Expression Of Interleukin-2 Gene Regulated By RU486 System

Animal experiment and clinical research indicate that Interleukin-2 (IL-2) exhibits the functions of activating immune system and therapeutic effect on tumor. So far, the main administration routes of IL-2 include systemic or local injection of recombinant protein and gene therapy. However, the tumor couldn't be eradicated by either systemic or local injection of recombinant IL-2 protein. Frequent injection caused toxic and side effects on the local organ or the whole body. Gene therapy is one of the promising strategies for cancer therapy. But the targeting and regulating of vector are the key problems to be solved, because unrestriced gene expression is unphysiologic, and also harmfull to organs. A new kind of gene switch—RU486 regulatory system is attractive because of low immunogenicity, fast response to induction, powerful inducibility.MethodsThe plasmid pRS-IL-2 which contains RU486 regulatory system and liver-specific promotor TTR encodes murine interleukin-2 (mIL-2) gene. The plasmid pRS-LacZ which contains RU486 regulatory system and liver-specific promotor TTR encodes LacZ gene. The plasmids were reacted with restriction enzymes to confirm the structure after large scale isolation. Two plasmids were transfected into the cells in vitro, or injected into the mice by hydrodynamic injection respectively. RU486 was added into culture medium to induce the transgene expression. Intraperitoneal injection of RU 486 or sesame oil was used to induce transgene expression in tissues. LacZ gene expression was examined in transfected cells and tissues of mice byχ-gal staining. The mIL-2 level of supernatant of cultured cells and serum of mice was detected by mIL-2 ELISA set. Total DNA or RNA was extracted from major organs of mice. PCR or RT-PCR was carried out to detect the distribution and expression of plasmid pRS-IL-2. The morphological changes of the liver were observed after HE staining.Results1. Plasmid identification The expected fragments were obtained after digestion of pRS-IL-2 and pRS-LacZ by different restriction enzymes respectively, which confirmed that the structure of amplified plasmids was correct.2. Tissue specific and RU486 dependent expression of LacZ genePlasmid pRS-LacZ was transfected into cells deriving from different tissues. LacZ expression was assayed in cells byχ-gal staining after induction with RU486(10-8M). In the presence of RU486, 15% of the SMMC-7721 cells were dyed blue, while less than 0.5% of SMMC-7721 cells were blue-stained in the absence of RU486. However, no blue-colored cells or less than 0.5% were discovered in other non-hepatic cells with or without RU486. The mice were treated with intraperitoneal injection of RU486(250μg/kg)or sesame oil(50μl) at 24h after receiving 5μg pRS-LacZ. Ten h after induction, the mice were sacirificed and the liver, spleen, kidney and lung were taken forχ-gal staining. Thirty percent of hepatocytes were dyed blue in the liver of mice with RU486 induction, but no or less than 0.5% of hepatocytes were stained blue in the liver of mice with sesame oil. No blue-colored cells were observed in the other organs of mice with or without RU486.3. RU486 dose-dependent expression of mIL-2 in vitroSMMC-7721 cells transfected with pRS-IL-2 were induced with different concentrations of RU486. Twenty four h later, the supernatant of cultured cells was collected to test mIL-2 level. The mIL-2 level of the supernatant of cells without RU486 was low, but increased following RU486 addition. The maximum mIL-2 level appeared in the supernatant of cells with 10-8M RU486, which was 1.35 fold-greater than that of 10-10M RU486 (q=5.954, P<0.01), and 1.78 fold-greater than that of 10-12M RU486 (q=9.999, P<0.001), 1.95 fold-greater than that of cells without RU486 (q=10.449, P<0.001).4. Kinetics expression of mIL-2 in vivoThe mice were treated with a single intraperitoneal administration of RU486 days 7 after injection of different doses of plasmids. The sera were taken to determine mIL-2 concentration at indicated time points. The kinetics expression pattern was similar although the plasmid doses were various, i.e. the mIL-2 level reached at peak at 4h after induction, declined abruptly to baseline at 10h, and was undetectable later. In mice receiving 2μg, 5μg or 10μg plasmid, 4h after RU486 induction, the mIL-2 level was 46.1, 320.4 and 478.6 fold-greater than that of before induction respectively.5. Plasmid and RU486 dose-dependent expression of mIL-2 in vivoThe mice were treated with a single intraperitoneal injection of RU486(250μg/kg) at days 7 after hydrodynamic injection of different doses of plasmids. The maximum level of mIL-2 in serum appeared in the mice receiving 10μg plasmid, which was 1.5 fold-greater than that of mice receiving 5μg plasmid (q=3.552, P<0.05) and 10.4 fold-greater than that of mice receiving 2μg plasmid (q=9.152, P<0.001).The mice were treated intraperitonealy with different doses of RU486 at days 7 after receiving 5μg pRS-IL-2. The maximum level of mIL-2 was observed in serum of mice with RU486 at 500μg/kg, which was 1.9 fold-greater than that of mice with RU486 at 100μg/kg (q=3.685, P<0.05) and 1.2 fold-greater than that of mice with RU486 at 250μg/kg (q=1.100, P>0.05). The mIL-2 level in mice with RU486 at 250μg/kg was 1.7 fold-greater than that of mice with RU486 at 100μg/kg (q=2.937, P<0.05).6. Expression of mIL-2 with different induction mannersFiveμg pRS-IL-2 was injected into the mice, 7 days later, RU486(250μg/kg) was administered into the mice once per day, once per 2 days, or once per 7 days. The expression of mIL-2 was assayed for consecutive 6 days in mice with induction once per day and once per 2 days, and was tested for 28 days in the mice with induction once per 7 days. The serum was taken before and after induction with RU486 to determine the mIL-2 level. Induction every 2 days resulted in a wavelike pattern of serum mIL-2 expression. In contrast, sustained level of mIL-2 could be observed by administering RU486 every day. Induction every 7 days resulted in high expression of mIL-2 at 4h and baseline level 6 days after the first induction. Until 28 days after plasmid administration, the high expression of mIL-2 could be achieved again after each induction.7. Analysis of PCR and RT-PCRFiveμg pRS-IL-2 was injected into mice. Seven d later, the mice were treated with RU486(250μg/kg) or sesame oil(50μl) once per 7 days over 28d. The mice were sacrificed at 4h after each induction. Liver, spleen, lung and kidney were obtained to extract DNA and RNA. PCR was used to detect plasmid distribution in these organs. The sequences of GLp65 and mIL-2 gene were found only in the liver by PCR. RT-PCR was applied to investigate the transcription of GLp65 and mIL-2. GLp65 mRNA was detected specifically in the liver of mice induced with RU486 or sesame oil, and was undetectable in the lung, spleen and kidney. However, mRNA of transgene mIL-2 was detected only in the liver of mice with RU486 induction, and was absent in the liver of mice with sesame oil. Plasmid DNA and GLp65 mRNA could be detected until days 28 which was the end of this experiment after plasmid administration.8. Morphological change of the liverAt 7d, 14d, 21d and 28d after injection of pRS-IL-2, the mice were induced via intraperitoneal injection of RU486(250μg/kg) or sesame oil(50μl). Four h later after induction, the mice were sacrificed and the livers were taken for HE staining. Microscopic images showed that only lymphocytic infiltration foci were observed occasionally in both groups, no other abnormal changes were found.Conclusions1. Tight temporal control of target gene mIL-2 expression can be mediated by RU486 regulatory system.Expression of mIL-2 gene in control of RU486 regulatory system is dependent on the dose of plasmid and RU486. The expression of transgene mIL-2 can be switched on or off at desired time by addition or deletion of RU486. Expression tendency is different with different induction manners. Therefore, the level and duration of mIL-2 gene expression can be regulated by changing the dose of RU486 and plasmid or RU486 induction manner.2. Strict spatial control of target gene mIL-2 expression can be mediated by RU486 regulatory system.The mIL-2 gene mediated by naked DNA was successfully delivered into the liver of mice by hydrodynamic injection. The specific expression of mIL-2 gene in the liver could be achieved by using RU486 system driven by liver specific promotor TTR.