Ⅹ型胶原蛋白α1通过激活黏着斑激酶通路影响前列腺癌的进展

Effect of collagen typeⅩalpha 1 chain on progression of prostate cancer through the focal adhesion kinase pathway

目的探讨Ⅹ型胶原蛋白α1(COL10A1)对前列腺癌细胞增殖、迁移的影响及其作用机制。方法分析TCGA数据库中497例前列腺癌组织和52例前列腺正常组织中基因COL10A1的表达差异。将人源前列腺癌细胞系22RV1和DU145作为研究对象, 分别转染空载质粒和COL10A1过表达质粒, 转染2 d后, 使用5-乙炔基-2’-脱氧尿苷(EdU)染色实验、平板克隆形成实验和细胞计数试剂盒(CCK-8)实验验证细胞的增殖能力;使用Transwell实验、划痕愈合实验和蛋白质印迹实验验证细胞的迁移能力, 采用蛋白质印迹实验探索COL10A1与p-DDR2之间的关系, 并验证其对黏着斑激酶(FAK)通路的影响。两实验组间比较采用独立样本t检验, 多实验组间比较采用方差分析。结果对照组细胞EdU着色细胞比例低于实验组[22RV1:(14.760±2.255)%比(53.850±4.463)%, t=7.816, P<0.05;DU145:(20.560±3.198)%比(65.470±2.857)%, t=10.470, P<0.05]、培养96 h后吸光度低于实验组(22RV1:2.076±0.090比2.538±0.075, F=27.820, P<0.05;DU145:2.194±0.078比2.856±0.087, F=190.700, P<0.05)、克隆形成数低于实验组(22RV1:48.330±5.364比162.700±13.120, t=8.067, P<0.05;DU145:132.000±12.100比297.300±17.320, t=7.825, P<0.05)、划痕愈合率低于实验组[22RV1:(21.330±1.202)%比(31.330±1.453)%, t=5.303, P<0.05;DU145:(25.330±0.882)%比(48.670±2.906)%, t=7.683, P<0.05]、迁移细胞数低于实验组(22RV1:55.670±4.910比154.000±6.557, t=12.000, P<0.05;DU145:28.330±5.608比90.330±11.050, t=5.003, P<0.05)。同时, COL10A1能显著增加p-DDR2蛋白的表达(22RV1:0.621±0.010比0.690±0.017, t=3.417, P<0.05;DU145:0.557±0.010比0.734±0.010, t=12.580, P<0.05)、升高FAK蛋白的表达(22RV1:0.487±0.008比0.608±0.005, t=13.530, P<0.05;DU145:0.441±0.010比0.511±0.011, t=4.658, P<0.05)。结论 COL10A1能够通过DDR2/FAK轴促进前列腺癌细胞的增殖和迁移。

Objective To explore the effect of collagen typeⅩalpha 1 chain(COL10A1)on the progression of prostate cancer and its action mechanism.Methods Differential expression of gene COL10A1 in prostate cancer and paracancerous tissues was compared using the TCGA database.The human prostate cancer cell lines 22RV1 and DU145 were divided into control group and experimental group,and the vector and COL10A1 plasmid were transfected.After 2 days,the proliferation ability of cells was verified by 5-Ethynyl-2’-deoxyuridine(EdU)staining experiment,plate cloning formation experiment and cell counting kit-8(CCK-8)method,and the migration ability of cells was verified by Transwell experiment,scratch healing experiment and Western blotting.The targeting relationship between COL10A1 and p-DDR2/focal adhesion kinase(FAK)axis was explored by Western blotting.The comparison between the two experimental groups was carried out using the independent sample t-test,and the comparison between the multi-experimental groups was analyzed by analysis of variance.Results The proportion of EdU-stained cells[22RV1:(14.760±2.255)%vs.(53.850±4.463)%,t=7.816,P<0.05;DU145:(20.560±3.198)%vs.(65.470±2.857)%,t=10.470,P<0.05],absorbance(22RV1:2.076±0.090 vs.2.538±0.075,F=27.820,P<0.05;DU145:2.194±0.078 vs.2.856±0.087,F=190.700,P<0.05),number of colony formation(22RV1:48.330±5.364 vs.162.700±13.120,t=8.067,P<0.05;DU145:132.000±12.100 vs.297.300±17.320,t=7.825,P<0.05),scratch healing rate[22RV1:(21.330±1.202)%vs.(31.330±1.453)%,t=5.303,P<0.05;DU145:(25.330±0.882)%vs.(48.670±2.906)%,t=7.683,P<0.05],and number of migrated cells(22RV1:55.670±4.910 vs.154.000±6.557,t=12.000,P<0.05;DU145:28.330±5.608 vs.90.330±11.050,t=5.003,P<0.05)were lower in the control group than in the experimental group.COL10A1 could significantly increase the expression of p-DDR2 protein(22RV1:0.621±0.010 vs.0.690±0.017,t=3.417,P<0.05;DU145:0.557±0.010 vs.0.734±0.010,t=12.580,P<0.05)and increase the expression of FAK protein(22RV1:0.487±0.008 vs.0.608±0.005,t=13.530,P<0.05;DU145:0.441±0.010 vs.0.511±0.011,t=4.658,P<0.05).Conclusion COL10A1 can promote prostate cancer proliferation and migration through the DDR2/FAK axis.