摘要
目的构建稳定过表达mir-101的SW620细胞亚株并鉴定mir-101的靶基因。方法运用荧光定量PCR技术检测结直肠癌细胞株中mir-101的表达水平。利用GV209-mir101慢病毒感染SW620细胞,建立稳定过表达mir-101的细胞株。扩增包含mir-101结合位点的RAC1 3'UTR基因片段并将其亚克隆至psiCHECK-2载体,对此重组载体进行定点突变构建psiCHECK-2-Rac1-Mut;并用双萤光素酶报告实验检测RAC1 3'UTR相对荧光素酶活性。结果稳定过表达mir-101的细胞株中,mir-101的表达明显高于对照组。双萤光素酶报告实验证实mir-101 inhibitors可以上调3'UTR相对荧光素酶活性。稳定过表达mir-101,RAC1表达下调;而干扰mir-101之后RAC1表达上调。结论成功构建稳定过表达mir-101的SW620细胞亚株。RAC1是mir-101的靶基因。
Objective To construct a colorectal cancer cell line stably expressing mir-101 and identify the target gene of mir-101. Methods Quantitative real-time PCR was used to detect mir-101 expression in colorectal cancer cell lines. The recombined lentiviral vector GV209-mir101 or the empty lentiviral vector GV209 was transfected into human colorectal cancer cells SW620. The recombinant psiCHECK-2-Rac1 vector containing RAC1 3＇UTR was constructed, and site-directed mutagenesis of RAC1 3＇UTR was induced to construct the psiCHECK-2-Rac1-Mut vector. In HEK293A and SW480 cells co-transfected with mir-101 inhibitors or negative control （NC） and these recombined vectors, luciferase activities was examined with a dual-luciferase reporter assay. Results SW620 cells transfected with GV209-mir101 lentivirus exhibited higher mir-101 expression level than cells transfected with GV209 lentivirus. Mir-101 inhibitors significantly increased the luciferase activities of RAC1 3＇UTR. Overexpression of mir-101 increased the expression of RAC1 while inhibition of mir-101 suppressed RAC1 expression. Conclusion We have successfully constructed a SW620 cell line stably overexpressing mir-101. mir-101 can suppress RAC1 gene expression by targeting the specific sequence of RAC1 3＇UTR.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2014年第7期928-933,共6页
Journal of Southern Medical University
基金
国家自然科学基金(81272758
81302158)~~
