Yang Hui, Zhang Chao, Lu Yan-Xia, Wu Xiao-Jin, Yuan Li, Zhou Chang, Zhou Chun-Ping, Liu Guo-Bing, Li Xue-Nong
Department of Pathology, College of Basic Medical Sciences, Key Laboratory of Molecule Tumor Pathology of Guangdong Province, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2012 Mar;32(3):306-11.
To construct a lentiviral vector of miR-335 gene and verify the target gene of miR-335.
The precursor sequence of miR-335 gene was amplified from the genomic DNA by PCR and cloned into the lentiviral vector PLVTHM labeled with GFP. Real-time quantitative RT-PCR was used to detect miR-335 and RASA1 expression in different colorectal cancer cell lines. A recombinant vector psiCHECK-2-RASA1 containing RASA1 3'UTR was constructed followed by site-directed mutagenesis of RASA1 3'UTR to establish the vector psiCHECK-2-RASA1-Mut. Co-transfection of hsa-mir-335 or a NC with these recombined vectors in HEK293A and SW480 cells was performed, and dual-luciferase reporter assay was utilized to examine the changes in luciferase activities. The recombinant PLVTHM-miR335 plasmid was packaged into mature lentivirus by 293FT cells and used to infect SW620 cells. Flow cytometry was employed for sorting the GFP+ cells. The expression of miR-335 and RASA1 were determined by qRT-PCR, and Western blotting was used to detect the expression of RASA1 protein in SW620 cell lines.
The recombinant lentiviral vector PLVTHM-miR335, psiCHECK-2-RASA1 and the mutation expression vector psiCHECK-2-RASA1-Mut were successfully constructed. Dual-luciferase reporter assay showed that miR-335 decreased luciferase activity in cells co-transfected with psiCHECK-2-RASA1. The expression of miR-335 in SW620 cells infected with the lentivirus PLVTHM-miR335 was significantly increased, but the expression of RASA1 showed only slight changes. Overexpression of miR-335 suppressed the expression of RASA1 protein in SW620 cells.
We have successfully constructed the lentiviral vector containing mir-335 gene and a SW620 cell line with miR-335 overexpression. MiR-335 can suppress RASA1 gene expression by targeting the specific sequence of RASA1 3'UTR.
构建miR-335基因的慢病毒载体并验证miR-335的靶基因。
通过PCR从基因组DNA中扩增miR-335基因的前体序列，并克隆到标记有GFP的慢病毒载体PLVTHM中。采用实时定量RT-PCR检测不同结直肠癌细胞系中miR-335和RASA1的表达。构建含有RASA1 3'UTR的重组载体psiCHECK-2-RASA1，随后对RASA1 3'UTR进行定点诱变以建立载体psiCHECK-2-RASA1-Mut。在HEK293A和SW480细胞中，将hsa-mir-335或阴性对照与这些重组载体共转染，并利用双荧光素酶报告基因检测法检测荧光素酶活性变化。将重组PLVTHM-miR335质粒通过293FT细胞包装成成熟的慢病毒，用于感染SW620细胞。采用流式细胞术分选GFP+细胞。通过qRT-PCR测定miR-335和RASA1的表达，并用蛋白质免疫印迹法检测SW620细胞系中RASA1蛋白的表达。
成功构建了重组慢病毒载体PLVTHM-miR335、psiCHECK-2-RASA1和突变表达载体psiCHECK-2-RASA1-Mut。双荧光素酶报告基因检测法显示，miR-335降低了与psiCHECK-2-RASA1共转染细胞中的荧光素酶活性。感染慢病毒PLVTHM-miR335的SW6₂0细胞中miR-335的表达显著增加，但RASA1的表达仅略有变化。miR-335的过表达抑制了SW620细胞中RASA1蛋白的表达。
我们成功构建了含mir-335基因的慢病毒载体及miR-335过表达的SW620细胞系。MiR-335可通过靶向RASA1 3'UTR的特定序列抑制RASA1基因表达。
