透明带蛋白3对肾透明细胞癌增殖和迁徙的影响
The impact of zona pellucida protein 3 on proliferation and migration in renal clear cell carcinoma
徐欢 1王婷 1罗翠 1王如婷 1程玉 1杨中华 1彭建平1
作者信息
- 1. 武汉大学中南医院泌尿外科,武汉 430071
- 折叠
摘要
目的 探究透明带蛋白3(ZP3)对肾透明细胞癌(ccRCC)增殖和迁移的影响.方法 通过加利福尼亚大学圣克鲁兹分校(UCSC)数据库分析ZP3在ccRCC中的差异表达.在肾癌细胞系(786-O和OS-RC-2)中使用CRISPR/Cas9技术过表达ZP3,通过蛋白质印迹法(Western blot)及实时荧光定量聚合酶链反应(qPCR)验证过表达情况.设置组别为对照组及ZP3过表达试验组,随后通过细胞计数试剂盒(CCK-8)实验、平板克隆形成实验、Transwell实验以及划痕实验观察过表达ZP3基因后ccRCC细胞增殖和迁移的变化.组间比较采用单样本t检验.结果 在UCSC数据库发现ZP3在多种癌症中表达上升,差异有统计学意义[KIRC(肿瘤:2.87±1.23,正常:1.67±1.66,P＜0.05)].ZP3高表达的ccRCC患者生存明显减弱[总生存期:(Logrank P=0.011);无病生存期(Logrank P=0.043)].构建过表达ZP3细胞系后Western blot结果显示过表达ZP3基因后其灰度值明显高于对照组[786-O 细胞(31066.13±1 078.634 比 841.78±117.99,t=39.39,P＜0.05);OS-RC-2 细胞(15 395.32±1 015.764 比 549.35±187.78,t=20.33,P＜0.05)],qPCR 结果显示过表达ZP3后其吸光度显著高于对照组[786-O细胞(14.5±0.52比1.0±0.09,t=35.8,P＜0.05);OS-RC-2 细胞(34.3±1.7 比 1.05±0.30,t=26.88,P＜0.05)].CCK8 结果显示,过表达 ZP3 的细胞增殖能力显著高于对照组细胞[786-O细胞的吸光度(3.65±0.11比1.8±0.06,t=40.63,P＜0.05);OS-RC-2 细胞的吸光度:(3.80±0.10 比 1.80±0.11,t=40.38,P＜0.05)].平板克隆形成实验结果显示,ZP3过表达细胞增殖显著高于对照组细胞[786-O细胞(94±7比59±2,t=6.05,P＜0.05);OS-RC-2 细胞(212±9 比 104±5,t=15.31,P＜0.05)].Transwell 实验结果显示,ZP3 过表达细胞迁移显著高于对照组细胞[786-O细胞:504±17比167±6,t=25.94,P＜0.05);OS-RC-2细胞:(943±17比316±28,t=26.59,P＜0.05)].细胞划痕实验结果显示,ZP3过表达细胞迁移距离显著高于对照组细胞[786-O 细胞:(0.56±0.07 比 0.39±0.01,t=2.91,P＜0.05);OS-RC-2 细胞:(0.42±0.02比0.29±0、008,t=9.63,P＜0.05)].结论 ZP3在肾透明细胞癌中表达上调,ZP3能促进肾透明细胞癌的增殖与迁移.
Abstract
Objective To explore the bioinformatics implications of zona pellucida protein 3(ZP3)in clear cell renal cell carcinoma(ccRCC),as well as to examine the effects of its expression on the prolif-eration and migration of ccRCC.Methods The UCSC database was utilized to analyze the differential ex-pression of ZP3 in renal clear cell carcinoma.ZP3 was overexpressed in renal clear cell carcinoma cell lines(786-O and OS-RC-2)by CRISPR/Cas9 technology,Western blotting and quantitative real-time poly-merase chain reaction(qPCR)were used to verify the overexpression.The groups were divided into control group and ZP3-overexpression group.Then,the cell counting kit-8(CCK-8)assay,plate clone formation assay,and Transwell assay were employed to observe the impact of ZP3 on the proliferation and migration in ccRCC.comparisons between groups were made by applying the one-sample t test.Results The Univer-sity of California,Santa Cruz(UCSC)database found that ZP3 was highly expressed in a variety of cancers(P＜0.05),including renal clear cell carcinoma,and the expression of ZP3 in tumor tissues was higher than normal tissues,with a statistically significant difference KIRC(Tumor:2.87±1.23,Normal:1.67±1.66,P＜0.05),ccRCC patients with high ZP3 expression had significantly poor survival[Overall surviv-al:(Logrank P＜0.05);Disease-free survival(Logrank P＜0.05)].The Western blotting results showed that overexpression of ZP3 resulted in a significantly higher absorbance than that of controls[786-O cells(31 066.13±1 078.634 vs.841.78±117.99,t=39.39,P＜0.05);OS-RC-2 cells(15 395.32±1 015.764 vs.549.35±187.78,t=20.33,P＜0.05)],and qPCR results showed that after overexpress-ing ZP3 Its absorbance was significantly higher than that of the control group[786-O cells(14.5±0.52 vs.1.0±0.09,t=35.8,P＜0.05);OS-RC-2 cells(34.3±1.7 vs.1.05±0.30,t=26.88,P＜0.05)].CCK-8 results showed that the cell proliferation capacity in ZP3-overexpressed cells was signifi-cantly higher than control cells[the absorbance of 786-O cells(3.65±0.11 vs.1.8±0.06,t=40.63,P＜0.05);the absorbance of OS-RC-2 cells(3.80±0.10 vs.1.80±0.11,t=40.38,P＜0.05)].P＜0.05)].The results of plate clone formation assay showed that cell proliferation was significantly enhanced after ZP3 overexpression[786-O cells(94±7 vs.59±2,t=6.05,P＜0.05);OS-RC-2 cells(212±9 vs.104±5,t=15.31,P＜0.05)].The results of Transwell assay showed that cell migration was signifi-cantly enhanced after ZP3 overexpression[786-O cells(504±17 vs.167±6,t=25.94,P＜0.05);OS-RC-2 cells(943±17 vs.316±28,t=26.59,P＜0.05)].The results of cell scratch assay showed that cell migration was significantly enhanced after ZP3 overexpression[786-O cells(0.56±0.07 vs.0.39±0.01,t=2.91,P＜0.05);OS-RC-2 cells(0.42±0.02 vs.0.29±0.008,t=9.63,P＜0.05)].Conclusion ZP3 expression is up-regulated in ccRCC,and ZP3 promotes the proliferation and migration of ccRCC.
关键词
透明带蛋白3/肾透明细胞癌Key words
Zona pellucida protein 3/Renal clear cell carcinoma引用本文复制引用
出版年
2024
NETL
NSTL
万方数据