Construction Of β-catenin-targeting RNAi Eukaryotic Vector And Investigation Of The Effect On The Proliferation Of Human Neuroblastom Cell In Vivo And Vitro

Backgrounds and Purpose:Neuroblastoma (NB), derived from peripheral sympathetic nervous system of the neural crest, is the most common malignant tumor in children and is confirmed as a kind of embryonal tumor. Wnt/β-catenin signaling pathway is one key regular way of embryonic development, involved in regulation of cell differentiation, cell proliferation, cell polarity and cell migration. In recent years, studies have found the extensive activation of β-catenin is common in many tumors. Thus, futher study the relationship between β-catenin and neuroblastoma and explain possible reasons for the occurrence from the perspective of abnormal embryonic development will help to find an useful target gene of this tumor. And these researches will provide a new idea of prevention and treatment of neuroblastoma. The aim of this study was to construct an expressive RNAi lentivirus specific to β-catenin in order to observe the post-transcriptional knockdown effect of β-catenin in human neuroblastoma cell line; and then we used this technical tool to additionally investigate the regulation of Wnt/β-catenin signaling on the proliferation aspect of cell line in vivo and in vitro. Preliminary study the role and possible mechanism of β-catenin in neuroblastoma development and progression provide a scientific theory of the targeted therapy of neuroblastoma.Materials and Methods:Choice and design of β-catenin gene specific shRNA expression vector of human neuroblastoma cell line:1. PCR and Western blot technology were used to test the expression of β-catenin in human neuroblastoma SH-SY5Y and BE2C cells on two levels of gene and protein.2. SH-SY5Y, BE2C cells were respectively performed plasmid liposome, lentiviral infection efficiency evaluation. Then we selected the appropriate shRNA expression vectors and the corresponding cell lines.3. We designed and synthesized four pairs of complementary double strand DNA oligos, which could express short hairpin RNA targeting the β-catenin mRNA, and linked them into the lentiviral plasmid vector pGCL-GFP. Experiment were divided into three groups, including negative control, nonsense-shRNA and β-catenin-shRNA group. And most effective interference sequences were screened in terms of RT-PCR and Western.Construction and dectionof a stable human neuroblastoma cell line with down-regulated expression of β-catenin:The filtered coding sequence of a lentiviral shRNA expression vector re-infected cells, and the stable cell clones were obtained by using the drug resistance carrying in the shRNA vector, selection and expansion of stable cell clones cultured strains Detection by RT-PCR β-catenin mRNA expression changes, westemblot β-catenin protein expression, Contrast with the negative control group and the blank group to verify its intracellular β-catenin gene inhibition effect, we observed the inhibition of β-catenin mRNA and protein level in terms of Real time PCR (RT-PCR) and Western blot.The regulation influence of silence expression of β-catenin in human neuroblastoma cell proliferation:1. CCk-8was detected toinvestigate the tumor cell proliferation after inhibition of β-catenin.2. The stable inhibition of β-catenin expression in tumor cells inoculated in nude mice, which establish an animal mode. We certificated the RNA interference inhibited the expression of β-catenin gene in vivo effect conpared to the negative control and blank control group.Statistical methodsSPSS18.0statistical software was used to analyze data difference between the groups by using ANOVA. when P<0.05, the difference was statistically significant.Results:Choice and design of β-catenin gene specific shRNA expression vector of human neuroblastoma cell line:1. PCR method and Western-blot analysis showed that the level of β-catenin in mRNA transcription and protein expression were well in human neuroblastoma cell lines BE2C.2. SH-SY5Y, BE2C cell transfection efficiency of plasmid liposome were very low. Compared with SH-SY5Y cells, BE2C cell had high lentiviral infection efficiency and stability of the follow-up culture.3. We successfully constructed RNAi lentiviral plasmid vectors targeting different sits of β-catenin mRNA. And an most effective interference sequence were filtered out by RT-PCR, Western-blot method.Construction and dectionof a stable human neuroblastoma cell line with down-regulated expression of β-catenin:1. A stable inhibition of β-catenin in human neuroblastoma cell line BE2C were successfully screened and established.2. We detected a highly efficient and stable inhibition to the expression of β-catenin gene through RT-PCR and Western blot after infecting the specific RNAi lentivirus to BE2C cell.The regulation influence of silence expression of P-catenin in human neuroblastoma cell proliferation:1. CCK-8showed stable inhibition of β-catenin gene expression, could suppress the human neuroblastoma cell proliferation of BE2C cell.2. Silencing of β-catenin gene by RNAi could inhibit the tumorigenesis of BE2C cell in nude mice.Conclusion:1. β-catenin expressed well in human neuroblastoma BE2C cells which suggests that β-catenin may be one of the mechanisms leading to neuroblastoma.2. Construction of a stable human neuroblastoma cell line with down-regulated expression of β-catenin can serve as Wnt/β-catenin signaling pathway important technical means.3. Blocking the expression of β-catenin gene can inhibit neuroblastoma cell line BE2C proliferation and tumor formation in nude mice, suggesting that β-catenin may an important regulatory factor which affect the proliferation of neuroblastoma.