Study Of A Lentiviral-based System With RNA Interference To Functionally Silence IKKα In MCF-7 Cells And To Generate A Transgenic Mouse Model

NF-κ B has been shown to be involved in the development of carcinomas of epithelial origin, such as breast cancer. Some studies have documented that NF-κB activity, both in mammary carcinoma cell lines and primary breast cancer cells of human and rodent origin, is elevated or constitutively active. Almost all chemically induced rat mammary carcinomas have high levels of NF-κB activity. In human breast cancer cells, initial studies demonstrated elevated NF-κB activity in correlation with oestrogen independence. However, subsequent studies have shown that NF-κB is activated regardless of hormone-dependency status. This indicates that constitutive NF-κB activation may be one of the early events in breast cancer pathogenesis.NF-κB stimulates cell proliferation by increasing transcription of cyclin D1, in mammary epithelial cells during pregnancy. As a positive regulator of cell cycle progression ,NF-κB activates target genes, such as cyclin D1, that stimulate proliferation of lymphoid cells in an autocrine manner. It has been shown that cyclin D1 expression requires a functioning NF-κB activation pathway during mammary gland development.Cyclin D1 is probably one of the most important NF-κB target genes for normal mammary gland development and mammary carcigenesis.Other studies have established the role of the IKKα-dependent RANK-NF-κB pathway in controlling the proliferation of the mammary epithelium during pregnancy. These findings raise another possibility that deregulation of RANKL, or constitutive RANK, or IKKα activation, might be the underlying cause of elevated in NF-κB activity in breast cancer.RNA interference technology provides us with a new tool for modulating gene expression and thus, is a good method for developing gene therapy strategies. RNA interference is a natural mechanism whereby metazoan cells suppress the expression of genes when they come across double-stranded RNA molecules of the same sequence.Transfection of siRNA-like transcripts known as short hairpin interfering RNAs (shRNAs) is transient, lasting only for a week or so. DNA-based vectors can also allow for long duration of transfection, but only with drug selection. These DNA-based vectors carry target gene knockdown fragments that can provide stable gene silencing. In mammalian cells, RNA interfence can be triggered by viral expression vectors that direct the synthesis of shRNAs. Stable expression of these shRNAs mediates persistent knockdown of the target gene. Therefore, a lentiviral-delivering RNA interference system is the best tool to target gene knockdown in cancer cells for longer lengths of time.The purpose of this research project is to establish a lentiviral-based system with RNA interference to functionally silence IKKa in MCF-7 cells and to use this system to generate a transgenic mouse model. This study will provide a new strategy to cure carcinomas of epithelial origin that overexpress of NF-kB. To date, no literature has been published concerning a lentiviral vector system delivery of shRNA into MCF-7 cells or/and transfer IKKa-knockdown lentivirus into mouse embryos.This study aims to: (1) Construct a pU6-IKKa-knockdown expression vector;(2) Study the effects of pU6-IKKa-knockdown expression vector on IKKa expression and NF-kB activity by transient transfection into MCF-7 cells in vitro;(3) Construct IKKa-knockdown Lentivirus and observe its effects on IKKa expression and NF-kB activity by stable transfection into MCF-7 cells in vitro and observe its effects on tumorigenesis, in vivo, by inoculating lentivirus transducing MCF-7 cells into nude mice;(4) Generate transgenic mouse by infecting fertilized mouse embryos with IKKa-knockdown lentivirus and observe the phenotype of IKKa gene knockdown in this mouse model.Methods1. Using DNA of pENT/L6, named as an entry vector, double-stranded oligos encodinga shRNA targeting IKKa mRNA, was inserted in order to generate the pENT/U6-IKKa vector. Then, the LR recombination reaction was performed between pENT/U6-IKKa with pLenti6/BLOCK-iT-DEST to produce pU6-IKKa knockdown expression vector. 2. IKKa mRNA knockdown was determined by semiquantitative RT-PCR, real time PCR, and Western blotting for IKKa protein at 24hours, 48hours and 72hours after transienttransfection of the pU6-IKKa knockdown expression vector into MCF-7 cells. Also, the effects of IKKa mRNA knockdown on NF-kB activity were observed by Western blotting for nuclear p65 protein and by NF-kB activity depedent Dual-Luciferase Reporter Assay System from lysates of MCF-7 cells transiently transfected with pU6-IKKa knockdown expression vector.3. Generation of lentiviral stock delivering IKKa RNA interfere was created by co-transfecting the optimized ViraPower Packaging Mix with pU6-IKKa-knockdown expression vector into the 293FT Producer Cell Line. This IKKa-knockdown lentiviral stock was further concentrated by ultra-centrifugation and titration.4. A MCF-7 cell line with stable expression of IKKa-knockdown shRNA was created (named IKKa-knockdown-MCF-7 cells) by Blasticidin resistant selection after IKKa knockdown by lentivirus infection. The effects of gene silencing in IKKa-knockdown-MCF-7 cells was studied by Western blot for IKKa protein or nucler p65, immunofluorescence staining and real time PCR and also by inoculating IKKa-knockdown-MCF-7 in nude mice.5. Instead of the traditional microinjection into a one cell embryo, a transgenic mouse model expressing IKKa-knockdown shRNA was generated by injection of IKKa-knockdown lentivirus into fertilized mouse embryos below the zona pellucida. The zona pellucida is a protective outer layer of the embr>o that is a physical barrier to lentiviral infection. The IKKa-knockdown viral particles were injected into the subzonal space between the cytoplasrnic membrane and the zona pellucida by using three different methods: (1) subzonal infection of IKKa-knockdown lentivirus for fertilized embryos;(2) subzonal infection of IKKa-knockdown lentivirus by PIEZO instrument;(3) subzonal infection of IKKa-knockdown lentivirus and subsequent incubation of infected embryos with lentiviral stock overnight. The genotype for transgenic mice was determined by PCR and assessment of IKKa gene knockdown was determined by real time PCR.Results and discussion1. The structure of pENT/U6 IKKa vector and pU6-IKKa-knockdown expression vector were confirmed by digestion with restriction endonucleases followed by DNA sequence analysis.2. IKKa m RNA and protein levels were significantly reduced by transient transfection with pU6-IKKa-knockdown expression vector in MCF-7 cells using RT-PCR, real time PCR and Western blot analysis. Furthermore, p65 protein, a subunit of NF-kB, was significantly down-regulated by transient transfection as IKKa. The Dual-Luciferase Reporter Assay System analysis for NF-kB activity demonstrated a reduction in NF-kB activity by approximately 34.9% and 37.0% as compared to controls at 48h and 72h, respectively, following transient transfection of pU6-IKKa-knockdown expression vector into MCF-7 cells.3. The IKKa-knockdown lentiviral titre was further raised by ultra-concentrifugation and the final titre was 2~4*107 TU/ml. Using Blascitidin selection, a IKKa-knockdown-MCF-7 cell line was generated.4. IKKa mRNA and protein levels in the cytoplasm were significantly reduced as demonstrated by RT-PCR, Western blotting analysis and immunofluorescence staining in IKKa-knockdown-MCF-7 cells. Furthermore, U6-IKKa-knockdown- MCF-7 cells inoculated into the flanks of nude mice showed significant reduction in tumor size as compared to control counterparts.5. Infection of IKKa-knockdown lentivirus into fertilized mouse embryos generated IKKa-knockdown transgenic mice. The percentage of IKKa knockdown lentivirus positive transgenic mice were 15% and 36.5% in the Founder and Fl mouse, respectively. Using real time PCR, the level of IKKa mRNA of blood samples collected from transgenic mice was markedly lower than samples from wild-type mice.Conclusions1. The pENT/U6 vector and pU6-IKKa-knockdown-vector were successfully constructed and the structure for both vectors were confirmed by digestion with restriction endonucleases, followed by DNA sequence analysis.2. It is indicated that expression of IKKa knockdown shRNA, designed from double stranded oligos in the pU6-knockdown expression vector, is able to knockdown IKKa at the mRNA and protein level. Additionally, we have shown that this IKKa shRNA can cause a reduction in nuclear p65 protein level and reduce promoter activity of NF-kB in both transient transfection and stable transfection of MCF-7 cells.3.The nude mouse model inoculated with IKKa-knockdown-MCF-7 cells reveals IKKa mRNA knockdown reduces the ability of turmorigenesis for MCF-7 cells. 4. IKKa knockdown shRNA was successfully produced in these IKKa knockdown transgenic mice. The lentivirus infection in fertilized mouse embryos proves to be a high efficacy and high efficiency method to generate transgenic mouse.The above results demonstrate RNA interference technology to be a beneficial application for gene therapy in breast cancer research. Furthermore, this model correlates a relationshiop between IKKa-NF-KB signal transduction and cancer. The IKKa-knockdown transgenic mouse model also proves to be a useful tool to study physiological function of IKKa gene knockdown.