过表达PKM2增加卵巢癌细胞的增殖和迁移

Up-regulation of Pyruvate Kinase Type M2 Contributes to Elevated Ovarian Cancer Cell Proliferation and Migration

目的探究人M2型丙酮酸激酶（pyruvatekinasetypeM2，PKM2）对卵巢癌SKOV3细胞增殖和迁移的影响。方法将PKM2cDNA以XhoI和NotI位点克隆入pLVX—Neo—IRES．ZsGreenlvector慢病毒载体中，得到的重组载体及空载体分别与慢病毒包装质粒psPAX2和pMD2．G共转染入293T包装细胞．以得到PKM2过表达的慢病毒表达载体；将其转导入SKOV3细胞后用G418筛选稳定转染的细胞，分别测定PKM2过表达前后SKOV3细胞的增殖和迁移的改变。结果测序鉴定表明PKM2重组慢病毒表达载体正确无误。Western印迹检测结果显示转导PKM2慢病毒表达载体可增加SKOV3细胞中PKM2的表达。MTT测定和细胞划痕实验结果表明过表达PKM2可增加SKOV3的增殖和迁移。结论PKM2过表达可增加卵巢癌SKOV3细胞的增殖和迁移。

Objective To construct human pyruvate kinase type M2 （PKM2） gene recombi- nant lentivirus expression vector and to investigate its effect on the ovarian cancer SKOV3 cell prolif- eration and migration. Methods XhoI and NotI restriction enzyme sites were introduced into 5＇ and 3＇ end of PKM2 cDNA, respectively by PCR amplification. PKM2 eDNA was inserted into pLVX-Neo-IRES-Zs Greenl lentivirus vector via these two enzyme sites. The recombinant ptasmid and empty plasmid were cotransfected with psPAX2 and pMD2. G packaging plasimds into 293T cells to form PKM2 over-expressing lentivirus vector, which were then used to transduce SKOV3 cells. C,418 was used to select the cell clones with stable PKM2 over-expression or empty vector, re- spectively. MT-F and cell wound healing assays were used to detect SKOV3 cell proliferation and mi- gration. Results Sequencing demonstrated that PKM2 recombinant lentivirus expressing vector was successfully constructed and Western blotting exhibited that transduced PKM2 over-expressing lentivirus vector was able to increase the expression of PKM2 in SKOV3 cells. MTT and cell wound healing assays showed that over-expressed PKM2 resulted in elevated SKOV3 cell proliferation and migration. Conclusion s The PKM2 gene recombinant lentivirus expressing vector was successfully constructed, and up-regulated PKM2 expression contributed to increased proliferation and migration in SKOV3 cells.