Effects Of Bcl10Gene Knockdown On The HCT116Cell’s Biological Characteristics

Objective:The occurrence and development of cancer involves a series of complexmolecular progress. In the process of carcinogenesis, there is a variety of oncogeneactivation or mutation and tumor suppressor gene inactivation.Bcl10gene, cloned from aMALT lymphoma t (1;14)(p22; q32) chromosomal translocation breakpoints, is a genewhich regulate cells apoptosis, and the protein encoded by Bcl10gene widelyparticipates in the development and progression of many diseases. Bcl10gene mutationor overexpression is closely related to the development of lymphoma[1,2]. In the study of avariety of solid tumors such as ovarian cancer, head and neck squamous cell carcinoma,hepatocellular carcinoma, cervical cancer, breast cancer, renal cell carcinoma, showedthat Bcl10widely participate in the invasion and infiltration of cancer cells and promotecancer cells growth[3-8]. Studies had shown that Bcl10expression in colorectal cancertissues was significantly higher than normal tissue[9], so did Bcl10high expression alsoinvolve in the biological behavior of colorectal cancer? we want to research and explorethe possible molecular mechanism of this issue through the Bcl10gene knockdownmediated by lentivirus.Methods: The caPO4coprecipitation method was used to package lentivirus whichcontains the Bcl10gene shRNA, then used it to infect HCT116cells and to build a stableBcl10gene silencing cell line after puromycin selective. Western Blot and EMSA wereused to detect the expression of Bcl10and NF-κB in colorectal cancer. Trypan blueexclusion assay and MTT were used to detect cells growth and colony formation assaywas used to detect cells clone formation. Analyze the influence of inhibition of Bcl10onHCT116cells’s invasion and metastasis and related signaling pathway by Migration andinvasion experiment in vitro(Transwell chamber culture system) and Wound scratch assay,Flow Cytometry assay was used to analyze cells cycle and Western Blot and EMSA wereused to detect molecular changes in related signaling pathways and explore its possiblemolecular mechanism. Results: HCT116cells were infected by lentivirus with shRNA and selected bypuromycin to establish negative control group HCT116/sh-eGFP and the experimentalgroup HCT116/shBcl10. Bcl10, detected by RT-PCR, Western Blot in gene and proteinlevels, was effectively suppressed. Using HCT116/shBcl10as cell model to continuefurther studies and get the following results:(1) Trypan blue exclusion and MTT assayshowed that Bcl10RNAi significantly inhibited HCT116cells proliferation(P<0.05).Theabilities of HCT116/shBcl10’s colony forming also decreased(P<0.05). Migration andinvasion experiment in vitro confirmed that the cells of HCT116and HCT116/sh-eGFPwhich passed through the membranes showed no difference(P>0.05),but that ofHCT116/shBcl10was statistical significance observed in contrast with control(P<0.05).Itshowed that Bcl10RNAi could inhibited the migratory and invasive abilities ofHCT116/shBcl10.(2)The results of WB and EMSA turned out that the inhibitory of Bcl10reduced the expression of Bcl10、MMP-7、MMP-9、Pin1、Cdc2P34、Cdc25c、cyclinB1and up-regulation the expression of p-Cdc2P34(Thr14/Tyr15), but no effect onNF-κB and JNK.Conclusions: Lentivirus mediated RNAi could highly silence the expression of Bcl10ofHCT116cells.Cell model(HCT116/shBcl10) was successfully established. The inhibitionof Bcl10could impact on cell proliferation、colony forming、migratory abilities and arrestcell cycle in G2/M, and reduce the expression of Bcl10、MMP-7、MMP-9、Pin1、Cdc2P34、Cdc25c、cyclinB1and up-regulation the expression of p-Cdc2P34(Thr14/Tyr15), butno effect on NF-κB and JNK.