Fluid Shear Stress-Induced IL-8/CXCR Signaling Promotes Epithelial Mesenchymal Transition of Ovarian Cancer Cells

Objective Epithelial mesenchymal transition(EMT)plays a very important role in ovarian cancer metastasis,and IL-8 released from mechanosensitive cancer cells may contribute to the EMT process of solid carcinomas.In this study,we have explored IL-8 and its receptors signal transduction process of human ovarian cancer cells under conditions of FSS,and simultaneously detected the EMT process of ovarian cancer.Methods After the fluid shear stress was loaded,LightCyclerTM system and ELISA were employed to assay the IL-8 mRNA expression and protein production,respectively.Meanwhile,IL-8 reporter gene pEGFP1-IL8USCS was constructed for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis.RT-PCR,Northern blot and immunofluorescence were used to determine the expression of IL-8 receptor CXCR2 at mRNA and protein levels.IL-8 downstream signaling molecule NF-κB nuclear translocation was observed by immunocytofluoresent staining.Western blot was used to examine IκB phosphorylation and EMT-related protein.Results(1)The increase of IL-8 mRNA expression by shear stress was time-dependent.The expression increased when SKOV3 cells exposed to fluid shear stress for 1 h,reached the summits at 2 h,gradually decreased at 3 h and remained at a constant level at 4~12 h.Additionally,IL-8 expression was negatively associated with the intensity of shear stress.After SKOV3 cells were exposed to low fluid shear stress(1.5 dyne/cm2)for 1 h and 2 h,IL-8 mRNA expression increased near 68 and 52 times respectively as that of SKOV3 cells exposed to a high fluid shear stress of 5.0 dyne/cm^2.(2)The productions of IL-8 protein in SKOV3 cells subjected to shear stress were time-dependent.The secretion reached the summit when SKOV3 cells exposed to fluid shear stress for 5 h,then IL-8 secretion gradually decreased at 8 h of stimulation by shear stress.IL-8 secretion increased obviously when fluid shear stress(0.5,1.5,or 2.0 dyne/cm2)was exerted on SKOV3 cells for 1 h.Notablely,the secretion of IL-8 was the highest when SKOV3 cells subjected to fluid shear stress 1.5dyne/cm^2,which was near 6 or 7 times as that of SKOV3 cells subjected to high fluid shear stress(5.0 dyne/cm^2).(3)There was an increase in enhanced green fluorescent protein expression in pEGFPI-IL8USCS-transfected SKOV3 cells subjected to a fluid shear stress of 1.5 dyne/cm2 for 2 h,suggesting a flow shear stress induced IL-8 gene transcriptional activation;(4)CXCR2,which was constitutively present on the surface of SKOV3 cells,increased following exposure to fluid shear stress for 60 min.(5)Following the application of a shear stress of 1.5 dyne/cm^2,NF-κB p65 became detectable in the cell nuclei and Phosphorylated IκB in cell lysates increased significantly;(6)Compared with the control group,critical EMT-related proteins vimentin was upregulated,E-cadherin was downregulated after the application of the 1.5 dyne/cm2shear stress for 2 h,which suggested the EMT of ovarian cancer.Conclusions FSS triggered IL-8/CXCR2 signaling of SK-OV3 cells represents an early gene activation and the activation can be mediated through NF-κB.When the fluid shear stress-induced IL-8/CXCR signaling activated,the expression of EMT-related proteins changed.This observation suggested that fluid shear stress-induced IL-8 activation and the downstream signal pathways may have important contribution to the EMT process of ovarian cancer cells.

Cyclic biaxial tensile strain enhances osteogenic differentiation in rat bone marrow-derived mesenchymal stem cells via activating ERα-Wnt3a/β-catenin pathway

The present study was designed to investigate the role of estrogen receptorα(ERα)in biaxial tensile strain(BTS)regulated osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells(rBMSCs).rBMSCs were derived fromrats and overexpressed ERα.The rBMSCs were subjected to BTS at 1Hz with a strain of 2%for 4 h per day,3 days,with or without ERαinhibitor ICI 182,780(ICI).Then,bone mineralization was performed by Alizarin Red Staining.The markers of osteogenic differentiation and downstream Wnt3a/β-catenin signaling were detected by western blotting.Results showed that BTS enhanced the osteogenic differentiation of rBMSCs,increased protein expression levels of alkaline phosphatase(ALP),runt-related transcription factor 2(Runx2),collagen type I(Col I)and osteocalcin(OCN),and it increased the protein expression levels of estrogen receptor(ER)α(ERα),Wnt3a,andβ-catenin.BTS The activated Wnt3a/β-catenin signaling pathway induced by BTS was abolished by ICI 182,780(ICI).In addition,overexpressing ERαin rBMSCs promoted the osteogenic differentiation by BTS.Taken together,BTS induced osteogenic differentiation of rBMSCs via the ERαand downstream canonical Wnt3a/β-catenin pathway.

Single-cell RNA sequencing reveals classical monocytes are the major precursors of rat osteoclasts

To dissect which subset of bone marrow monocyte is the major precursor of osteoclast,3-month-old rat bone marrow was obtained for single-cell RNA sequencing.A total of 6091 cells were acquired for detailed analysis,with a median number of 1206 genes detected per cell and 17,959 genes detected in total.A total of 19 cell clusters were recognized,with the main lineages identified as B cells,Granulocytes,Monocytes,T cells,Erythrocytes and Macrophages.Monocytes were further divided into classical monocytes and non-classical monocytes.Compared with non-classical monocytes,classical monocytes highly expressed osteoclast differentiation related genes Mitf,Spi1,Fos and Csf1r.Additionally,biological processes of classical monocytes were related to osteoclast differentiation.qPCR revealed differentially expressed genes of classical monocytes played a role in osteoclast differentiation.In conclusion,classical monocytes were identified as the main precursors of osteoclasts in rats,and may contribute to osteoclast differentiation by regulating S100a4,S100a6,S100a10,Fn1,Vcan and Bcl2a1.The results of this study contribute to the understanding of the origin of osteoclasts and may provide potential biomarkers for early diagnosis of bone metabolic diseases,as well as molecular and cellular targets for clinical intervention in bone metabolic diseases.