摘要
目的:探究HOXC10通过靶向调控CST1的表达对肺癌细胞干细胞样特性的作用。方法:肺癌细胞系A549在无血清培养条件下形成肿瘤球,WB分别检测HOXC10及干性标志蛋白CD44、CD133、Nanog、SOX2、OCT4的表达。构建ShNC、ShHOXC10 A549细胞,检测干性标志蛋白CD44、CD133、Nanog、SOX2、OCT4的表达,肿瘤成球实验检测细胞自我更新能力,CCK8法检测细胞增殖能力。构建ShNC、ShHOXC10肺癌小鼠皮下瘤模型,测定肿瘤体积和重量,免疫组织化学法检测HOXC10、CST1和干性蛋白标志蛋白CD44、CD133在肿瘤组织中的表达。qPCR和WB法检测ShNC、ShHOXC10组A549细胞中CST1 mRNA和蛋白表达,ChIP-PCR和ChIP-qPCR检测HOXC10和CST1启动子序列结合,双荧光素酶报告基因验证HOXC10对CST1启动子活性的调控。在ShHOXC10细胞中回补pcDNA3.1-CST1,检测CST1过表达对干性标志蛋白CD44、CD133、Nanog、SOX2、OCT4表达及肿瘤成球和细胞增殖的影响。结果:HOXC10在A549细胞肿瘤球中高表达,敲低HOXC10显著抑制干性蛋白标志CD44、CD133、Nanog、SOX2、OCT4的表达,抑制A549细胞的增殖和肿瘤成球能力,皮下瘤实验显示,敲低HOXC10抑制A549细胞的成瘤能力(P<0.001),肿瘤重量(P<0.0001)和体积(P<0.01)显著降低。HOXC10直接结合CST1上游启动子序列,敲低HOXC10抑制CST1的mRNA及蛋白表达水平,显著抑制CST1启动子活性(P<0.01)。在ShHOXC10细胞中过表达CST1显著上调干性蛋白标志CD44、CD133、Nanog、SOX2、OCT4的表达,增强A549细胞的增殖和肿瘤成球能力。结论:在肺癌细胞中HOXC10通过CST1调控肺癌细胞干细胞样特性。
Objective:To investigate the effect of HOXC10 on the stem cell-like characteristics of lung cancer cells through targeted regulation of CST1 expression.Methods:TLung cancer cell line A549 was cultured under serum-free conditions to form tumour spheres.WB detected the expression of HOXC10 and stemness marker proteins CD44,CD133,Nanog,SOX2 and OCT4 respectively.ShNC and ShHOXC10 A549 cells were constructed to detect the expression of stemness marker proteins CD44,CD133,Nanog,SOX2 and OCT4,tumour sphere-forming assay to detect cell self-renewal ability and CCK8 assay to detect cell proliferation ability.ShNC and ShHOXC10 lung cancer mouse subcutaneous tumour models were constructed,tumour volume and weight were measured,and the expression of HOXC10,CST1 and stemness marker proteins CD44 and CD133 were detected in tumour tissues by immunohistochemistry.CST1 mRNA and protein expression in A549 cells of ShNC and ShHOXC10 groups were detected by qPCR and WB.ChIP-PCR and ChIP-qPCR were used to detect HOXC10 and CST1 promoter sequence binding,and dual luciferase reporter genes were used to verify the regulation of CST1 promoter activity by HOXC10.The effect of CST1 overexpression on the expression of stemness marker proteins CD44,CD133,Nanog,SOX2,OCT4 and tumour spheroid formation and cell proliferation was examined by back-complementing pcDNA3.1-CST1 in ShHOXC10 cells.Results:HOXC10 was highly expressed in the tumour spheres of A549 cells,and knockdown of HOXC10 significantly inhibited the expression of stemness protein markers CD44,CD133,Nanog,SOX2,and OCT4,inhibiting the proliferation and tumour sphere-forming ability of A549 cells.Subcutaneous tumour assays showed that knockdown of HOXC10 inhibited the tumour-forming ability of A549 cells(P<0.001)and tumour weight(P<0.0001)and volume(P<0.01)were significantly reduced.Conclusion:HOXC10 regulates the stem cell-like properties of lung cancer cells through CST1.
作者
康建军
高乐
席俊峰
KANG Jianjun;GAO Le;XI Junfeng(The First Hospital of Yulin City,Shaanxi Suide 718000,China)
出处
《河北医学》
CAS
2022年第7期1071-1076,共6页
Hebei Medicine
基金
陕西省自然科学基础研究计划项目,(编号:2018JM7068)。
