Hepatic Stellate Cells Affect The Invasion Ability Of Hepatocellular Carcinoma Through Exosome-derived CircWDR25 And Interaction With ΓΔT Cells

Objective: this study was to investigate the effect of circ WDR25 derived from HSCs exosomes on the invasion and proliferation of hepatocellular carcinoma cells and the relationship between the interaction of paracancerous activated hepatic stellate cells(HSCs)and γδT lymphocytes(the ratio of the two)and the prognosis of patients with hepatocellular carcinoma(HCC)after radical resection.Methods:Hepatic stellate cell line LX-2 was co-cultured with hepatocellular carcinoma cell lines Hep3 B,SMMC-7721,and HCCLM3 by Transwell method to establish tumour-activated HSCs.Comparison of gene chip detection showed that circ WDR25 was significantly upregulated and the difference was the most obvious,so circ WDR25 was selected as the target gene for this study.After circ WDR25 in LX-2 was knocked down by lentivirus,exosomes derived from NC-LX-2-circ WDR25 and sh-LX-2-circ WDR25 were extracted by gradient hypercentrifugation.Transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA)were used to observe the morphology of exosomes and analyze their size and concentration.Western Blot(WB)was used to identify the expression of exosome surface markers.Exosomes derived from NC-LX-2-circ WDR25 and sh-LX-2-circ WDR25 were co-cultured with Hep3 B and SMMC 7721,respectively.Wounld healing assay,transwell cell migration and invasion assay,and CCK-8 assay were used to detect the changes of migration,invasion and proliferation of HCC cells.The ratio of HSCs to γδT lymphocytes was calculated by immunohistochemistry.Kaplan Meier method was used for survival analysis.Univariate analysis and multivariate analysis were used to compare the relationship between HCC and prognosis after radical hepatectomy.Results: The expression of circ WDR25(hsa-circ RNA-0004310)was most significantly upregulated by gene microarray assay using tumor activated h SCs.The exosomes of NC-LX-2-circ WDR25 and sh-LX-2-circ WDR25 were successfully isolated and identified after lentivirus transfection with LX-2.The co-culture of NC-LX-2-circ WDR25-isolated exosomes with Hep3 B and SMMC-7721 cells could promote the migration,invasion and proliferation of Hep3 B and SMMC-7721 cells.In addition,the expression of HSCs in paracancerous tissues were significantly higher than that in γδT lymphocytes by immunohistochemical analysis.X-tile software was used to calculate the optimal cut-off value of 18.Patients were divided into low ratio group(ratio≤18,n=222)and high ratio group(ratio > 18,n=98)according to the ratio of HSCs to γδT lymphocytes in paracancerous tissues.In multivariate analysis,preoperative albumin>40g/L(HR=0.553,95%CI: 0.371～0.824),multiple tumors(HR=1.895,95%CI: 1.155-3.108),microvascular invasion(HR=1.665,95%CI: 1.104～2.512),tumor diameter > 5cm(HR=2.400,95%CI: 1.603～3.594),HSCs to γδT lymphocyte ratio >18(HR=1.880,95%CI:1.257 to 2.810)were independent risk factors for overall survival.Preoperative alpha-fetoprotein > 20μg/L(HR=1.631,95%CI: 1.151-2.311),microvascular invasion(HR=2.145,95%CI: 1.346-2.994),tumor diameter>5 cm(HR=1.866,95%CI: 1.342-2.592),HSCs to γδT lymphocyte ratio >8(HR=1.517,95%CI: 1.084-2.122)were independent risk factors for tumor-free survival in HSCs.The cumulative survival rate and tumor-free survival rate in the low ratio group of paracancerous HSCs and γδT lymphocytes were better than those in the high ratio group,and the differences were statistically significant(P < 0.05).Conclusions: Circ WDR25 was significantly overexpressed in tumor-activated HSCs.After down-regulating circ WDR25 in LX-2,exosomes were successfully isolated and inhibited the migration,invasion and proliferation of HCC cells in vitro.The ratio of HSCs and γδT cells was negatively correlated with overall and tumor-free survival after radical resection of HCC.