基于M2型巨噬细胞来源的Siglec15对食管鳞癌细胞恶性生物学行为影响的生物信息学分析及实验验证

Bioinformatics analysis based on effect of M2 macrophagederived Siglec15 on malignant biological behaviour of esophageal squamous cell carcinoma cells and its experimental validation

目的:采用生物信息学方法分析M2型肿瘤相关巨噬细胞(M2-TAMs)来源唾液酸结合免疫球蛋白样凝集素15(Siglec15)促进食管鳞状细胞癌(ESCC)恶性生物学行为的作用,并通过细胞实验对其进行验证。方法:应用肿瘤免疫评价资源(TIMER)数据库分析Siglec15在泛癌和癌旁正常组织中的表达差异及免疫浸润情况,实时荧光定量PCR(RT-qPCR)法检测M2-TAMs和ESCC EC109及KYSE150细胞中Siglec15 mRNA表达水平。在M2-TAMs与ESCC细胞非接触性共培养基础上,分别设置EC109/KYSE150组、EC109/KYSE150+si-NC组(转染si-NC序列)和EC109/KYSE150+si-Siglec15组(分别转染si-Siglec15#1和si-Siglec15#2序列),采用CCK-8法检测各组细胞增殖活性,细胞划痕实验检测各组细胞划痕愈合率,Transwell小室实验检测各组细胞中迁移和侵袭细胞数,流式细胞术检测各组细胞凋亡率。结果:生物信息学分析,与癌旁正常组织比较,食管癌、结肠癌和头颈部鳞状细胞癌等泛癌组织中Siglec15 mRNA表达水平升高(P<0.05或P<0.01),且食管癌组织中Siglec15 mRNA表达水平与巨噬细胞浸润呈明显正相关关系(P<0.05);与EC109细胞和KYSE150细胞比较,M2-TAMs中Siglec15 mRNA表达水平明显升高(P<0.01)。EC109/KYSE150组、EC109/KYSE150+si-NC组和EC109/KYSE150+si-Siglec15组细胞增殖率比较差异均无统计学意义(P>0.05)。与EC109/KYSE150组比较,24和48 h时EC109/KYSE150+si-NC组细胞划痕愈合率升高(P<0.01),迁移和侵袭细胞数增加(P<0.05),细胞凋亡率降低(P<0.01);与EC109/KYSE150+si-NC组比较,EC109/KYSE150+si-Siglec15#1组和EC109/KYSE150+si-Siglec15#2组细胞划痕愈合率降低(P<0.05),迁移和侵袭细胞数减少(P<0.05),细胞凋亡率差异无统计学意义(P>0.05)。结论:M2-TAMs来源Siglec15可能是促进ESCC细胞迁移和侵袭的关键因子。

Objective:To discuss the effect of sialic acid-binding immunoglobulin-like lectin-15(Siglec15)derived from M2 tumor-associated macrophages(M2-TAMs)on promoting the malignant biological behavior of the esophageal squamous cell carcinoma(ESCC)through bioinformatics analysis,and to validate the findings through cell experiment.Methods:The Tumor Immune Estimation Resource(TIMER)online Database was used to analyze the expression differences and immune infiltration of Siglec15 in pan-cancer and adjacent normal tissues.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of Siglec15 mRNA in M2-TAMs and ESCC EC109 and KYSE150 cells.Based on the non-contact co-culture of M2-TAMs and ESCC cells,the following groups were set up,such as EC109/KYSE150 group,EC109/KYSE150+si-NC group(transfected with si-NC sequence),and EC109/KYSE150+si-Siglec15 group(transfected with si-Siglec15#1 and si-Siglec15#2 sequences).CCK-8 method was used to detect the proliferation activities of the cells in various groups;wound healing assay was used to detect the wound healing rates of the cells in various groups;Transwell chamber assay was used to detect the numbers of migration and invasion cells in various groups;flow cytometry was used to detect the apoptotic rates of the cells in various groups.Results:The bioinformatics analysis results showed that compared with adjacent normal tissue,the expression levels of Siglec15 mRNA in pan-cancer tissues such as esophageal cancer,colon cancer,and head and neck squamous cell carcinoma tissues were increased(P<0.05 or P<0.01),and the expression level of Siglec15 mRNA in esophageal cancer tissue was significantly positively correlated with the infiltration of the macrophages(P<0.05).Compared with the EC109 cells and KYSE150 cells,the expression level of Siglec15 mRNA in M2-TAMs was significantly increased(P<0.01).There was no significant difference in the proliferation rate of the cells among EC109/KYSE150 group,EC109/KYSE150+si-NC group,and EC109/KYSE150+si-Siglec15 group(P>0.05).Compared with EC109/KYSE150 group,after treated for 24 and 48 h,the wound healing rate of the cells in EC109/KYSE150+si-NC group was increased(P<0.01),the numbers of migration and invasion cells were increased(P<0.05),and the apoptotic rate was decreased(P<0.01).Compared with EC109/KYSE150+si-NC group,the wound healing rates of the cells in EC109/KYSE150+si-Siglec15#1 group and EC109/KYSE150+si-Siglec15#2 group were decreased(P<0.05),the numbers of migration and invasion cells were decreased(P<0.05),and the apoptotic rates of the cells had no significant difference(P>0.05).Conclusion:Siglec15 derived from M2-TAMs may be a key factor in promoting the migration and invasion of the ESCC cells.