Lv-shRNA-Hsa-microRNA-691慢病毒表达载体的构建与鉴定

Inhibition of miR-691 through the regulation of VIM on effects of invasion and metastasis of human pancreatic cancer cell line BXPC-3

目的构建Lv-shRNA-hsa-microRNA-691慢病毒表达载体。方法 PCR扩增pri-miR-691-2前体序列,克隆至plenti-GFP慢病毒表达载体;双酶切及测序鉴定正确后进行慢病毒包装与滴度检测。构建成功后感染人胰腺癌细胞Panc-1,48h后Real-time Q-PCR检测miR-691的表达。结果酶切、测序鉴定证明插入序列正确,测定病毒滴度为1×109TU/m L,病毒感染48h后的Panc-1胰腺癌细胞在倒置荧光显微镜下观察可见绿色荧光,Real-time Q-PCR显示被感染细胞的miR-691表达量较未感染细胞显著增高。结论建立了高效稳定表达Lv-shRNA-hsa-miR-691的慢病毒转染系统。

Objective To study the small RNA-691 （ miR-691 ） expression in human pancreatic cancer cell lines and inhibits the miR-691 effects on the biological behavior of human pancreatic cancer cell line BXPC -3. Methods miR-691 used real-time fluo- rescent quantitative PCR for detection. Artificial chemical synthesis inhibition of transfected by miR-691 and BXPC-3 ceils. Detection of cell apoptosis by flow cytometry. By Transwel - x room experiment of cell invasion and metastasis detection BXPC-3 cell invasion and metastasis. Structure. Results BXPC-3 for miR-691 in human pancreatic cancer cell line expression; transfection inhibits miR-691, BXPC-3 decline in the miR-691 expression, no significant change in apoptosis （P 〉 0. 05）, transfection inhibits the ceils of miR-691 group by Transwel small room above the blank in the number groups and the control group （P 〈 0. 01 ）. Conclusion miR-691 can be expected to be a candidate target for gene therapy of pancreatic cancer.