Construction Of Notch1-targeting RNAi Lentivirus Vector And The Effect Of Notch1 Pathway On Proliferation In Neuroblastoma Cells

Objective:The aim of this project was to construct RNAi lentivirus expressive vector specific to Notch1 gene and to observe its gene knockdown effect on the expression of Notch1. Secondly, we also wanted to explore the role of Notch1 pathway on the regulation of proliferation in neuroblastoma cells using the RNAi lentivirus vector.Methods:The first step was to design and synthesize two pairs of complementary single-strand DNA oligos which targeting the various sites of Notch1 mRNA. Annealling was used to generate double-strand oligos(ds oligos), and then the ds oligos were cloned into pENTRTM/U6, the entry vector, to generate the Entry clone named pENTR. Recombination reaction in vitro with the pENTR and pLenti6/BLOCK-iTTM-DEST, the lentivirus backbone vector, was used to creat the lentivirus plasmid which contains the RNAi cassette. Then, we transfected the lentivirus plasmid into 293FT cells to product lentivirus stock, and titered the lentivirus stock. We transducted Neuroblastoma cells SH-SY5Y with lentivirus, the Notch1 gene kockdown effect was detected by RT-PCR and western blot analysis. In the last part, we transducted the RNAi lentivirus to inhibit the expression of Notch1 protein and to explore the role of Notch1 pathway on the regulation of proliferation in neuroblastoma cells SH-SY5Y with MTT assay.Results:1,Two RNAi lentivirus expression vectors targeting to two various sites of Notch1 gene were produced, and the sequence and correct site of ds oligos inserted were confirmed by PCR and sequencing assay;2,The lentivirus were packaged in 293FT cells with high titer. The results showed that the titer of lentivirus range from1.5×10~5TU to 2.3×10~5TU;3,The results of RT-PCR and Western blot showed the expression of Notch1 mRNA and protein in neuroblastoma cells SH-SY5Y could be inhibited efficiently by two various RNAi lentivirus;4,The results of MTT assay suggested that blocking the Notch1 pathway with the RNAi lentivirus could decrease the proliferation of neuroblastoma cells SH-SY5Y efficiently.Conclusion:The Notch1-targeting RNAi lentivirus is an important tool that can inhibit the expression of Notch1 gene efficiently, used in studying the function of Notch1 pathway. The Notch1 pathway plays an important role in the regulation of proliferation of neuroblastoma cells SH-SY5Y.