Hsa miR-7慢病毒表达载体的构建及其在人卵巢癌细胞HO8910-PM中表达的有效性

目的：构建Hsa miR-7的慢病毒表达载体，感染人卵巢癌细胞HO8910-PM后检测微小RNA 7（miR-7）及其靶基因血管内皮生长因子受体（EGFR）的表达，为研究miR-7在HO8910-PM中的生物学功能奠定基础。方法：将聚合酶链反应（PCR）扩增得到的miR-7前体序列pre-miR-7和慢病毒载体pLVX-IRES-ZsGreen1经酶切后连接产生pLVX-miR-7-IRES-ZsGreen1，经酶切及测序鉴定正确后在HEK293T细胞中包装病毒，病毒浓缩后感染HO8910-PM用实时荧光定量PCR（qPCR）方法检测感染后的HO8910-PM中pre-miR-7和miR-7的表达，qPCR及蛋白质印迹（Western blot）方法检测其靶基因EGFR的表达。结果：酶切及测序结果示慢病毒表达载体pLVX-miR-7-IRES-ZsGreen1构建正确，病毒浓缩液感染HO8910-PM后能有效提高pre-miR-7的表达（t=17.909，P=0.004）和miR-7的表达（t=35.320，P=0.024），并能有效降低其靶基因EGFR的mRNA的表达（t=8.83，P=0.005）及蛋白的表达（t=22.14，P=0.002）。结论：成功构建了pLVX-miR-7-IRES-ZsGreen1慢病毒表达载体及稳定表达miR-7的HO8910-PM细胞亚系。

Construction of the Hsa miR-7 Lentiviral Expression Vector and its Expression in Human Ovarian Cancer Cell Line HO8910-PM

Objective： To construct the Hsa miR-7 lentiviral vector, and to detect its effectiveness in human ovarian cancer cell line HO8910-PM. It is the bases of functional study of Hsa miR-7 in ovarian cancer. Methods：Hsa miR-7 was amplified from the genomic DNA and inserted into pLVX-IRES-ZsGreen1 vector after double digestion to generate pLVX-miR7-IRES-ZsGreen1 vector. The vector was then confirmed by PCR and DNA sequencing. HO8910-PM was infected by the concentrated lentivirus 293T produced. Pre-miR-7，miR-7 and gene EGFR were assessed by qPCR. Results：The recombinant lentiviral vector was successfully established and confirmed by PCR and DNA sequencing. Pre-miR-7 （t=17.909，P=0.004） was integrated into the genome of HO8910-PM and miR-7 （t=35.320，P=0.024） expression was enhanced effectively by qPCR. Moreover，miR-7 target gene EGFR was also reduced both in mRNA （t=8.83，P=0.005） and protein （t=22.14，P=0.002） levels after miR-7 overexpression in HO8910-PM. Conclusions：The Hsa miR-7 lentiviral expression vector was successfully constructed，and the Hsa miR-7 overexpressed in HO8910-PM cell line.