低氧环境下甲状腺乳头状癌细胞中期因子表达的调节机制研究  

Regulatory mechanism of Midkine secretion in papillary thyroid carcinoma cells under hypoxic environment

作　　者：李吉 张建辉 谢中意 李君强 LI Ji;ZHANG Jianhui;XIE Zhongyi;LI Junqiang(Department of Surgery,Beilun People's Hospital,Ningbo 315800,China;不详)

机构地区：[1]宁波市北仑区人民医院外科,315800 [2]宁波市北仑区人民医院病理科,315800

出　　处：《浙江医学》2023年第9期931-938,共8页Zhejiang Medical Journal

基　　金：浙江省医药卫生科技计划项目(2020KY920)。

摘　　要：目的探讨低氧环境下甲状腺乳头状癌(PTC)细胞中期因子(Midkine)表达的调节机制。方法以人PTC细胞系BCPAP和TPC1为材料,使用低氧、缺氧诱导因子-1α(HIF1-α)稳定剂DMOG刺激,使用HIF1-α小发夹RNA(HIF1-αshRNA)转染,采用Western blot法分别检测不同细胞HIF1-α、CD10与半乳糖凝集素3(gal3)蛋白表达的变化。采用双荧光素报告酶法检测HIF1-α与CD10、gal3 mRNA的靶向关系,并在公共数据库中检测HIF1-α与CD10、gal3 mRNA表达的关联。将BCPAP和TPC1为对照组、CD10的编码基因膜金属肽内切酶(MME)敲低组(转染MME shRNA)、gal3的编码基因人可溶性半乳糖凝集素3(LGALS3)敲低组(转染LGALS3 shRNA)、MME+LGALS3共敲低组(共转染MME shRNA和LGALS3 shRNA),采用Western blot法检测各组HIF1-α、CD10和gal3蛋白相对表达量,采用ELISA法检测各组Midkine水平。结果低氧、DMOG刺激下,细胞CD10和gal3蛋白相对表达量均上升(均P<0.01),且呈时间依赖性。转染HIF1-α后,细胞CD10和gal3蛋白相对表达量均显著下降(均P<0.01);双荧光素报告酶实验显示,HIF1-αcDNA与CD10和gal3启动子靶向结合,增强两者的转录表达;HIF1-α与MME mRNA和LGALS3 mRNA表达的相关系数分别为0.43和0.58,均呈正相关(均P<0.01)。随缺氧时间延长,细胞中Midkine表达水平升高;相比对照组,MME敲低组、LGALS3敲低组和实验组Midkine表达水平显著降低,差异均有统计学意义(均P<0.01)。结论低氧环境下PTC细胞系能够通过HIF1-α调节CD10、gal3的表达,进而调节Midkine表达。Objective To explore the regulatory mechanism of Midkine secretion in papillary thyroid carcinoma(PTC)cells under hypoxic conditions.Methods Human PTC BCPAP and TPC1 cells were exposed to hypoxia,stimulated with hypoxia inducible factor1-α(HIF1-α)stabilizer DMOG or transfected with HIF1-αshort hairpin RNA(HIF1-αshRNA),respectively.After the various treatment,the changes of HIF1-α,CD10 and galectin-3(gal3)protein expression in cells were detected with Western blot assay.The targeting relationship of HIF1-αto CD10 and gal3 mRNA was examined with dualluciferase reporter enzyme assay,and the association between HIF1-αand CD10,gal3 expression based on public databases was also analyzed.The cells were divided into control group,membrane metalloendopeptidase endonuclide(MME)knockout group(transfected with MME shRNA),human soluble galactol 3(LGALS3)knockout group(transfected with LGALS3 shRNA),and MME+LGALS3 co-knockout group(co-transfected with MME shRNA and LGALS3 shRNA),the relative expression levels of HIF1-α,CD10 and gal3 in each group were detected by Western blot,and Midkine levels in each group were detected by ELISA.Results Under hypoxia and DMOG stimulation,the relative expressions of CD10 and gal3 protein increased(both P<0.01)in a time-dependent manner.And the relative expressions of CD10 and gal3 protein were decreased significantly(all P<0.01)after transfection with HIF1-α.The results of dual-luciferase reporter enzyme assay showed that HIF1-αcDNA was target-bound to CD10 and gal3 promoters to enhance their transcriptional expression.The correlation coefficients between HIF1-αand the expression of MME mRNA and LGALS3 mRNA were 0.43 and 0.58,respectively(P<0.01).Meanwhile,the concentration of Midkine increased with the extension of hypoxia time.Compared with the control group,the concentration of Midkine in MME knockdown group,LGALS3 knockdown group and experimental group was significantly decreased(all P<0.01).Conclusion PTC cells under hypoxic conditions can further regulate the expression of CD

关 键 词：甲状腺乳头状癌 缺氧诱导因子-1Α CD10 半乳糖凝集素3 中期因子 

分 类 号：R736.1[医药卫生—肿瘤]