Utilize RNAi Technology Interrupting The Immunosuppression Effects Of TCDD Mediated By AhR

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a member of polychlorinated biphenyls (PCBs) family, which contains many potent environmental pollutants. TCDD was found exihibiting the highest toxicity among PCBs family members. It could produce a variety of adverse effects in laboratory animals including hepatotoxicity, immune suppression, thymic atrophy, reproductive toxicity, wasting syndrome, endocrine disruption, developmental toxicity, and carcinogenicity. Researchers also discovered multi-system dysfunction occurrences in people on TCDD exposure. The health-disrupted potential of TCDD impel more researchers to study its bio-effects and the underlying mechanisms. Immune suppression is one of the most sensitive and severe consequences of TCDD exposure. Humoral and cell-mediated immune responses were found greatly suppressed in exposed animals. Tremendous research works had gradully discovered some mechanisms to explain the way that TCDD exerts its toxic effects. Several of them, including toxicity mediation pathway by aryl hydrocarbon receptor (AhR), TCDD interference on kinase signaling pathway, TCDD interference on metabolism of Ca2+ and radical, TCDD interaction with estrogen hormone receptor, were considered play key roles in the way, especially the first one, AhR mediation pathway. The aryl hydrocarbon receptor (AhR) was identyfied to be an important receptor of TCDD and mediated most of its toxicities, including the immonotoxocity. The signal transduction pathway is considered started from TCDD binding. The binding will stimulate AhR and make AhR-TCDD complex translocate into nuclear, where they bind to AhR nuclear translocator (ARNT) and form a heterodimer. The complex then interacts with specific element in DNA, such as DRE, which locates in the promoters or enhancers of TCDD-responsive genes. Finally the gene transcriptional status is altered and leads to impairments. Previeous data of our lab showed that: (1)TCDD significantly suppressed B lymphocyte function; (2)B lymphocyte expressed AhR protein; (3)The TCDD suppression effects on B lymphocyte was AhR-dependent; (4)Indirect evidence implicated the existence of AhR-independent pathway. The last point was also supported by other labs publications. Based on the materials shown above, we hypothesized that depletion of AhR in TCDD signaling pathway could elicit two consequences. First, provide a target for TCDD toxic syndrome therapy and its theory background. Second, further understand the role of AhR in TCDD toxicitiy mediation pathway. In the last few years, the research foci RNA interference (RNAi) was considered an important mechanism in post-transcriptional gene silencing (PTGS). It has been discovered that the RNAi is triggered by small interference RNA (siRNA), which came from short double strand RNA processed by Dicer. SiRNA can bind to other partners in cytoplasma and form a complex named RNA induced silencing complex (RISC). Then RISC recognizes and cleaves cognate mRNA of siRNA, finally leads to the silencing fo target gene. Along with the application of siRNA specific promoters, such as hH1, hU6 and mU6, RNAi has been rendered the ability to silence gene for a long period. It makes RNAi becoming a more and more powerful biological tool in specific gene silencing, as well as a potential gene therapy means. Researchers had demonstrated the host persistence of TCDD and its chonic toxic effects. Baccarelli and his coworkers found that, even after 20 years, TCDD exposure victims still displayed high blood TCDD level with concomitant plasma immunoglobulin and complement levels decrease. Considering this concern and the mechanisms about how TCDD works, it's rational to use RNAi technology, especially a long term one, to knock down the expression of AhR gene, and further to interrupt its function in mediation of TCDD toxicities. The study may lead to an effective method in healing patients who suffer from chronic TCDD toxicities. Based on materials mentioned above, three objectives of present study were carefully set:The second objective was to search for a vector permeable to CH12.LX cell line. It willhelp to express siRNA in cells for a long time and provide a possible tool for genetransfer therapy. The last objective was to optimize some defective aspects in RNAitechnology application encountered in research. The main results and conclusions out of present study were briefly manifestedbelow. 1. The immunotoxicity of TCDD on CH12.LX cell line (1) TCDD significantly inhibits the IgM secretion of CH12.LX cells induced by LPS CH12.LX cell line was derived from murine B lymphocyte leukemia andcharacterized as mature B cell line. The results from ELISA showed that high levels ofIgM was determined in supernatants when CH12.LX cells were treated with 5 μg/ml LPSand the secretion was significantly suppressed by TCDD (≥0.03 nM). Besides,quantitative PCR and western blot analysis detected AhR mRNA and protein inCH12.LX cells total RNA and protein samples. These results suggested that the TCDDimmunotoxicity mediated by AhR was successfully reproduced in CH12.LX cells. It isfeasible to carry out RNAi study in this cell line. (2) TCDD induced the synthesis of Cyp1a1 mRNA in CH12.LX cells Cyp1a1 is one of the most sensitive cytochrome P450 isoenzymes to TCDD and itsmRNA synthesis induced by TCDD is mainly mediated by AhR. The results ofquantitative PCR showed that Cyp1a1 mRNA synthesis in CH12.LX cells wassignificantly induced by TCDD treatment, which convinced us to use it as a indirectbiomarker of AhR protein level in RNAi study. 2. The effects of transient RNAi in AhR gene expression and its consequences inTCDD immunotoxicity mediation (1) Evaluation of self-summarized principles for siRNA design Based on publications, a set of principles for effective siRNA design weresummarized and tested. The test results demonstrated that these principles were reliable.No.2 AhR siRNA, designed following these principles, ever showed 98% silencingefficiency in mRNA level in a single experiment. These principles will greatly benefitfuture RNAi study. (2) Selection of siRNA transfection reagentsThree kind of siRNA transfection reagents were chosed to screen. Reagents Amine and Lipid were excluded because the results of cytotoxicity experiments suggested that they exhibited obvious growth inhibition effects on CH12.LX cells. Reagent CodeBreaker was finally selected based on two proofs. One, CodeBreaker only induced moderate growth toxicity to CH12.LX cells. The other, GAPDH siRNA transfected by CodeBreaker downregulated both mRNA and protein levels of GAPDH within 72 h in CH12.LX cells, although in the early stage, the transfection induced the stress response in cells and lead to the temporary increase of GAPDH mRNA and protein level. (3) Gene silencing effecs of AhR siRNAs Total 5 AhR siRNAs were tested by transient tranfection experiments. The results of quantitative PCR suggested that No.2 AhR siRNA resulted in highest knock down effects on AhR mRNA level. However, it was also found that the silencing of AhR mRNA only last about 48 hours. Two reasons might be responsible for the short silencing period of AhR siRNAs. One, the dividing cycle of CH12.LX cells only took as short as 14 hours. Thus the effective siRNA concentration in cells would soon be diluted by cell dividing and lost silencing effects. Two, the half life of AhR siRNA in CH12.LX cell might be relatively short, because the increasing siRNA concentration didn't elongate the functioning time. The results of western blot analysis showed that the reduction of AhR protein level induced by No.2 AhR siRNA was not statistically significant. It might relate to the metabolism of AhR protein was adjusted by cell to maintain homeostasis under stress, as well as the short functioning time of AhR siRNA. (4) The effects of transient transfection of AhR siRNA on TCDD immunotoxicity Y Results of ELISA analysis showed that the IgM secretion of CH12.LX cellsinduced by LPS was significantly suppressed by TCDD with or without No.2 AhRsiRNA treatment. T Results of quantitative PCR showed that the induction of Cyp1a1mRNA synthsis by TCDD was not interuptted by No.2 siRNA treatment. Collectively, these results suggested that the reduction of AhR protein induced bytransient transfection of AhR siRNA was not enough to produce any obvious effects onTCDD immunotoxicity mediation. It's necessary to apply long term RNAi to knock downAhR protein efficiently.3. The primary investigation to apply long term RNAi of AhR gene in CH12.LXcells Previous data of our lab showed the efficiency of plasmid transfection reagents wasvery low to CH12.LX cell line. In order to express siRNA in CH12.LX cells for a longtime, firstly we need to seek for a vector permeable to CH12.LX cells. (1) Investigation with plasmid vector The plasmid vector used in this investigation was termed pEGFP-C2, which is 4.7kbin length and able to highly express green fluorescent protein (GFP) in mammalian cells.Two methods of transfection, i.e. reagent transfection and electroporation, wereemployed and the GFP expression level was examined with fluorescent microscope. YResults of reagent transfection showed that Lipofectamine 2000 successfully deliveredpEGFP-C2 into HEK293 cells, whereas failed with CH12.LX cells. T Results ofelectroporation showed that electroporated CH12.LXcells expressed GFP 24h afterelectroporation. ? However, two adverse side-effects were also observed inelectroporation experiments, first was electroporation caused severe lethal effects onCH12.LX cells, second was the few GFP(+) CH12.LX cells lost dividing ability, so thatthe ratio of transfected cells among the population was too low to undergo further RNAistudy. It seemed that plasmid vector was inappropriate for present study. (2) Investigation with adenoviral vector The adenoviral vector used in this investigation was termed Ad-CMV-eNOS and thereporter gene in it was endothelial nitric oxide synthase (eNOS) gene. The final titrationof adenovirus used in infection experiments was about 1×1010 pfu/ml, and the control cellline was HEK293. Results of RT-PCR analysis showed that the adenovirus successfullyinfected HEK293 cells whereas failed with CH12.LX cells. The inefficiency ofadenovirus was probably due to the lack of specific receptor on the surface of CH12.LXcells. Thus this kind of adenoviral vector was excluded from the candidate lists. (3) Investigation with retroviral vector The retroviral vector used in this investigation was termed RevLuc, which wasderived from Molony Murine Leukemia Virus (MoMuLV) and packaged with 10A1envelope protein. The reporter gene in RevLuc was luciferase gene and the expression ofreporter gene was under the control of Tet-ON system. Unpurified retroviruses were usedin the infection experiments and the control cell line was HEK293. Results of luciferase activity determination suggested that the retrovirus successfully infected HEK293 cells whereas failed with CH12.LX cells. The inefficiency of retrovirus was also due to the lack of specific receptor on the surface of CH12.LX cells. Thus this kind of retroviral vector was also excluded from the candidate lists. (4) Investigation with lentiviral vectors Three lentiviral vectors were used in this investigation, including 2 vectors derived from human immunodeficiency virus termed pHOX-GFP and pWOX-GFP, 1 vector derived from feline immunodeficiency virus termed pVC-GFP. The reporter gene in lentiviral vectors was GFP gene and the envelope protein was Vesicular Stomatitis Virus G glycoprotein (VSV-G). Purified lentiviruses were used in the infection experiments and the control cell line was HEK293. Results of GFP expression in infected cells suggested that the lentiviruses successfully infected both HEK293 and CH12.LX cells. It implicated that the VSV-G packaged lentiviruses were permeable to CH12.LX cells. However, the infection of lentiviruses induced severe apoptosis of CH12.LX cells 4-7 days later, and it killed all the cells in the experiments within 2 weeks. This tragedy didn't happen to HEK293 cells. Thus, the lentivirus induced apoptosis might be CH12.LX cell line specific. Collectively, the results in present investigation implied the application of viral vectors in long-term RNAi study was expectable, although needed to be further optimized to find effective as well as non-apoptotic-inducible vectors.