The Effect of TRIM25over Expression on Drug Sensitivity to Cisplatin of Human Lung Cancer Cells H1299
|School||Chongqing Medical University|
|Keywords||overexpression plasmid vector TRIM25 PKM2 drugsensitivity|
Objective: To investigate the effects of TRIM25overexpression on thechemotherapeutic drug sensitivity to cisplatin of human lung cancer cell lineH1299preliminarily, and to provide an experimental basis for furtherresearch on tumor drug sensitivity.Methods: The TRIM25overexpression plasmid vector wasconstructed by gene cloning techniqucs and used to transform competenceE.coli DH5α，then positive clones were confirmed and verified bynucleotide sequencing and enzyme digestion. We named the plasmidspGCMV-trim25. After pruifing and extracting plasmids, liposome mediatedpGCMV-trim25vector were transfected into H1299cells as experimentalgroup. Empty vector pGCMV/neo was transfected as control group, theuntransfected cells as blank group. The efficiency of transfecion wasobserved by fluorescence microscope. Western Blot were used to determinethe TRIM25protein expression and the changing of PKM2protein levelwhen TRIM25was overexpressed. After treating Untransfected andtransfected H1299cells with cisplatin for24h, inhibition of proliferation ability was detected by MTT assay and apoptosis was detected by AnnexinV/PI.Results: By using transient transfection in this assay, transfection ratecan be more than80%. Compared with control group and blank group, theTRIM25protein level in transfected H1299cells was significantly increased(P<0.05). Moreover, when up-regulating the TRIM25, PKM2expressionwas reduced (P<0.05).After treating with cisplatin, in transfected group thegrowth inhibition rates as well as the apoptosis rates were significantlydecreased (P<0.05). Drug resistance index (RI) to DDP of experimentalgroup was8.889, drug sensitivity to cisplatin decreased.Conclusions:1The eukaryotic plasmid vector pGCMV-trim25was successfullyconstructed, and the TRIM25protein level was specifically up-regulatedwhen transfected with pGCMV-trim25vector.2TRIM25and PKM2expression were negatively correlated.3Overexpression of TRIM25could decrease the drug sensitivity tocisplatin and inhibit the apoptosis of lung cancer cells H1299.