CAJ | 학술논문

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RAC13'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RAC13'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。
목적：구건은정과표체mir-101적SW620세포아주병감정mir-101적파기인。방법운용형광정량PCR기술검측결직장암세포주중mir-101적표체수평。이용GV209-mir101만병독감염SW620세포，건립은정과표체mir-101적세포주。확증포함mir-101결합위점적RAC13'UTR기인편단병장기아극륭지psiCHECK-2재체，대차중조재체진행정점돌변구건psiCHECK-2-Rac1-Mut；병용쌍형광소매보고실험검측RAC13'UTR상대형광소매활성。결과은정과표체mir-101적세포주중，mir-101적표체명현고우대조조。쌍형광소매보고실험증실mir-101 inhibitors가이상조3'UTR상대형광소매활성。은정과표체mir-101， RAC1표체하조；이간우mir-101지후RAC1표체상조。결론성공구건은정과표체mir-101적SW620세포아주。RAC1시mir-101적파기인。
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.