies against red palm weevil, a primary pest of palm trees.Recent attacks by the red palm weevil,  Rhynchophorus ferrugineus (Olivier) (Coleoptera: Dryophthoridae) has strong invasion capability and within the last few decades have become invasive in more than 27 countries around the globe [Phoenix dactylifera, coconut palm Cocos nucifera, and African oil palm Elaeis guineensis [The red palm weevil (RPW), he globe . The RPWineensis . The femineensis . The larineensis . The repineensis . The VgsIn recent decades, scientists have studied the basic ecology and biology of RPW , 10–12 ade novo transcriptome sequencing technology has been successfully demonstrated in several insects, including the migratory locust Locusta migratoria [Bactrocera dorsalis [Spodoptera littoralis [R. ferrugineus [Nilaparvata lugens [Ephestia cautella [The rapid progress and convergence of modern techniques from different areas of science have resulted in the enrichment of the fields of genetics and molecular sciences. Next-generation sequencing (NGS) is an efficient and economical technology used for identification of large numbers of expressed genes in a specific tissue, and confirms the biological, physiological, and molecular properties of the tissue. The use of NGS is very effective for discovering novel genes and determining gene structures and functions , 17. Thigratoria , orientadorsalis , noctuidttoralis , RPW R. rugineus , brown pa lugens , and almcautella .Vg and other important genes such as: apolipophorin III, low-density lipoprotein receptor, and the chorion protein. The analysis of the fat body transcriptome provides extensive information about the genes involved in biological, physiological, and metabolic processes of the RPW and may facilitate future molecular studies, especially of Coleopteran animals, and even promote development of control tactics against invasive species, particularly the RPW.Thus, to isolate RPW reproduction control genes, the transcriptome of female fat body tissue was sequenced and analyzed using an Illumina Hiseq 2000 NGS platform. Although transcriptome and genome resources are accessible from several Coleopteran insects –28, the The red palm weevil adults were collected directly from the date palm orchard, Riyadh region, Saudi Arabia. We declare that red palm weevil was not collected from the public parks or protected areas. Moreover, it is not an endangered species.RPW different stages  were initially collected from infested date palm trees in Dirab, Kingdom of Saudi Arabia . Adult RPW were kept in plastic boxes containing a piece of cotton saturated with 10% sugar solution [A total of 25 (1–5 days old) virgin adult females were selected from the colony for fat body tissue preparations. The insects were dissected using fine microscissors in phosphate-buffered saline (pH 8.0) . The fatTotal RNA was extracted from RPW fat body tissues (~800 mg) using Tri-RNA reagent . The RNA integrity number 28S/18S ratio and sample size were determined using an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit and DNase treatment was done to elude genomic DNA contamination. Finally, the purity was assessed by using NanoDrop. The total volume of 80 μL RNA samples with a concentration of 488 ng/μL were used to synthesize cDNA. The Superscript II Reverse Transcription kit (Invitrogen) was used to generate first-strand complementary DNA from mRNA. To synthesize second-strand cDNA, second-strand master mix was added in the first-strand cDNA; the mixture incubated at 16°C for 1 h, and cDNA was purified using Ampure XP Beads . The purified cDNA was supplemented with End-Repair Mix, incubated at 20°C for 30 min, and purified. The repaired cDNA was supplemented with A-Tailing mix and incubated at 37°C for 30 min. Then, ligation reaction was done by combining adenylated cDNA with adapters and ligation mix at 20°C for 20 min. Finally, PCR products were purified using Ampure XP Beads. The resulting cDNA library was quantified using an Agilent 2100 Bioanalyzer, Agilent DNA 1000 Reagent, and quantitative PCR (TaqMan Probe) .Competent libraries were amplified using cBot, and clusters generated on TruSeq PE Cluster Kit V3-cBot-HS; Illumina, a flow cell, were sequenced using Illumina HiSeq 2000 system. Read lengths were 50 bp and were sequenced via a paired-end strategy. The raw reads produced by the sequencing machine contained unclean reads ; the raw reads were cleaned using filter-fq to generate high-quality transcriptome data. The assembly was then created from the clean reads using assembling program Trinity (Version: release-20130225) [Assembled unigenes were aligned to different protein databases such as: non-redundant (NR), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Groups (COG) databases through the BLASTX tool  and to the nucleotide database (NT) using the BLASTN tool . The alignment results with the best sequence similarity were selected and annotated to the unigene. For unigenes that failed to align with the aforementioned databases, ESTScan software was used to detect the coding region sequence and to find the sequence direction . Blast2GAll types of SSR sequences (mono to penta-nucleotide repeats) were identified using the software program MicroSAtellite (MISA), and only the SSRs in unigenes of >150 bp in length were retained. Similarly, all potential SNPs were identified and classified using the software program SOAPsnp .The genes involved in RPW reproduction were acknowledged from RPW fat body transcriptome data. The sequences of these genes were downloaded separately, and their identification was confirmed using the BLASTX tool from NCBI.Five Vg transcripts were identified from RPW transcriptome data with different FPKM values and validated via RT-PCR using following gene-specific primers: RfVgF1, RfVgR1, RfVg1F1, RfVg1R1, RfVg2F1, RfVg2R1, RfVg3F1, RfVg3R1, RfVg4F1, and RfVg4R1; actin primers RfActF1 and RfActR1 were used for normalization control . Total RRfVg was checked against the NCBI GenBank database using the BLASTX tool. The Vg amino acid sequences acquired from different insect species were used to construct a phylogenetic tree. Similarity analyses of the protein sequences were conducted. A multiple-sequence alignment was performed using the ClustalW program [The sequence of  program , and a n program .In this study, 105,938,182 raw reads were generated from the RPW fat body cDNA library using the Illumina Hiseq 2000. After trimming adapter sequences and eliminating low-quality reads, the raw data yielded 102,645,544 clean reads and 9,238,098,960 clean nucleotides (nt), with a GC content of 40.31% . From thThe assembled unigene transcripts were annotated using public databases NR, Swiss-Prot, KEGG, and COG  with BLASTX, and the nucleotide database  with BLASTN. Of the 43,789 unigenes, a total of 23,880 (54.53%) were annotated, including 23,178 (52.93%) using NR, 12,589 (28.74%) using NT, 18,706 (42.71%) using Swiss-Prot, 16,512 (37.7%) using KEGG, 9604 (21.93%) using COG, and 10,300 (23.52%) using GO databases . Among 2Dendroctonus ponderosae (59.9%), followed by the red flour beetle Tribolium castaneum (26.9%)  .The COG database classifies orthologous gene products, and each COG protein is presumed to come from an ancestral protein. In this study, unigenes were mapped to to predict possible functions using COG database. COG analysis permitted the functional classification of 9,604 unigenes .The most frequently identified class was “general function” , followed by “replication, recombination and repair” , “translation, ribosomal structure and biogenesis” , and “transcription” . In GO analysis, unigenes were separated into three ontologies: molecular function, cellular component, and biological process. GO analysis categorized 10,300 unigenes  into 60 functional groups. “Cellular process” and “metabolic process” were the 2 largest groups, containing 6,255 and 5,879 unigenes, respectively. However, “developmental process” and “reproduction” contained 1,540 and 536 unigenes, respectively .The unigene pathway analysis mapped 16,512 unigenes (37.7%) to 258 KEGG pathways. Metabolic pathways comprised 2,298 unigenes (13.91%), which was significantly more than the number mapped to other pathways, such as the pathways for the regulation of the actin cytoskeleton , focal adhesion , purine metabolism , vascular smooth muscle contraction , RNA transport , cancer , and the spliceosome . According to KEGG classifications, there were six major annotational categories for the identified unigenes .Unigenes were first aligned by BLASTX  to protein databases. The 22,861 coding DNA sequences (CDS) were mapped to protein databases, whereas, the EST scans predicted that 1,446 unigenes were related to CDS. However, 24,307 total numbers of CDS were obtained in the study Figs  and 11.Transcriptome analysis is an important source to develop genetic markers. In our study, a total of 2,060 SSRs were recognized . The larIn the transcriptome database, the present study identified a total of 59,012 high-quality SNPs, including 41,100 (69.64%) transitions and 17,912 (30.35%) transversions . TogetheVgs, with maximum mean length of 5361 bp (unigene 12151). A variety of putative hormonal proteins that are involved in reproduction were also identified, including juvenile hormone-inducible protein, juvenile hormone epoxide hydrolase-like protein 5 precursor, juvenile hormone esterase, and ecdysone response nuclear receptor , and were designated as RfVg, RfVg1, RfVg2, RfVg3, and RfVg4, respectively. The incongruity in the FPKM values of all five Vg transcripts was verified by RT-PCR, and expression was only confirmed of RfVg  [Tribolium castaneum (27.2%).The Illumina sequencing of the RPW fat body yielded 102,645,544 clean reads, comprising 64,046 contigs and 43,789 unigenes . Almost  (56.7%)  and the In this research, most of the unigenes were annotated with COG and GO databases . The general function prediction class  was the largest COG class, showing similarity to other insects transcriptome data , 43. AmoVg is one of the major gene which is highly expressed in RPW female fat bodies during reproductive phase and retain a substantial role in in oviparous organism’s reproduction [Vg production and accumulation in oocytes by membrane-bound receptors (the VgRs) via receptor-mediated endocytosis [Periplaneta americana [Blattella germanica [Aedes aegypti [From the RPW fat body transcriptome, nearly 25 annotated genes were predicted to be involved in reproduction. Among these, oduction , 45–51. ocytosis , 52–55. ocytosis . The accocytosis –59. In omericana , German ermanica , and yel aegypti , Vn contde novo transcriptome sequence data in the present study demonstrate substantial homology to sequences in publicly available NCBI databases. This indicates that the Illumina-based transcriptome data of the present study were correctly assembled and that a significant fraction of exclusive genes was transcribed in RPW fat body tissues. From the present transcriptome data, five partial Vg gene transcripts were obtained; however, based on the FPKM values and RT-PCR results, it is very clear that RfVg is the only functional Vg gene in RPW. This is also in accordance with other Coleopteran species such as the boll weevil Anthonomus grandis [Tenebrio molitor [Octodonta nipae [Colaphellus bowringi [Vg gene has been reported. Presence of different numbers of Vg genes in insect species have been recorded from several insect including A. aegypti [Plautia stali [P. americana [Leucophae maderae [Vg gene has been depicted so far by members of the order Coleoptera [Vg gene. This current transcriptome data from RPW fat body tissues have delivered a surplus strong evidences regarding the genes involved in RPW physiological functions, especially in the reproduction. Reproduction control genes identification will make available a reference line for characterization of these genes. In particular, Vg gene characterization would be of great worth to understand RPW reproduction mechanism at molecular level and may encourage the biotech-based control strategies development against this pest species.In general, the  grandis , mealwor molitor , nipa pata nipae , and cabbowringi , where a aegypti , 68, theia stali , P. amermericana , 46, and maderae , 70, onlleoptera . Thus, oS1 File(DOCX)Click here for additional data file.


Subcutaneous fat thickness and intramuscular fat content are closely related to meat production and quality in the pig industry. Adipogenesis in adipocytes from subcutaneous and intramuscular fat tissues involves different genes and regulatory mechanisms. Analyzing the data of mRNA and miRNA transcriptomes during the differentiation of adipocytes from these two sources will help identify the different mechanisms of subcutaneous and intramuscular fat deposition. In this study, RNA sequencing technology was used to analyze the differential expression of genes and miRNAs in subcutaneous and intramuscular adipocytes at days 0, 2, 4, and 8 of differentiation. We mainly attributed the difference between fat depositions of the two types of adipocytes to variations in the expression patterns of related genes. Through combined weighted gene co-expression network analysis and K-MEANS, we identified 30 and 22 genes that mainly regulated the differentiation of subcutaneous adipocytes and intramuscular adipocytes, respectively. A total of 17 important candidate miRNAs were identified. This study provides valuable reference for the study of different mechanisms of adipogenesis among subcutaneous and intramuscular fat and contributes to improving pig breeding. Adipose tissue is one of the largest and most vigorous organs in humans and animals and plays a major role in the control of energy homeostasis . Many huSLC27A1, ACAA2, ssc-miR-132, ssc-miR-146b, and ssc-miR-221-5p, were screened that had regulative functions on fat deposition [POSTN and FGFR4 that might regulate intramuscular fat depositin were identified from transcriptomic profiles of intramuscular adipocytes in pigs [MicroRNAs (miRNAs) are small non-coding RNAs that regulate the gene expression at the post-transcriptional level and adipogenesis in animals ,15. Tranposition . The gen in pigs . The deslongissimus dorsi (LD) tissues and differentiation was induced in vitro. Adipocytes were collected at four time points  to generate transcriptomic data of mRNAs and miRNAs using RNA-seq. The key functional genes and miRNAs involved in subcutaneous adipocytes or intramuscular adipocytes were identified to understand the regulatory mechanisms of fat deposition in the two tissues of pigs.In this study, porcine preadipocytes were isolated from subcutaneous fat and The animals were reared and handled in accordance with the Guide for the Care and Use of Laboratory Animals in China. All experimental guidelines were approved by the Committee on the Ethics of Animal Experiments of the China Agricultural University (permit number: SKLAB-2012-04-07).2. All culture medium components were obtained from Gibco .Three six-day-old Yorkshire piglets were provided by the Beijing Shunyi Pig Breeding Farm. Intramuscular preadipocytes were isolated from theLD and subcutaneous adipocytes were stripped from the back subcutaneous fat (BF) tissues. First, BF tissues were cut into pieces and digested with 1 mg/mL collagenase type I  at 37 °C for 60 min and then filtered through 70 and 200 mesh sieve to remove the undigested part. LD tissues were digested for 1.5–2 h with 2-mg/mL collagenase type I. Preadipocytes were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM-F12) growth medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C in an atmosphere of 5% COWhen the cell confluency reached 90%, the standard culture medium was replaced with adipogenic induction medium   for two days. Next, the cells were cultured in maintenance medium (growth medium supplemented with 5-μg/mL insulin) for an additional two days. The growth medium was then changed every alternate day until adipocyte maturation.For Oil Red O staining, the culture medium was removed, and adipocytes were washed thrice with PBS and fixed in 4% formaldehyde for 30 min. Subsequently, 4% formaldehyde was removed, and cells were washed thrice with PBS and stained with Oil Red O for 20 min. Cells were washed thrice with PBS and photographed under a microscope . After the oil red O dye was extracted with isopropanol for 20 min, the absorbance was measured at a wavelength of 490 nm to quantify the lipid accumulation.Total RNA was isolated from adipocytes after differentiation on days 0, 2, 4, and 8 using the TRIzol reagent . RNA concentration and purity were measured using NanoDrop 2000 . RNA integrity was assessed using the RNA Nano 6000 Assay Kit on the Agilent Bioanalyzer 2100 system . All RNA samples had RNA integrity number (RIN) values exceeding 8.6. High-throughput sequencing was performed using NovaSeq 6000, and 150 paired-end reads were generated.For small RNA sequencing, first, 3′ SR and 5′ SR adaptors were ligated, and then reverse transcription of the synthetic first chain was performed. Afterward, PCR amplification was performed, and PAGE gel was used for electrophoresis fragment screening. The rubber was cut and recovered as fragments and purified to obtain a small RNA library. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v4-cBot-HS  according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on a NovaSeq 6000, and 50 single-end reads were generated.ftp://ftp.ensembl.org/pub/release-99/fasta/sus_scrofa/dna/Sus_scrofa.Sscrofa11.1.dna.toplevel.fa (4 December 2021) using HISAT2(2.0.4) [Clean reads were filtered by removing adapters, reads containing more than 10% unknown nucleotides (N), and low-quality sequences to obtain clean reads. Clean reads were mapped to the pig reference genome  ,19.2 of the fold change |log2FC| ≥ 1.StringTie (1.3.4 d) was used to assemble the transcript and calculate the fragments per kilobase million mapped read (FPKM) values . DESeq2(Clean reads were mapped to the pig reference genome (scrofa. Sscrofa11.1) with perfect matches using Bowtie software. Annotated tRNA, rRNA, snoRNA, and snRNA sequences were filtered out, and conserved miRNAs were identified using BLAST against miRbase. miRDeep2 software was used to predict new miRNAs based on the characteristics of the miRNA precursor hairpin structure. A differential expression analysis between the two groups was performed using the DESeq2 R package. Differentially expressed miRNAs (DE-miRNAs) were screened with a fold change criterion of  >1.5 and FDR < 0.05 using DESeq2. Target genes of the DE-miRNAs were predicted using miRanda and TargetScan. The miRNA–mRNA signaling pathway interaction network was generated using Cytoscape software .Gene Ontology (GO) enrichment analysis of the DEGs was implemented using the GOseq R packages based on Wallenius noncentral hypergeometric distribution , and KOBAll annotated genes (FPKM > 0.01) were analyzed using the weighted gene co-expression network analysis (WGCNA) package in R studio software. The parameters of the WGCNA program were as follows: minimum module size = 30, minimum height for merging modules = 0.25, and soft threshold = 15. Gene significance (GS) and module membership (MM) refer to the correlation between gene expression and each trait and the correlation between gene expression and each module eigengene, respectively. The correlation between GS and MM was performed using Pearson’s correlation in the WGCNA package. For the genes with eigengene-based connectivity values (|KME|) ≥ 0.7, |MM| ≥ 0.8, and |GS| > 0.3, the pivot gene was selected as the key module.AGT, GK, and CAT) and four miRNAs  were used for qRT-PCR verification. The primers used for quantitative real-time RT-PCR (qRT-PCR) are presented in −ΔΔCt method.Three genes  in SPSS software . Statistical significance was set at The induced differentiation process is illustrated in Compared to subcutaneous preadipocytes, intramuscular preadipocytes slowly produce lipid droplets. Only a small part of these droplets was formed after two days of differentiation, and their amount on the fourth day was similar to that on the second day. However, on the eighth day, the accumulation of lipid droplet accumulation increased significantly d. In genThe transcriptomes of the 24 samples generated 160.63 Gb of clean data, and the percentage of clean Q30 bases ranged from 90.40% to 96.24%. The clean reads of each sample were aligned with the reference genome (Scrofa 11.1), and the mapping ratio ranged from 90.40% to 96.24% , indicatThe statistics for the number of DEGs and DE-miRNAs are shown in A total of 2365 DEGs and 68 DE-miRNAs were found in subcutaneous adipocytes during differentiation, and 24 DEGs and two DE-miRNAs were shared by all three pairs between stages a,e. DuriUpon comparing all DEGs and miRNAs in the differentiation process of the two adipocytes, 1116 overlapping genes and 22 overlapping miRNAs were identified. During the differentiation of subcutaneous adipocytes, 1249 specific DEGs and 46 specific DE-miRNAs were identified, whereas 1204 DEGs and 23 DE-miRNAs were specific differentially expressed in intramuscular adipocytes c,g. UponWe performed a functional enrichment analysis of the overlapping DEGs and specific DEGs in the differentiation process of the two adipocytes. Overlapping DEGs enriched 278 signaling pathways. Subcutaneous and intramuscular adipocyte-specific DEGs were enriched in 274 and 282 pathways, respectively. The main enriched pathways were glycolysis/gluconeogenesis, rheumatoid arthritis, metabolic pathways, steroid biosynthesis, amino acid biosynthesis, HIF-1 signaling pathway, focal adhesion, interaction of viral proteins with cytokines and cytokine receptors, carbon metabolism, fatty acid metabolism, and other pathways . In totaThe DEGs between the two adipocyte groups may explain the differences between the production mechanisms of the two adipocytes. The KEGG analysis showed that, on day 0 of the differentiation, although the induction of differentiation had not yet begun, the highly expressed genes of intramuscular fat cells were enriched in the cell cycle, DNA replication, carbohydrate digestion and absorption, starch and sucrose metabolism, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), and biosynthesis of the unsaturated fatty acids pathway, while the highly expressed genes in subcutaneous fat participate in the Hippo signaling pathway, multiple species, butanoate metabolism, propanoate metabolism, various amino acid metabolism, fatty acid biosynthesis, and other signaling pathways a.On days 2 and 4 of differentiation, the genes highly expressed in intramuscular adipocytes were mainly involved in DNA replication, synthesis, the degradation of ketone bodies, other glycan degradation, maturity onset diabetes of the young, and other pathways. The highly expressed genes in subcutaneous adipocytes were mainly involved in fatty acid biosynthesis, the adipocytokine signaling pathway, the MAPK signaling pathway, ether lipid metabolism, sphingolipid metabolism, and alpha-linolenic acid metabolism b,c. Aftep < 0.05) and 18 modules for intramuscular fat differentiation  in the four periods may be genes that specifically regulate the differentiation of the two adipocytes. We performed the KEGG analysis on the DEGs between the groups and found 466 genes involved in the lipid metabolism-related pathways . Combining these 466 genes with genes related to the differentiation of BF and IMF analyzed by the WGCNA, we found 40 genes that may specifically regulate subcutaneous fat deposition, 35 genes that specifically regulate intramuscular fat deposition, and 7 genes that coregulate the differentiation of these two adipocytes . PCK1, STAT1, IGF1, CYP26B1, CAT, AGT, SOAT1, ENO2, GK, and FABP4  were predicted, and the negative regulatory relationship between the target genes and DEGs was analyzed. A total of 17 DE-miRNAs targeted 187 DEGs in comparison of the adjacent time groups during the differentiation process of the subcutaneous adipocytes, and 12 DE-miRNAs targeted 255 DEGs in intramuscular adipocytes. For group BF0d vs. IMF0d, we found 19 DE-miRNAs negatively regulated 177 DEGs, BF2d vs. IMF2d group, found that three DE-miRNAs negatively regulated four DEGs, the BF4d vs. IMF4d group, found 17 DE-miRNAs negatively regulated 462 DEGs, and for group BF8d vs. IMF8d, 48 DE-miRNAs were found to target 522 genes .The KOBAS online software was used to perform the GO and KEGG functional enrichment analyses of the target genes. In total, 157 KEGG signaling pathways were enriched in 187 genes targeted by 17 DE-miRNAs during subcutaneous adipocyte differentiation. The main pathways included the pentose phosphate pathway, glucagon signaling pathway, fructose and mannose metabolism, Hippo signaling pathway, and propanoate metabolism . For intThe target gene enrichment analysis of the DE-miRNAs compared between two adipocytes at the same differentiation stage showed that the main enrichment pathways of the target genes of the DE-miRNAs downregulated in intramuscular adipocytes were peroxisome, alanine, aspartate, and glutamate metabolism; fatty acid degradation; valine, leucine, and isoleucine degradation; glycerolipid metabolism; renin secretion; fatty acid metabolism; fluid shear stress and atherosclerosis; and aldosterone synthesis and secretion. The target genes of the DE-miRNAs upregulated in intramuscular adipocytes were mainly enriched for insulin resistance, glycosaminoglycan biosynthesis–keratan sulfate, starch and sucrose metabolism, aldosterone-regulated sodium reabsorption, staphylococcus aureus infection, retinol metabolism, and fructose and mannose metabolism .PRKAG2, CA13, FOS, SFRP1, and FGF1. The novel-miR-126 and novel-miR-667 of DE-miRNAs in intramuscular adipocytes target a large number of genes related to glycerolipid metabolism, carbohydrate metabolism, and insulin signaling pathways, such as ENO2 involved in glycolysis/gluconeogenesis, cholesterol synthesis gene SOAT1, lipogenic gene FAS, glyceride metabolism gene GK, etc. To identify the key miRNAs that regulate lipid metabolism, we constructed a co-expression network diagram of miRNAs, targeted mRNAs, and related signaling pathways involved in targeted mRNA. In the subcutaneous adipocyte group, 32 miRNA–mRNA pairs were constructed from 13 miRNAs and 23 targeted mRNAs a. In theAGT, GK, and CAT) and four miRNAs , were selected for qRT-PCR to validate their expression differences in RNA-Seq. After comparison with the RNA-Seq data, it was found that the expression trend of qRT-PCR was similar to that of RNA-Seq . This is consistent with the phenotype where subcutaneous adipocytes accumulate more lipid droplets than intramuscular adipocytes. This also confirms that fats accumulated by the two adipocytes have different carbon precursors ,23.GK, CAT, FABP4, AGT, PCK1, STAT1, IGF1, PDE3B, APOA1, SOAT1, CYP26B1, ENPP2, and HGF) have been reported to play a role during adipogenesis or in differentiated adipocytes [GGT5, LOC100521789, LPCAT2, PTPR, HHAT, GADD45G, PRKG1, ENSSSCG00000035293, GRIN3A, JAK2, CCDC180, NFATC1, CCBN2, CCBN3, ENO2, PIK3C2B, and ADH1C. When adipocytes are induced to differentiate, preadipocytes re-enter the cell cycle synchronously and undergo mitotic clonal expansion [CCBN2) and cyclin B3 (CCBN3) in subcutaneous adipocytes confirmed this. The expression of these genes changes significantly during the differentiation of subcutaneous adipocytes, and expression levels of most of them exceeded those of intramuscular adipocytes, indicating that these genes may be related to fat deposition in the subcutaneous adipocytes of pigs.The gene expression profile of subcutaneous adipocytes and the intramuscular adipocyte differentiation process provides a lot of information for studying the regulatory mechanism of specific fat depositions in the differentiation of different adipocytes in pigs. We identified 30 genes in subcutaneous adipocytes that may have a specific effect on the differentiation of Yorkshire subcutaneous adipocytes. Half of the genes  is a pleiotropic growth factor that controls cell proliferation, migration, and differentiation [FGF1 gene promoter region combined with the PPARγ gene plays a role in regulating adipogenesis. The PPARγ–FGF1 axis, a key regulator of adipose tissue remodeling and systemic metabolic homeostasis, is functionally conserved in mammals [FGF gene. The ssc-miR-874-, novel-miR-779, and novel-miR-126 genes targeting SFRP1 are endogenous regulators of Wnt/β-catenin signaling and participate in the paracrine regulation of human adipogenesis [Sfrp1 aggravates weight gain, glucose homeostasis, and inflammation in mice with diet-induced obesity [HHAT and GRIN3A, respectively, which are considered to be important candidates for the regulation of porcine subcutaneous adipocyte differentiation.miRNAs are small non-coding RNAs that mainly target the 3′ untranslated region of mRNAs to reduce the expression of encoded proteins . Certainntiation . The res mammals ,47. In oogenesis . Loss of obesity . In addiARRB1 and ssc-miR-199a-5p-FOXO4 are important miRNA–mRNA pairs that regulate intramuscular fat. The transgene overexpression of ARRB1 inhibited diet-induced obesity and improved glucose tolerance and systemic insulin sensitivity [FOXO) proteins are a family of transcription factors, and FOXO4 is a member of this family. It inhibits the biosynthesis of cholesterol by inhibiting the expression of CYP51, leading to the accumulation of dihydrolanosterol [In intramuscular adipocytes, we identified two new miRNAs (novel-miR-667 and novel-miR-126) through the function of target genes. In addition to the two newly identified miRNAs, ssc-miR-6782-3p-sitivity . Forkheanosterol .In conclusion, our results demonstrated that intramuscular and subcutaneous adipocytes had different ways of generating lipid droplets, i.e., different carbon sources. The DEGs and DE-miRNAs in the differentiation process were not the same. We identified 30 genes that play key roles in the differentiation process of subcutaneous adipocytes. Using the constructed miRNA–mRNA signaling pathway network diagram, 10 miRNAs, novel-miR-1018, novel-miR-326, novel-miR-433, novel-miR-930, ssc-miR-185, ssc-miR-370, ssc-miR-874, ssc-miR-615, novel-miR-779, and ssc-miR-214-3p, were found to regulate the differentiation process of subcutaneous adipocytes. In intramuscular adipocytes, a total of 22 genes that may specifically regulate the differentiation process of intramuscular adipocytes were observed. Using the constructed miRNA–mRNA signaling pathway network diagram, ssc-miR-339-5p, ssc-miR-199a-5p, and novel-miR-667, three miRNAs that may regulate the differentiation process of intramuscular adipocytes, were identified.There is a limitation in the research that needs to be explained. We identified important mRNA–miRNA pairs by predicting the targeting relationship between candidate genes and miRNAs. However, we have not verified their targeting relationship, and future research should focus on the verification of these results.