GAPDH expression as a measurement of transfection efficiency for p^16INK4agene silencing (siRNA) in senescent human diploid fibroblasts

Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent HDFs showed several molecular and cytological changes such as large flat morphology, expression of senescence-associated β-galactosidase (SA β-gal) activity and altered gene expression. Small interfering RNA (siRNA) has been demonstrated to be a potential research tool to analyse gene function and pathway. Expression of an appropriate housekeeping or reference gene can be used as a measurement of transfection efficiency in siRNA. Therefore this study was designed to determine the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model. GAPDH knockdown with an appropriate transfection reagent was measured by quantitative real time RT-PCR while cellular senescence was characterized based on morphological changes, expression of SA β-gal and p16INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16INK4a in senescent transfected HDFs caused morphological alterations which results in the formation of spindle shaped fibroblasts. This study demonstrated the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model.

Culture of Bovine Fetal Skin Fibroblasts in vitro and Ipr1 Gene Transfection

In order to generate transgenic donor cells for nuclear transfer, bovine fetal fibroblasts were isolated in vitro and transfected with the eukaryotic expression vector pSRA-EGFP-Ipr1. The mouse Ipr1 gene and human SR-A promoter were successfully cloned and then used to construct this macrophage-specific eukaryotic expression vector. Bovine fetal fibroblasts in stable primary culture (4th passage) were transfected with pSRA-EGFP-Ipr1 by electroporation. Fluorescence from GFP was observed after 24h. Transgenic cells were selected using G418 and the resultant monoclones were picked and expanded. The transgenic cells, at the 9 th passage, were evaluated by PCR and flow cytometry. The inserted Ipr1 was confirmed by PCR, indicating stable integration of the transgene into the genome and cells had normal karyotypes and very good appearance, which indicate no deleterious result of the transgenesis. In conclusion, the cells obtained could be used as donor cells for nuclear transfer for further research of transgenic cattle.

Expression of MMP-3 mRNA in hypertrophic scar fibroblasts and MMP-3 gene transfection

To investigate the molecular mechanism of extracellular matrix overdeposition in hypertrophic scar tissues and to explore MMPs gene therapy for hypertrophic scar. Methods: Hypertrophic scarderived and normal skin-derived fibroblasts were cultured and a recombinant retrovirus vector containing MMP-3 gene was constructed and then transfected into hypertrophic scar fibroblasts. Expressive level of MMP-3 mRNA was detected by dot blotting, and the activity of MMPs was determined by DNP-peptide.Results: Lower expression of MMP-3 mRNA and fewer DNP-peptide hydrolyzed fragments were observed in hypertrophic scar-derived fibroblasts compared with normal skin-derived fibroblasts. Transfection of MMP-3gene into hypertrophic scar-derived fibroblasts could enhance the expression of MMP-3 mRNA (3. 4 fold)and the de novo capacity to hydrolyze DNP-peptide (2. 1 fold). Conclusion: Overdeposition of extracellular matrix in hypertrophic scar tissue was related to low expression of MMP-3 due to its down-degradation of extracellular matrix. MMP-3 gene transfection could be a better way to treat hypertrophic scars by degrading extracellular matrix.

The Combined Effect of Lumenato and Ceramide in the Protection of Collagen Damage Induced by Neutrophils in Normal Human Dermal Fibroblasts

Introduction: Collagen is the primary structural protein fibroblasts produce in the skin’s extracellular matrix. Infiltration of neutrophils into the epidermis and dermis by exposure to UV causes collagen damage and contributes to photoaging. Methods: To study the combined effect of Lumenato and ceramide in preventing collagen-1 damage induced by phagocytes, we used co-cultures of normal human dermal fibroblasts (fibroblasts) and activated human neutrophils. The present study aimed to determine the protective effect of the combination of Lumenato and ceramide on fibroblast collagen-1 damage induced by neutrophils. Results: Lumenato (in the range of 6.5 - 208 μg/ml) or ceramide (in the range of 0.1 - 50 μM) inhibited the production of superoxides and MPO by TNFα-stimulated neutrophils, as well as the production of NO by LPS-stimulated macrophages in a dose-dependent manner. The combinations of Lumenato and ceramide, in low concentrations, caused synergistic prevention of fibroblasts’ collagen-1 damage induced by TNFα-activated neutrophils, detected by fluorescence immunostaining and WB analysis. MPO activity in the supernatants of the co-cultures was also synergistically inhibited. Adding Lumenato or ceramide singly or in combinations in these low concentrations to the fibroblast cultures did not affect the expression of collagen-1. The combinations of Lumenato or ceramide in these concentrations also caused a synergistic inhibition of NO production by activated macrophages. Conclusions: The results suggest that combining low concentrations of Lumenato and ceramide results in synergistic protection against fibroblasts’ collagen-1 damage induced by neutrophils, thus indicating their possible potential for enhanced skin health.

Lysine demethylase 5B transcriptionally regulates TREM1 in human cardiac fibroblasts

Background:A differential gene,triggering receptor expressed on myeloid cells 1(TREM1),was identified in blood sequencing datasets from myocardial infarction patients and healthy controls.Myocardialfibrosis following myocardial infarction significantly contributes to cardiac dysfunction.Objectives:This study aimed to unveil the intrinsic regulatory mechanism of TREM1 in myocardialfibrosis.Methods:Mimicking pathology by angiotensin II(Ang II)treatment of human cardiacfibroblasts(HCFs),the impacts of TREM1 knockdown on its proliferation,migration,and secretion of the pro-fibrotic matrix were identified.Using the Human Transcription Factor Database(HumanTFDB)website,lysine-specific demethylase 5B(KDM5B)was found to bind to the TREM1 promoter,which was further validated through luciferase reporter and chromatin immunoprecipitation(ChIP).By promoting KDM5B overexpression,its effect on the regulation of TREM1 was examined.Results:TREM1 knockdown suppressed the proliferation,migration,and secretion of the pro-fibrotic matrix in HCFs upon Ang II treatment.KDM5B bound to the TREM1 promoter and upregulated its transcriptional expression.Furthermore,KDM5B overexpression reversed the regulation of the above cellular phenotypes by TREM1 knockdown.Conclusion:This study sheds light on the positive regulation of TREM1 by KDM5B,demonstrating their role in promoting myocardialfibrosis.Thisfinding provides a theoretical foundation for understanding disease pathology and potentially advancing the development of new targeted therapies.

CRABP2 regulates infiltration of cancer-associated fibroblasts and immune response in melanoma

Finding biomarkers for immunotherapy is an urgent issue in cancer treatment.Cellular retinoic acid-binding protein 2(CRABP2)is a controversial factor in the occurrence and development of human tumors.However,there is limited research on the relationship between CRABP2 and immunotherapy response.This study found that negative correlations of CRABP2 and immune checkpoint markers(PD-1,PD-L1,and CTLA-4)were observed in breast invasive carcinoma(BRCA),skin cutaneous melanoma(SKCM),stomach adenocarcinoma(STAD)and testicular germ cell tumors(TGCT).In particular,in SKCM patients who were treated with PD-1 inhibitors,high levels of CRABP2 predicted poor prognosis.Additionally,CRABP2 expression was elevated in cancer-associated fibroblasts(CAFs)at the single-cell level.The expression of CRABP2 was positively correlated with markers of CAFs,such as MFAP5,PDPN,ITGA11,PDGFRα/βand THY1 in SKCM.To validate the tumor-promoting effect of CRABP2 in vivo,SKCM xenograft mice models with CRABP2 overexpression have been constructed.These models showed an increase in tumor weight and volume.Enrichment analysis indicated that CRABP2 may be involved in immunerelated pathways of SKCM,such as extracellular matrix(ECM)receptor interaction and epithelial-mesenchymal transition(EMT).The study suggests that CRABP2 may regulate immunotherapy in SKCM patients by influencing infiltration of CAFs.In conclusion,this study provides new insights into the role of CRABP2 in immunotherapy response.The findings suggest that CRABP2 may be a promising biomarker for PD-1 inhibitors in SKCM patients.Further research is needed to confirm these findings and to explore the clinical implications of CRABP2 in immunotherapy.

Unraveling the role of cancer-associated fibroblasts in colorectal cancer

Within the intricate milieu of colorectal cancer(CRC)tissues,cancer-associated fibroblasts(CAFs)act as pivotal orchestrators,wielding considerable influence over tumor progression.This review endeavors to dissect the multifaceted functions of CAFs within the realm of CRC,thereby highlighting their indispensability in fostering CRC malignant microenvironment and indicating the development of CAFs-targeted therapeutic interventions.Through a comprehensive synthesis of current knowledge,this review delineates insights into CAFsmediated modulation of cancer cell proliferation,invasiveness,immune evasion,and neovascularization,elucidating the intricate web of interactions that sustain the pro-tumor metabolism and secretion of multiple factors.Additionally,recognizing the high level of heterogeneity within CAFs is crucial,as they encompass a range of subtypes,including myofibroblastic CAFs,inflammatory CAFs,antigen-presenting CAFs,and vessel-associated CAFs.Innovatively,the symbiotic relationship between CAFs and the intestinal microbiota is explored,shedding light on a novel dimension of CRC pathogenesis.Despite remarkable progress,the orchestrated dynamic functions of CAFs remain incompletely deciphered,underscoring the need for continued research endeavors for therapeutic advancements in CRC management.

Molecular diagnostic approaches in detecting rearranged during transfection oncogene mutations in multiple endocrine neoplasia type 2

Different types of neuroendocrine cancer,including medullary thyroid cancer(MTC)and thyroid C-cell hyperplasia,are part of multiple endocrine neoplasia type 2(MEN2).A proto-oncogene mutation of the rearranged during transfection(RET)gene changes the way that receptor tyrosine kinases work.Multiple endocrine neoplasia,a pathological condition,involves these kinases.When the RET protooncogene changes,it can cause endocrine adenomas and hyperplasia to happen at the same time or one after the other.Pheochromocytoma,medullary thyroid carcinoma,and hyperparathyroidism,alone or in combination,are present in MEN2A patients.Some patients may also have skin lichen amyloidosis or Hirschsprung's disease.Patients with MEN2A often present with MTC.MTC is aggressive and has the worst prognosis,as most patients exhibit lymph node metastasis.MTC is one of the important causes of death in patients with MEN2A.RET mutation analysis aids in identifying MEN2A symptoms and monitoring levels of calcium,thyroid hormones,calcitonin,normetanephrine,fractionated metanephrines,and parathyroid hormone.The earlier diagnosis of MTC significantly improves survival and prompts better management of MEN2A.In this editorial,we will discuss the significance of molecular diagnostic approaches in detecting RET oncogene mutations in MEN2A.

Impact of STAT-signaling pathway on cancer-associated fibroblasts in colorectal cancer and its role in immunosuppression

Colorectal cancer(CRC)remains one of the most commonly diagnosed and deadliest types of cancer worldwide.CRC displays a desmoplastic reaction(DR)that has been inversely associated with poor prognosis;less DR is associated with a better prognosis.This reaction generates excessive connective tissue,in which cancer-associated fibroblasts(CAFs)are critical cells that form a part of the tumor microenvironment.CAFs are directly involved in tumorigenesis through different mechanisms.However,their role in immunosuppression in CRC is not well understood,and the precise role of signal transducers and activators of transcription(STATs)in mediating CAF activity in CRC remains unclear.Among the myriad chemical and biological factors that affect CAFs,different cytokines mediate their function by activating STAT signaling pathways.Thus,the harmful effects of CAFs in favoring tumor growth and invasion may be modulated using STAT inhibitors.Here,we analyze the impact of different STATs on CAF activity and their immunoregulatory role.

Resveratrol inhibits pancreatic cancer proliferation and metastasis by depleting senescent tumor-associated fibroblasts

BACKGROUND Pancreatic cancer,a formidable gastrointestinal neoplasm,is characterized by its insidious onset,rapid progression,and resistance to treatment,which often lead to a grim prognosis.While the complex pathogenesis of pancreatic cancer is well recognized,recent attention has focused on the oncogenic roles of senescent tumor-associated fibroblasts.However,their precise role in pancreatic cancer remains unknown.Resveratrol is a natural polyphenol known for its multifaceted biological actions,including antioxidative and neuroprotective properties,as well as its potential to inhibit tumor proliferation and migration.Our current investigation builds on prior research and reveals the remarkable ability of resveratrol to inhibit pancreatic cancer proliferation and metastasis.AIM To explore the potential of resveratrol in inhibiting pancreatic cancer by targeting senescent tumor-associated fibroblasts.METHODS Immunofluorescence staining of pancreatic cancer tissues revealed prominent coexpression ofα-SMA and p16.HP-1 expression was determined using immunohistochemistry.Cells were treated with the senescence-inducing factors known as 3CKs.Long-term growth assays confirmed that 3CKs significantly decreased the CAF growth rate.Western blotting was conducted to assess the expression levels of p16 and p21.Immunofluorescence was performed to assess LaminB1 expression.Quantitative real-time polymerase chain reaction was used to measure the levels of several senescence-associated secretory phenotype factors,including IL-4,IL-6,IL-8,IL-13,MMP-2,MMP-9,CXCL1,and CXCL12.A scratch assay was used to assess the migratory capacity of the cells,whereas Transwell assays were used to evaluate their invasive potential.RESULTS Specifically,we identified the presence of senescent tumor-associated fibroblasts within pancreatic cancer tissues,linking their abundance to cancer progression.Intriguingly,Resveratrol effectively eradicated these fibroblasts and hindered their senescence,which consequently impeded pancreatic cancer progression.CONCLUSION This groundbreaking discovery reinforces Resveratrol's stature as a potential antitumor agent and positions senescent tumor-associated fibroblasts as pivotal contenders in future therapeutic strategies against pancreatic cancer.

Effects of Serum Concentration, Synchronization Time and Confluence on the Cell-Cycle Synchronization Efficiency of Goat Fibroblasts

This study aims to evaluate the effect of serum concentration, synchronization time, and confluence degree on the synchronisation efficiency of goat fibroblast cycle. The results indicated that there was no difference in the percentage of nucleated fibroblasts in the G0/G1 stage between serum concentrations of 0.3% and 0.4% (83.89% and 82.69%, respectively, P > 0.05) as well as between serum concentrations of 0.2% and 0.5% (76.95% and 75.46%, respectively, P > 0.05). The percentage of nucleated fibroblasts in the G0/G1 stage was highest at the concentration of 0.3% and lowest in the control group (83.89% vs. 62.67%, P 0.05). The beneficial effect of high confluence was confirmed by the large percentage of nucleated fibroblasts at the G0/G1 stage. The 60% confluency was significantly lower than the 80% and 100% confluency (73.44%, 86.63%, and 87.17%, respectively, P < 0.05). The results indicate that the goat fibroblast cycle synchronization is the most effective at the serum concentration of 0.3%, 72 hours of synchronization and 100% confluency.

Exosomes from umbilical cord mesenchymal stromal cells promote the collagen production of fibroblasts from pelvic organ prolapse

BACKGROUND Pelvic organ prolapse(POP)involves pelvic organ herniation into the vagina due to pelvic floor tissue laxity,and vaginal structure is an essential factor.In POP,the vaginal walls exhibit abnormal collagen distribution and decreased fibroblast levels and functions.The intricate etiology of POP and the prohibition of trans-vaginal meshes in pelvic reconstruction surgery present challenges in targeted therapy development.Human umbilical cord mesenchymal stromal cells(hucMSCs)present limitations,but their exosomes(hucMSC-Exo)are promising therapeutic tools for promoting fibroblast proliferation and extracellular matrix remodeling.suppressed inflammation in POP group fibroblasts,stimulated primary fibroblast growth,and elevated collagen I(Col1)production in vitro.High-throughput RNA-seq of fibroblasts treated with hucMSC-Exo and miRNA sequencing of hucMSC-Exo revealed that abundant exosomal miRNAs downregulated matrix metalloproteinase 11(MMP11)expression.CONCLUSION HucMSC-Exo normalized the growth and function of primary fibroblasts from patients with POP by promoting cell growth and Col1 expression in vitro.Abundant miRNAs in hucMSC-Exo targeted and downregulated MMP11 expression.HucMSC-Exo-based therapy may be ideal for safely and effectively treating POP.

17β-estradiol inhibits TGF-β-induced collagen gel contraction mediated by human Tenon fibroblasts via Smads and MAPK signaling pathways

AIM:To investigate the impact of 17β-estradiol on the collagen gels contraction(CGC)and inflammation induced by transforming growth factor(TGF)-βin human Tenon fibroblasts(HTFs).METHODS:HTFs were three-dimensionally cultivated in type I collagen-generated gels with or without TGF-β(5 ng/mL),17β-estradiol(12.5 to 100μmol/L),or progesterone(12.5 to 100μmol/L).Then,the collagen gel diameter was determined to assess the contraction,and the development of stress fibers was analyzed using immunofluorescence staining.Immunoblot and gelatin zymography assays were used to analyze matrix metalloproteinases(MMPs)and tissue inhibitors of metalloproteinases(TIMPs)being released into culture supernatants.Enzyme-linked immunosorbent assay(ELISA)and reverse transcription-quantitative polymerase chain reaction(RT-PCR)were used to detect interleukin(IL)-6,monocyte chemoattractant proteins(MCP)-1,and vascular endothelial growth factor(VEGF)in HTFs at the translational and transcriptional levels.The phosphorylation levels of Sma-and Mad-related proteins(Smads),mitogen-activated protein kinases(MAPKs),and protein kinase B(AKT)were measured by immunoblotting.Statistical analysis was performed using either the Tukey-Kramer test or Student’s unpaired t-test to compare the various treatments.RESULTS:The CGC caused by TGF-βin HTFs was significantly inhibited by 17β-estradiol(25 to 100μmol/L),and a statistically significant difference was observed when comparing the normal control group with 17β-estradiol concentrations exceeding 25μmol/L(P<0.05).The suppressive impact of 17β-estradiol became evident 24h after administration and peaked at 72h(P<0.05),whereas progesterone had no impact.Moreover,17β-estradiol attenuated the formation of stress fibers,and the production of MMP-3 and MMP-1 in HTFs stimulated by TGF-β.The expression of MCP-1,IL-6,and VEGF mRNA and protein in HTFs were suppressed by 100μmol/L 17β-estradiol(P<0.01).Additionally,the phosphorylation of Smad2 Smad3,p38,and extracellular signal-regulated kinase(ERK)were downregulated(P<0.01).CONCLUSION:17β-estradiol significantly inhibits the CGC and inflammation caused by TGF-βin HTFs.This inhibition is likely related to the suppression of stress fibers,inhibition of MMPs,and attenuation of Smads and MAPK(ERK and p38)signaling.17β-estradiol may have potential clinical benefits in preventing scar development and inflammation in the conjunctiva.

Optimization of Culture Medium and Transfection Method for Head and Neck Squamous Cell Carcinoma Organoids

[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term suspension transfection for organoid in matrigel. [Methods] Advanced DMEM/F12 medium, GlutaMax and HEPES buffer, nicotinamide, N-acetylcysteine, B27, A83-01, EGF, Y-27632 and Primocin primary cell antibiotics were prepared. On this basis, fibroblast growth factor 10(FGF10), Neuregulin 1, Noggin and R-spondin-1 were added in turn to prepare the selection medium, and the organoid diameter was used as the evaluation index to evaluate the effect of organoid medium. Using lentivirus, mCherry red fluorescent protein was transfected into HNSCC—PDO in different ways, and the transfection effect was evaluated by the fluorescence intensity of organoid sphere. [Results] Nrg1 Noggin and R-Spondin-1 promoted the growth of head and neck squamous cell carcinoma sphere(P<0.05) while FGF10 did not significantly promote the growth of head and neck squamous cell carcinoma sphere(P>0.05). Compared with direct transfection, short-term suspension transfection had higher transfection efficiency for HNSCC—PDO in matrigel. [Conclusions] R-Spondin-1 Nrg1 and Noggin may be the key cytokines in culture of HNSCC—PDO whereas FGF10 played an insignificant role in this study. Short-term suspension transfection could improve the transfection efficiency of lentivirus to HNSCC—PDO.

Calcitriol Suppressed Isoproterenol-induced Proliferation of Cardiac Fibroblasts via Integrinβ3/FAK/Akt Pathway

Objective Cardiac fibroblasts(CFs)proliferation and extracellular matrix deposition are important features of cardiac fibrosis.Various studies have indicated that vitamin D displays an anti-fibrotic property in chronic heart diseases.This study explored the role of vitamin D in the growth of CFs via an integrin signaling pathway.Methods MTT and 5-ethynyl-2′-deoxyuridine assays were performed to determine cell viability.Western blotting was performed to detect the expression of proliferating cell nuclear antigen(PCNA)and integrin signaling pathway.The fibronectin was observed by ELISA.Immunohistochemical staining was employed to evaluate the expression of integrinβ3.Results The PCNA expression in the CFs was enhanced after isoproterenol(ISO)stimulation accompanied by an elevated expression of integrin beta-3(β3).The blockade of the integrinβ3 with a specific integrinβ3 antibody reduced the PCNA expression induced by the ISO.Decreasing the integrinβ3 by siRNA reduced the ISO-triggered phosphorylation of FAK and Akt.Both the FAK inhibitor and Akt inhibitor suppressed the PCNA expression induced by the ISO in the CFs.Calcitriol(CAL),an active form of vitamin D,attenuated the ISO-induced CFs proliferation by downregulating the integrinβ3 expression,and phosphorylation of FAK and Akt.Moreover,CAL reduced the increased levels of fibronectin and hydroxyproline in the CFs culture medium triggered by the ISO.The administration of calcitriol decreased the integrinβ3 expression in the ISO-induced myocardial injury model.Conclusion These findings revealed a novel role for CAL in suppressing the CFs growth by the downregulation of the integrinβ3/FAK/Akt pathway.

In vitro antioxidant and wound healing activity of Sargassum polycystum hydroethanolic extract in fibroblasts and keratinocytes

Objective:To investigate the in vitro antioxidant and wound healing properties of the hydroethanolic extract of Sargassum polycystum,and elucidate the mechanism of its wound healing activity.Methods:Human dermal fibroblast and HaCaT cells were used to evaluate the proliferation by sulforhodamine B and dsDNA assay after treatment with Sargassum polycystum extracts.Scratch wound healing and phalloidin-rhodamine staining were employed to observe migratory activity and filopodia formation,respectively.Western blot and real-time RT-PCR assays were performed to determine the protein and gene expressions related to wound healing activities.Results:The phytochemical analysis found a higher level of flavonoid than phenolic compound in Sargassum polycystum extracts.In human dermal fibroblast cells,Sargassum polycystum extracts at 50 and 100μg/mL significantly increased fibroblast proliferation and the gene expressions of hyaluronic acid synthase 1(HAS1),HAS2,HAS3,collagen type 1 alpha 1 chain(COL1A1),collagen type 3 alpha 1 chain(COL3A1),and elastin.The phosphorylation of Akt,ERK1/2,and p38 MAPK was also significantly upregulated after treatment with Sargassum polycystum extracts.Additionally,50 and 100μg/mL of the extracts prominently enhanced the proliferation,migration,and filopodia formation of HaCaT cells,as well as the protein levels of pFAK/FAK,pSrc/Src,pAkt/Akt,pERK1/2/ERK1/2,Rac1 and Cdc42.Conclusions:Sargassum polycystum extracts show promising wound healing activities in human dermal fibroblasts and keratinocytes.

Effects of areca nut consumption on cell differentiation of osteoblasts, myoblasts, and fibroblasts

Areca nut is used worldwide as a hallucinogenic addicting drug along the tropical belt.Arecoline,a toxic compound,is the most important alkaloid in areca nuts.The adverse effects of oral uptake and chewing of areca nut are well known.For example,the possibility of cancer caused by chewing areca nuts is widely discussed.Chewing areca nut has other adverse effects on other organs,including abnormal cell differentiation,oral cancer,and several other diseases.The use of areca nut is also associated with low birthweight.Skeletal musculature is the largest organ in the body and is attached to the bones.During embryo development,the differentiation of bone and muscle cells is critical.In this article,we reviewed the effects of areca nut and arecoline on embryonic cell differentiation,particularly osteoblasts,myoblasts,and fibroblasts.

Bone marrow mesenchymal stem cell-derived exosomal microRNAs target PI3K/Akt signaling pathway to promote the activation of fibroblasts

BACKGROUND Fibroblast plays a major role in tendon-bone healing.Exosomes derived from bone marrow mesenchymal stem cells(BMSCs)can activate fibroblasts and promote tendon-bone healing via the contained microRNAs(miRNAs).However,the underlying mechanism is not comprehensively understood.Herein,this study aimed to identify overlapped BMSC-derived exosomal miRNAs in three GSE datasets,and to verify their effects as well as mechanisms on fibroblasts.AIM To identify overlapped BMSC-derived exosomal miRNAs in three GSE datasets and verify their effects as well as mechanisms on fibroblasts.METHODS BMSC-derived exosomal miRNAs data(GSE71241,GSE153752,and GSE85341)were downloaded from the Gene Expression Omnibus(GEO)database.The candidate miRNAs were obtained by the intersection of three data sets.TargetScan was used to predict potential target genes for the candidate miRNAs.Functional and pathway analyses were conducted using the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases,respectively,by processing data with the Metascape.Highly interconnected genes in the protein-protein interaction(PPI)network were analyzed using Cytoscape software.Bromodeoxyuridine,wound healing assay,collagen contraction assay and the expression of COL I andα-smooth muscle actin positive were applied to investigate the cell proliferation,migration and collagen synthesis.Quantitative real-time reverse transcription polymerase chain reaction was applied to determine the cell fibroblastic,tenogenic,and chondrogenic potential.RESULTS Bioinformatics analyses found two BMSC-derived exosomal miRNAs,has-miR-144-3p and hasmiR-23b-3p,were overlapped in three GSE datasets.PPI network analysis and functional enrichment analyses in the GO and KEGG databases indicated that both miRNAs regulated the PI3K/Akt signaling pathway by targeting phosphatase and tensin homolog(PTEN).In vitro experiments confirmed that miR-144-3p and miR-23b-3p stimulated proliferation,migration and collagen synthesis of NIH3T3 fibroblasts.Interfering with PTEN affected the phosphorylation of Akt and thus activated fibroblasts.Inhibition of PTEN also promoted the fibroblastic,tenogenic,and chondrogenic potential of NIH3T3 fibroblasts.CONCLUSION BMSC-derived exosomes promote fibroblast activation possibly through the PTEN and PI3K/Akt signaling pathways,which may serve as potential targets to further promote tendon-bone healing.

Cancer-associated fibroblasts of colorectal cancer: Translationalprospects in liquid biopsy and targeted therapy

Colorectal cancer (CRC) is a major global health concern. Accumulation of cancer-associated fibroblasts(CAFs) in CRC is associated with poor prognosis and disease recurrence. CAFs are the main cellular component ofthe tumor microenvironment. CAF-tumor cell interplay, which is facilitated by various secretomes, drives colorectalcarcinogenesis. The complexity of CAF populations contributes to the heterogeneity of CRC and influences patientsurvival and treatment response. Due to their significant roles in colorectal carcinogenesis, different clinicalapplications utilizing or targeting CAFs have been suggested. Circulating CAFs (cCAFs) which can be detected inblood samples, have been proposed to help in determining patient prognosis and enables the detection of cancerthrough liquid biopsy. Liquid biopsy is gaining traction as it is non-invasive, allows frequent and easy sampling, andshows concordance to tissue biopsy analysis. In addition, CAF-targeted therapy is currently being studied extensivelyto be used as one of the treatment avenues for CRC. Various mechanisms of CAF-targeted therapy have beenreported, including blocking the signaling pathways involving CAFs and cancer cells, thus abolishing the CAF-tumorcell crosstalk and subsequently hindering tumorigenesis. These translational applications of cCAFs and utilization ofCAFs as key targets for CRC therapy, although still in the early phases of development, will potentially improve CRCpatient management in the future.