LXA4 And Its Analog Inhibit The Promoting Effects Of CAFs On The Migration And Invasion Of HCC Cells Based On The JAK2/STAT3 Pathway

Aims:Hepatic stellate cells(HSCs)are also known as liver fibroblasts,which are the main components of normal liver tissue interstitial cells and the precursors of carcinoma-associated fibroblasts(CAFs).It can promote the growth and development of liver tumors.In this study,we investigated whether the endogenous bioactive lipid lipoxygen A4(LXA4)and its analog BML-111 could inhibit the transformation of liver fibroblasts to CAFs and whether fibroblasts treated with LXA4 and its analogue BML-111 impair the progression of hepatocellular carcinoma.Methods:1.The surgical resected specimens of 20 patients with hepatocellular carcinoma,10 patients with cirrhosis and 5 patients with intrahepatic bile duct calculi who were admitted to the Second Affiliated Hospital of Nanchang University on August 1,2017,and solstice on October 1,2019 were collected.Immunohistochemical assay was used to detect the difference of immunophenotype between tumor stromal cells and normal hepatic stromal cells.2.In vitro,we set five cell experiment groups:(1)group 1:HSC-T6;(2)group2:HSC-T6+TGF-β;(3)group 3:HSC-T6+LXA4;(4)group 4:HSC-T6+TGF-β+LXA4;(5)gruop 5:HSC-T6+TGF-β+LXA4+BOC-2.The changes of the immunophenotype and protein level of HSC-T6 were detected by immunofluo-rescence,quantitative real time PCR and western blot.At the same time,the conditioned medium for HSC-T6 production was collected.The cell proliferation,migration and invasion abilities of tumor cells were detected by MTT,wound healing assay and transwell migration assay.The protein changes of JAK2/STAT3 pathway in tumor cells were detected by immunofluorescence assay and western blot.A 3D co-culture model of HSC-T6 and HCCLM3 was established to detect the influence of HSC-T6 on the growth activity of HCCLM3 and whether LXA4 can inhibit this effect.3.A H22 tumor-bearing mouse model was established in vivo.The effect of LXA4 analogue BML-111 on tumor and tumor stroma in vivo was detected by immunohi-stochemical assay.Results:1.LXA4 inhibited TGF-β-induced HSC-T6 activation in vitro,and decreased the expression of CAFs-related markers(α-SMA,FAP and CollagenⅠ)in HSC-T6 cells treated with LXA4(P<0.05).2.LXA4 inhibits the regulation of HSC-T6 paracrine on tumor cells.The ability of HSC-T6 treated with LXA4 to promote the migration and invasion of Hep3 b and HCCLM3 cells in vitro was significantly weaker than that of HSC-T6 treated with TGF-β(P<0.05),but had no significant effect on Hep3 b proliferation(P>0.05).3.LXA4 inhibits the expression of JAK2/STAT3 signaling pathway protein in HCCLM3 and Hep3 b cells.The effect of LXA4-pretreated fibroblasts on the JAK2/STAT3 pathway in HCC cells was significantly lower than that of TGF-βpretreated fibroblasts(P<0.05).4.LXA4 inhibits the growth of HSC-T6/HCCLM3 3d spheres.After the formation of tumor spheres,the growth rate of tumor spheres treated with LXA4 was significantly lower than that of tumor spheres treated with PBS(P<0.05)).5.Inhibitory effect of BML-111 on CAFs markers in H22 tumor-bearing mouse model and protein expression of JAK2/STAT3 pathway in tumor tissue.In the H22tumor-bearing mouse model,the expression of CAFs markers in the mesenchymal cells and the JAK2/STAT3 pathway protein in tumor tissues in the BML-111 treatment group were lower than those in the control group(P<0.05).Conclusion:1.In liver cancer,the immunophenotype of normal fibroblasts was changed after activation of CAFs,and increased α-SMA expression was the most significant.2.Activated fibroblasts can abnormally activate the JAK2/STAT3 signaling pathway in tumor cells and promote the migration and invasion of HCC cells.3.LXA4 and its analog BML-111 can inhibit the activation of fibroblasts into CAFs.4.LXA4 and its analog BML-111 can inhibit the promoting effects of CAFs on the migration and invasion of HCC cells based on the JAK2/STAT3 signaling pathway.