Vector-mediated RNA Interference In Human Tumor Cells

Objective:RNAi is the mechanism of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNAs (dsRNA) homologous to the gene being suppressed. In order to offer a new approach of tumor gene therapy, we want to study the RNAi effect in tumor cells by constructing plasmid and lentiviral vectors which mediate RNAi. Methods:We construct the plasmid vectors pLasRiGFP1/2/3 driven by human H1 RNA promoter and pU6RiGFP1/2 driven by mouse U6 promoter using the methods of molecular cloning, which target different GFP gene sequences. We transfer the vectors and pcdna3.1EGFP into HEK293 and Hela cells by calcium phosphate mediated- cotransfection, and into SPCA1 and SPCA1-GFP cells by liposome, and then analysis GFP gene expression inhibition effect of both single vector and combination of different vectors using FACS and observe the effects using inverted microscope.After that, we construct lentiviral vectors plentiRiGFP1/2/3 driven by inserting the shRNA expression structures from pLasRiGFP1/2/3. We transfer plentiRiGFP1/2/3 and pcdna3.1EGFP into SPCA1 and Hela cells by calcium phosphate mediated- cotransfection, and then analysis GFP gene expression inhibition effect. Next, we choose plentiRiGFP1, and transfer it into HEK293T cells along with pCMVΔR8.2 and pCMVVSVG by calcium phosphate mediated- cotransfection. Meanwhile, we construct the lentiviral vector LentiGFP expressing GFP and the control lentiviral vector Lenti. Titration of lentiviral vectors is measured by real time RT-PCR directly. We optimize the conditions of transduction through transducing SPCA1 cells with LentiGFP. Subsequently, we use LentiRiGFP1 to transduce SPCA1-GFP cells and analyse effects of lentiviral vector induced RNAi. Results: 1. pLasRiGFP1/ 2/3 can knock down GFP gene expression, and the inhibition rate is 97.5%, 97%, 89% in HEK293 cell, 83%, 90%, 90% in Hela cells and 65%, 55%, 40% in SPCA1 cells respectively; the inhibition rate of pU6RiGFP1/2 is 47%, 65% in Hela cells and 60%, 37% in HEK293 cell.2. The GFP gene inhibition rate of pLasRiGFP1/2/3 was most efficient at 48h～96h,and became declining with time going on. The rate was increasing with the dose of plasmid, but was not increasing when the dose reached the highest level.3.The inhibition rate of different combination of the plasmid vectors pLasRiGFP1+2/1+3/2+3/1+2+3 is 97.5%, 96.5%, 98.5%, 99% in HEK293 cell and 93%, 82%, 80%, 88% in Hela cells respectively.4. The inhibition rate of pLasRiGFP1/2 is 53%, 58% in SPCA1-GFP cells. The rate was increasing with the dose of plasmid, but was not increasing when the dose reached the highest level.5. plentiRiGFP1/2/3 can knock down GFP gene expression, and the inhibition rate is 60%, 60%, 20% in Hela cells and 58%, 65%, 70% in SPCA1 cells respectively.6. The inhibition rate of LentiRiGFP1 is increasing with the increasing of MOI of LentiRiGFP1, but the rate is stabilized when MOI is 5. The RNAi effect can last 15 days.Conclusions:Plasmid vectors expressing shRNA driven by H1 RNA promoter can induce gene-specific knockdown for high efficiency and have dose- and time- dependent effects; the inhibition rates exsit differences between the combination of the plasmid vectors and the single plasmid vector, but they have no great significance; the vectors have different effects in various mammalian cells, and different vectors targeting different sites of gene have different effect in the same cell line. Plasmid vectors expressing shRNA driven by U6 promoter cannot induce the gene-specific knockdown for high efficiency. Lentiviral vectors expressing shRNA can be used successfully to transduce tumor cells and the specific gene can be down-regulated for a long-lasting time.