The Effects Of Stable Silence Of CARMA1 By Lentivirus On K562 Cell's Invasion And Metastasis

Objective: Chronic myelognous leukemia(CML) was a malignant disease of proliferation which originated from the bone marrow multipotent hematopoietic stem cell.More than 90% cases had marker chromosome of CML (Ph chromosome),which was based on the rearrangement of fusion protein BCR-ABL. BCR-ABL could couse activation of NF-κB to lead to the characteriseics changes of growth,metastasis and durg resistant. CARMA1 was highly expressed in CML and likely to play a significant role in activing NF-κB. EGR-1 was a cancer suppressor gene and had NF-κB binding site in its promoter sequence which could co-regulate tumor invasion and metastasis.In this study, we approached whether inhibition of CARMA1 by lentivirus could impact K562 cells's invasion and metastasis and related signaling pathway ,discussing the likely mechanism.Methods: Use three plasmids of lentivirus system,package cell 293T and CaPO4 transfection to produce shCARMA1 lentivirus particles.K562 cells were infected by lentivirus and selected by puromycin to pick put clones stably expressing shCARMA1. CARMA1 mRNA level was detected by PT-PCR and Western Blot was used to measure the contents of CARMA1 protein. Analyse the influence of inhibition of CARMA1 on K562 cells's invasion and metastasis and related signaling pathway by PT-PCR,Western Blot,Trypan blue exclusion assay,Colony forming assay,Migration and invasion experiment in vitro(Transwell chamber culture system and Matrigel).Results: K562 cells were infected by lentivirus with shRNA and selected by puromycin to establish six cells models:one was negative control (K562/sh-eGFP),others were experimental groups(K562/shCARMA 1-90,K562/shCARMA 1-91,K562/shCARMA 1-92,K562/shCARMA 1-93,K562/shCARMA 1-94).Compared with K562 and K562/sh-eGFP, the expression of CARMA1 in K562/shCARMA 1-93 was inhibited greatest obviously.Using K562/shCARMA 1-93 as cell model to contiute future studies:(1) Trypan blue exclusion assay showed that CARMA1 RNAi significantly inhibited K562 cells proliferation(P<0.01).After one week cluture,compared with blank control K562 and negative control K562/sh-eGFP,the growth inhibition rates were respectively 29.3% and 28.6%; The abilities of K562/shCARMA 1-93's colony forming also decreased(P<0.01). Compared with K562 and K562/sh-eGFP,the colony forming inhibition rates were respectively 37.6% and 34.1%; Migration and invasion experiment in vitro confirmed that the cells of K562 and K562/sh-eGFP which passed through the membranes showed no difference(P>0.05),but that of K562/shCARMA1-93 was statistical significance observed in contrast with control(P<0.01).It showed that CARMA1 RNAi could inhibited the migratory and invasive abilities of K562/shCARMA 1-93.(2)The resules of WB and RT-PCR. turned out that the inhibitory of CARMA1 reduced the expression of BCL10,NF-κB and EGR1.Conclusions: Lentivirus mediated RNAi could highly silence the expression of CARMA1 of K562 cells.Cell model(K562/shCARMA1-93) was successfully established. The inhibition of CARMA1 could impact on cell proliferation,colony forming,migratory and invasive abilities and reduce the expression of BCL10,NF-κB,EGR1.