The Research of Radionuclide Imaging and Therapy for Cervical Cancer Mediated by Egr-1Promoter-driven hNIS Gene Transfection
|Course||Medical Imaging and Nuclear Medicine|
|Keywords||Egr-1 sodium iodide symporter cervical cancer radionuclide|
ObjectiveTo construct a recombinant plasmid containing early growth response gene1（Egr-1）promoter-driven the human sodium iodide symporter （hNIS） gene to express specificallyon human cervical cancer Hela cell line, and to investigate the feasibility of radionuclideimaging and therapy mediated by hNIS gene controlled with Egr-1promoter.Methods1. To extract the FL*-hNIS/pcDNA3plasmids, amplify of Egr-1promoter sequenceby PCR from the template Egr-1/pMR18T, then insert in the FL*-hNIS/pcDNA3plasmid,constitute the recombinant plasmid Egr-1-hNIS/pcDNA3, and then verified by restrictionenzyme electrophoresis.2. Egr-1-hNIS/pcDNA3plasmid was used to transfect human cervical cancer Helacell line. Cell clones were isolated after selective growth under G418and evaluated forhNIS expression by flow cytometry （FCM）. Clone Hela-Egr-1-hNIS was identified by itsability to expand in selection media and its highest level of expression efficiency of hNIS.Based on the same method, the FL*-hNIS/pcDNA3plasmid transfected Hela-FL*-hNIScell line was used as a positive control, untransfected Hela cell line was used as a negativecontrol. The expression of hNIS gene of the three cell lines were detected by RT-PCR.3. The biologic functions of the three cell lines were investigated by measuring125Idynamic uptake, perchlorate （NaCLO4） inhibition and dynamics of125I efflux experiments.Therapeutic effects of131I were evaluated by in vitro clonogenic assay.125I dynamic uptakeand125I efflux experiments were finished after extra different doses of X-ray ionizingradiation were performed to Hela-Egr-1-hNIS cells to evaluate the radiation-induced Egr-1promoter of hNIS protein function role. 4. The cervical cancer xenograft models of nude mice were established withHela-Egr-1-hNIS cells and control cells. In vivo distribution of Na125I was carried out, andthe%ID/g and tumor/non-tumor tissue radioactivity count ratio （T/NT） of transplantedtumor and the main organs or tissues were calculated at the different time points. TheNa131I and99mTcO4-imaging were performed to observe the transplanted tumor imaging,analyze the radioactivity count ratio （T/B） of xenograft and corresponding contralateralparts of the normal base tissue.Results1. The Egr-1promoter was amplified successfully, and the recombinant plasmidEgr-1-hNIS/pcDNA3was constructed. The results of electrophoretic bands of restrictionenzyme electrophoresis were correct.2. Egr-1-hNIS/pcDNA3and FL*-hNIS/pcDNA3plasmid were successfullytransfected into Hela cell line. From FCM, the hNIS cell lines with stable and highestefficient expression were gained, expression rates were11.2%and10.14%. RT-PCR resultsshowed that Hela-Egr-1-hNIS group and positive control group all had hNIS geneexpression, negative control group had no expression.3.125I dynamic uptake experiments showed that the iodine uptake capacity ofHela-Egr-1-hNIS cells and Hela-FL*-hNIS cells were increased by13times and21timesthan the negative control, and that capacity can be inhibited by NaClO4. After X-rayionizing radiation, iodine uptake of Hela-Egr-1-hNIS cells increased in roughlydose-dependent manner but iodine outflow process had no significant changes.131I in vitrotreatment of clonogenic experimental showed that the colony formation ofHela-Egr-1-hNIS, Hela-FL*-hNIS and Hela were （50.49±9.75）%,（6.70±1.57）%and（86.32±6.56）%, respectively.4. Na125I in vivo distribution experiments of Hela-Egr-1-hNIS cells in tumor-bearingnude mice model showed that transplanted tumors can obviously uptake Na125I. During1～12h, tumor%ID/g significantly higher than that of other organs except for the thyroidand stomach, T/NT values reached their highest values at3h. Na131I imaging showed thatthe transplanted tumor significantly uptake the radioactivity. The T/B value reached topeak at4h to4.05.99mTc4-imaging showed that transplanted tumor continued to bevisulized to48h, the T/B peak value was9.13at19h. T/B values of two types of imaging ateach time point were higher than that of the positive control group. Conclusions1. Successfully established the Hela-Egr-1-hNIS cell line, and the Egr-1promoter-driven hNIS protein was stably expressed. The cell line showed a strongcapability of iodide uptake, and had a certain effect of radiation inducement.2. Hela-Egr-1-hNIS cells tumor-bearing nude mice were found to obviously uptakeNa125I. The results of Na131I and99mTc4-imaging suggested that Hela-Egr-1-hNIS cellsprimarily formed a positive feedback chain of in vivo iodide uptake, and this is promisingto have better therapeutic response in vivo treatment.