Has-mir-335慢病毒表达载体的构建及其靶基因鉴定

Construction of has-miR-335 lentiviral vector and verification of the target gene of miR-335

目的构建过表达miR-335的稳定细胞株SW620并筛选和初步鉴定miR-335的靶基因。方法扩增包含hsa-miR-335前体序列在内的基因片段,并将其亚克隆至PLVTHM慢病毒载体;扩增包含RASA1 3'UTR种子区域的基因片段并将其亚克隆至psiCHECK-2载体,并对此重组载体进行定点突变构建psiCHECK-2-RASA1-Mut;检测萤火虫荧光素值(F)和海肾荧光素值(R),以R/F表示相对荧光素酶活性。流式细胞仪筛选建立稳定表达miR-335的细胞株,利用荧光定量PCR检测miR-335及其靶基因RASA1的表达,Western Blot检测RASA1蛋白水平的表达。结果质粒双酶切以及测序鉴定PLVTHM-miR335、psiCHECK-2-RASA1、psiCHECK-2-RASA1-Mut重组质粒构建成功。荧光定量PCR显示结直肠癌细胞中miR-335与RASA1表达趋势成负相关。双荧光素酶报告基因分析证实hsa-miR-335能够作用于RASA1的3'UTR。稳定过表达miR-335的细胞中,miR-335的表达明显高于对照组和未处理组,蛋白质水平有所下降。结论成功构建了PLVTHM-miR-335慢病毒重组质粒,构建过表达miR-335的细胞株SW620,初步证实RASA1是miR-335的靶基因。

Objective To construct a lentiviral vector of miR-335 gene and verify the target gene of miR-335.Methods 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.Results 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.Conclusion 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.