miR-125b慢病毒表达载体的构建及其对卵巢癌SKOV3细胞增殖和迁移的影响

Construction of Lentiviral Vector of miR-125b and the Effect of miR-125b on Cell Proliferation and Migration of Ovarian Cancer SKOV3 Cells

构建并鉴定miR-125b慢病毒过表达载体,研究miR-125b对卵巢癌细胞增殖和迁移的影响及其可能机制。将PCR扩增的miR-125b前体序列与经过酶切后的GP-Supersilencing Vector进行连接,产生miR-125b重组慢病毒表达载体。将重组慢病毒载体质粒、pGag/Pol、pRev和pVSV-G共转染293T细胞,包装产生慢病毒。使用收获的病毒颗粒感染卵巢癌SKOV3细胞,嘌呤霉素筛选稳定感染细胞株;实时荧光定量PCR(Real-time qPCR)检测miR-125b在SKVO3细胞中的表达;Western blot检测其潜在靶基因HER-2的表达;MTT实验和Transwell侵袭实验分别观察miR-125b过表达后SKOV3细胞增殖和迁移能力的改变。该研究成功构建miR-125b慢病毒过表达载体,感染卵巢癌SKOV3细胞后,能够过表达miR-125b,并抑制SKOV3细胞的增殖及迁移,降低潜在靶基因HER-2的表达。该研究证明,miR-125b能够抑制SKOV3细胞的增殖及迁移,并可能通过降低潜在靶基因HER-2的表达而实现。

We constructd the lentiviral vector expressing miR-125b and observed the effect ofmiR-125b on ovarian cancer cell proliferation and migration. The PCR amplification of miR-125b precursor sequence was linked with linearized GP-Supersilencing Vector to generate the recombinant lentiviral vector expressing miR-125b. The 293T cells were trans- fected with the plasmids of recombinant lentiviral vector, pGag/Pol, pRev and pVSV-G plasmids to package the lentivirus. SKOV3 cells were infected by obtained lentivirus and screened with puromycin. The expression of miR-125b in SKOV3 cells was detected by Real-time qPCR and the expression of potential target gene HER-2 was tested by Western blot. Cell proliferation and cell migration were assessed using the MTT assay and transwell migration assay. The results showed that the lentiviral vector expressing miR-125b was successfully constructed and achieved the over-expression of miR-125b after infected ovarian cancer SKOV3 cells. The over-expression of miR-125b inhibited cell proliferation and migration of SKOV3 cells and decreased the expression of HER-2 gene. We concluded that miR-125b could inhibit the proliferation and migration of SKOV3 cells through down-regulation of HER-2 expression.