摘要
目的探讨长链非编码RNA(lncRNA)MMP12-001在胆囊癌(GBC)细胞中的表达及对GBC细胞生物学行为的影响。方法采用qRT-PCR法检测lncRNA MMP12-001在GBC癌组织、癌旁组织、GBC细胞株及胆囊上皮细胞中的相对表达量。采用RNA核质分离实验确定lncRNA MMP12-001的亚细胞定位。构建特异性靶向lncRNA MMP12-001的沉默序列及过表达慢病毒载体,分别转染多株GBC细胞株。通过细胞存活率检测、克隆形成实验分析细胞增殖能力的变化。采用Transwell实验观察细胞迁移、侵袭能力的变化。通过RNA互作蛋白拉下实验结合蛋白质谱、RNA结合蛋白免疫共沉淀(RIP)方法筛选lncRNA MMP12-001在GBC中的相互作用蛋白。结果qRT-PCR结果显示癌组织及GBC细胞株中lncRNA MMP12-001相对表达量显著高于癌旁组织和胆囊上皮细胞。RNA核质分离实验发现lncRNA MMP12-001主要位于细胞质内。过表达lncRNA MMP12-001明显促进GBC细胞的增殖、克隆形成、迁移和侵袭能力,差异均有统计学意义(均P<0.05)。沉默lncRNA MMP12-001表达则导致相反的结果(均P<0.05)。RNA互作蛋白拉下实验结合蛋白质谱鉴定结果显示lncRNA MMP12-001在GBC中可与去乙酰化酶沉默调节蛋白3(SIRT3)直接结合。SIRT3 RIP实验也发现SIRT3能够富集lncRNA MMP12-001。结论lncRNA MMP12-001能够促进胆囊癌细胞的增殖、迁移和侵袭。
Objective To investigate the expression of long non-coding RNA(lncRNA)MMP12-001 in gallbladder cancer(GBC)cells and its impact on the biological characteristics of GBC.Methods The expression of lncRNA MMP12-001 in GBC cancer tissues,adjacent tissues,GBC cell lines,and gallbladder epithelial cells was verified using qRT-PCR.The subcellular localization of lncRNA MMP12-001 was determined using RNA nuclear-cytoplasmic separation experiments.shRNA and overexpression lentiviral vectors specifically targeting lncRNA MMP12-001 were constructed and transfected into multiple GBC cell lines.The changes in cell proliferation ability were analyzed by cell survival rate detection and colony formation experiments.The changes in cell migration and invasion ability were observed using Transwell experiments.The interaction proteins of lncRNA MMP12-001 in GBC were initially screened using RNA pull down experiments combined with protein profiling and RNA binding protein immunoprecipitation(RIP).Results qRT-PCR verification showed that the expression level of lncRNA MMP12-001 in cancer tissues and GBC cell lines was significantly higher than that in adjacent tissues and gallbladder epithelial cells.RNA nuclear-cytoplasmic separation experiments showed that lncRNA MMP12-001 was mainly located in the cytoplasm.Overexpression of lncRNA MMP12-001 significantly promoted the proliferation,colony formation,migration,and invasion of GBC cells,with statistically significant differences(all P<0.05).Silencing the expression of lncRNA MMP12-001 resulted in the opposite effect(all P<0.05).RNA pull down combined with protein profiling identification showed that lncRNA MMP12-001 could directly bind to Sirtuin 3(SIRT3)in GBC.SIRT3 RIP experiments also found that SIRT3 IP could enrich lncRNA MMP12-001.Conclusion lncRNA MMP12-001 can promote the proliferation,invasion,and migration of GBC cells.
作者
尹磊
张津瑜
蔡炜龙
汪伟民
YIN Lei;ZHANG Jinyu;CAI Weiong;WANG Weimin(Department of General Surgery,Huzhou Central Hospital,Huzhou 313000,China)
出处
《浙江医学》
CAS
2024年第14期1474-1480,共7页
Zhejiang Medical Journal
基金
湖州市科技局公益性应用研究项目(2022GY17)。
