环状RNA-SEC31A对胰腺癌细胞侵袭、迁移的影响及其机制研究  

Effects of circular RNA-SEC31A on the invasion and migration of pancreatic cancer cells and molecular mechanism

作　　者：阴艺娜 苏民 林梓航 陈亚鹏 陈汝福 李志花[1] Yin Yina;Su Min;Lin Zihang;Chen Yapeng;Chen Rufu;Li Zhihua(Department of Medical Oncology,Sun Yat-sen Memorial Hospital,Guangzhou 510120,China;Department of General Surgery,Guangdong Provincial People′s Hospital,Guangzhou 510080,China)

机构地区：[1]中山大学孙逸仙纪念医院肿瘤内科,广州510120 [2]广东省人民医院普外科,广州510080

出　　处：《中华胰腺病杂志》2023年第2期99-107,共9页Chinese Journal of Pancreatology

基　　金：国家自然科学基金面上项目(82173271)。

摘　　要：目的明确环状RNA-SEC31A(circSEC31A)在胰腺癌中的表达水平,探讨其对胰腺癌细胞侵袭、迁移能力的影响及潜在的分子机制。方法采用实时荧光定量PCR检测人正常胰腺细胞株HPDE及胰腺癌细胞株BXPC-3、PANC1、CaPan-2、SW1990 circSEC31A的表达水平,选用表达量较高的胰腺癌细胞株进行后续实验。针对circSEC31A设计两条siRNA(#1、#2),采用脂质体将siRNA转染胰腺癌细胞沉默circSEC31A表达(siR-circSEC31A组),以不针对circSEC31A设计的siRNA转染胰腺癌细胞为对照组(siR-NC组),Transwell实验及划痕实验检测circSEC31A对胰腺癌细胞侵袭、迁移能力的影响。采用circSEC31A探针及oligo阴性对照探针进行RNA Pull-down实验,筛选与circSEC31A结合的miRNA,并验证该miRNA对胰腺癌细胞侵袭、迁移的影响。荧光定量PCR检测沉默miR-200c-3p及circSEC31A对胰腺癌细胞PDK1 mRNA表达的影响。蛋白质印迹法检测circSEC31A沉默对PDK1蛋白及下游相关蛋白Akt、磷酸化Akt(p-Akt)表达量的影响。结果胰腺正常细胞株HPDE circSEC31A表达量(1.000±0.120)显著低于胰腺癌细胞株BXPC-3(1.920±0.130)、SW1990(2.93±0.528)、PANC1(4.557±0.692)和CaPan-2(5.247±0.194)的circSEC31A表达量,差异有统计学意义(P<0.001)。Transwell实验结果显示,与PANC1 siR-NC组穿膜细胞数(1301.3±94.6)个/100倍视野和CaPan-2 siR-NC组穿膜细胞数(1835.0±70.1)个/100倍视野比较,PANC1 siR-circSEC31A#1组、siR-circSEC31A#2组和CaPan-2 siR-circSEC31A#1组、siR-circSEC31A#2组穿膜细胞数显著下降,分别为(727.3±92.9)、(792.0±18.1)、(718.0±90.6)、(692.7±84.8)个/100倍镜视野;划痕实验结果显示,与PANC1 siR-NC组(55.000±4.359)%和CaPan-2 siR-NC组(69.000±3.606)%比较,PANC1 siR-circSEC31A#1组(20.667±3.215)%、siR-circSEC31A#2组(20.000±4.583)%和CaPan-2 siR-circSEC31A#1组(28.000±8.185)%、siR-circSEC31A#2组(29.667±5.686)%的划痕区细胞覆盖率显著降低;RNA Pull-down实验表明,与PANC1 oligo探针组(1.000±0.091Objective To determine the expression of circular RNA-SEC31A(circSEC31A)in pancreatic cancer and investigate the effects on the invasion and migration of pancreatic cancer cells and the underlying molecular mechanism.Methods Differentially expressed circRNAs between pancreatic cancer cells(BXPC-3,PANC1,CaPan-2,SW1990)and human normal pancreatic cells(HPDE)were identified by qRT-PCR.Then,two cell lines with high circSEC31A expression were selected to conduct next experiments.According to the sequence of the back-splicing site in circSEC31A,siRNAs for downregulation of circSEC31A were designed and transfected by liposome to silence circSEC31A in pancreatic cancer cells,and grouped as followed siR-circSEC31A#1 and siR-circSEC31A#2.Meanwhile,siR-NC group transfected with non-specific siRNA served as control.Transwell assays and wound healing assays were operated to evaluate the functional role of circSEC31A on the invasion and migration of pancreatic cancer cells.RNA Pull-down assay with circSEC31A probe and oligo control probe was used to screen the miRNA combining with circSEC31A and the effects of miRNA on cell invasion and migration of pancreatic cancer cells were validated.The effect of miR-200c-3p and circSEC31A silencing on the expression of PDK1 mRNA was identified by qRT-PCR.The protein expression of PDK1,downstream Akt and p-Akt after circSEC31A silencing was verified by Western blotting assays.Results The relative expression level of circSEC31A in HPDE(1.000±0.120)was obviously lower than that in BXPC-3(1.920±0.130),SW1990(2.93±0.528),PANC1(4.557±0.692)and CaPan-2(5.247±0.194),and all the differences were statistically significant(P<0.001).Compared with the PANC1 siR-NC group(1301.3±94.6)and CaPan-2 siR-NC group(1835.0±70.1)per 100 high power field,transwell assays showed that the numbers of invasive pancreatic cancer cells was highly decreased in PANC1 siR-circSEC31A#1 group(727.3±92.9),siR-circSEC31A#2 group(792.0±18.1),CaPan-2 siR-circSEC31A#1 group(718.0±90.6),siR-circSEC31A#2 group(692.7±84

关 键 词：胰腺肿瘤 环状RNA circSEC31A miR-200c-3p PDK1 

分 类 号：R735.9[医药卫生—肿瘤]