SOX7靶向ERK1/2/PD-L1通路抑制结直肠癌血管生成

SOX7 targeting the ERK1/2/PD⁃L1 pathway inhibits angiogenesis in colorectal cancer

目的探讨性别决定区Y框蛋白7(SOX7)对结直肠癌血管生成的影响及潜在作用机制。方法应用免疫荧光检测结直肠癌患者组织样本中SOX7表达水平,之后通过裸鼠、转染SOX7 mimic的人结直肠癌细胞系SW480细胞和人脐静脉内皮细胞(HUVEC)共培养进一步研究。用Western-blot验证SOX7与ERK1/2/PD-L1对结直肠癌细胞的相关蛋白表达的影响。用CCK8检测SOX7与ERK1/2/PD-L1对HUVEC增殖的影响。通过体外内皮细胞成管实验测定SOX7与ERK1/2/PD-L1对肿瘤血管生成的影响。结果SOX7在人结直肠癌组织中表达被抑制(P<0.01),同时SOX7的过表达抑制了小鼠体内肿瘤生长(P<0.01)。SW480细胞中SOX7的过表达抑制了ERK1/2、c-Jun的表达,并在ERK1/2的激动剂Senkyunolide I的作用下上调了SW480细胞的ERK1/2、c-Jun蛋白表达(P<0.01),逆转了SOX7对SW480细胞中ERK1/2、c-Jun蛋白表达的影响(P<0.01)。HUVEC中SOX7抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,Senkyunolide I上调了HUVEC的PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,并逆转了SOX7对HUVEC中上述相关蛋白表达的影响(P<0.01)。PD-1/PD-L1 Inhibitor 3抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,SOX7过表达在PD-1/PD-L1 Inhibitor 3的影响下并没有表现出抑制作用。CCK8实验结果显示SOX7过表达显著抑制了HUVEC的增殖能力,Senkyunolide I作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显上升,PD-1/PD-L1 Inhibitor 3作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显下降,以上均有明显统计学差异(P<0.01)。成管实验结果显示SOX7过表达抑制了HUVEC的血管生成,Senkyunolide I强烈加速了血管生成,而PD-1/PD-L1 Inhibitor 3血管生成则被显著抑制,以上均有明显统计学差异(P<0.01)。结论SOX7通过ERK1/2/PD-L1通路抑制结直肠肿瘤的增殖和血管生成,SOX7可能是晚期CRC患者临床治疗中潜在的抗血管生成靶点。

Objective To investigate the effects and potential mechanisms of SOX7 on the angiogenic functions of colorectal cancer cells.Methods Immunofluorescence was applied to detect the expression level of SOX7 in tissue samples from colorectal cancer patients,followed by further study by co⁃culture of nude mice,human colorectal cancer cell line SW480 cells and human umbilical vein endothelial cells(Human Umbilical Vein Endothe⁃lial Cells,HUVEC).Western⁃blot was applied to verify the effect of SOX7 and ERK1/2/PD⁃L1 on the expression of related proteins in colorectal cancer cells.CCK8 was applied to detect the effect of SOX7 with ERK1/2/PD⁃L1 on the proliferation of colorectal cancer cells.The effects of SOX7 with ERK1/2/PD⁃L1 on tumour angiogenesis were deter⁃mined by endothelial cell tube⁃forming assay in vitro.Results SOX7 expression was suppressed in human colorec⁃tal cancer tissues(P<0.01),while overexpression of SOX7 inhibited tumour growth in mice in vivo(P<0.01).Over⁃expression of SOX7 in SW480 cells suppressed the expression of ERK1/2 and c⁃Jun,and at the same time up⁃regu⁃lated the expression of ERK1/2 and c⁃Jun proteins in SW480 under the action of Senkyunolide I(P<0.01),an ago⁃nist of ERK1/2,and reversed the effect of SOX7 on the expression of ERK1/2 and c⁃Jun proteins in SW480(P<0.01).In HUVEC,SOX7 inhibited the protein expression of PD⁃L1,V⁃EGFR2,p⁃PI3K and HIF⁃1ɑ,while Senkyu⁃nolide I up⁃regulated the protein expression of PD⁃L1,V⁃EGFR2,p⁃PI3K and HIF⁃1ɑin HUVEC.The effect of SOX7 on the expression of the above related proteins in HUVEC was reversed(P<0.01).PD⁃1/PD⁃L1 Inhibitor 3 in⁃hibited the protein expression of PD⁃L1,V⁃EGFR2,PI3K and HIF⁃1α,while SOX7 overexpression did not show in⁃hibitory effect under the influence of PD⁃1/PD⁃L1 Inhibitor 3.CCK8 results revealed that SOX7 overexpression signif⁃icantly inhibited the proliferative capacity of HUVEC.The proliferation ability of HUVEC in the two groups under the effect of Senkyunolide I was significantly increased compared with the SOX7 NC group and SOX7 mimic group,and the proliferation ability of HUVEC in the two groups under the effect of PD⁃1/PD⁃L1 Inhibitor 3 was significantly de⁃creased compared with the SOX7 NC group and SOX7 mimic group,all of the above have significant statistical differ⁃ences(P<0.01).The results of tube⁃forming experiments revealed that SOX7 overexpression inhibited angiogenesis in HUVEC,Senkyunolide I strongly accelerated angiogenesis,and PD⁃1/PD⁃L1 Inhibitor 3 angiogenesis was signifi⁃cantly inhibited,all of the above have significant statistical differences(P<0.01).Conclusion SOX7 inhibits tu⁃mour proliferation and angiogenesis through the ERK1/2/PD⁃L1 pathway,and SOX7 may be a potential anti⁃angio⁃genic target in the clinical treatment of advanced CRC patients.