Experimental Study On The Effect Of Lentiviral Vector Functionalized Graphene Oxide Particles Mediated RNA Interference HIF-1α Gene On The Uptale Of 18F-FDG And Other Biological Behaviors In Human Pancreatic Cancer Patu 8988 Cells

Part Ⅰ Experimental Study on the Effect of Lentiviral Vector-Mediated RNA Interference Hypoxia-Inducible Factor 1α Gene on the Uptake of 18F-FDG in the Human Pancreatic Cancer Cell Line,Patu8988Objective To investigate the effect of lentiviral vector-mediated RNA interference(RNAi)for Hypoxia-Inducible Factor 1α(HIF-1α)on the uptake of 18F-fluorodeoxyglucose(18F-FDG)in human pancreatic cancer cell line Patu8988.Methods Lentiviral vector for RNAi targeting HIF-1α gene(LV-HIF-1αRNAi)was constructed and used to treat cells at various concentrations(25-200 nM).The expression changes of HIF-1α in hypoxic Patu8988 cells after RNAi treatment was determined using fluorescent quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)and Western blotting.The expression of glucose transporter 1(Glut-1)was also detected with RT-PCR.The inhibition rate of cell proliferation 48 hr after the addition of 10 μL of different concentrations of LV-HIF-1αRNAi(25-200 nM)was assayed using MTT method.Meanwhile,the cell uptake of 18F-FDG was also assessed.Results After RNAi transfection,for cells treated respectively with 25,50,100,and 200 nM of LV-HIF-laRNAi,the relative expression levels of HIF-1α mRNA under hypoxia were reduced to 0.426±0.036,0.334±0.031,0.262±0.022,and 0.223±0.018,respectively;correspondingly,the relative expression levels of HIF-1α protein also decreased to 0.672±0.026,0.348±0.022,0.226±0.016,and 0.164±0.012,respectively;compared with the control group,the inhibition rates of cell proliferation were 20.98%,25.90%,31.19%,and 37.81%,respectively,exhibiting a positive correlation with the viral doses(r=0.628,P<0.05).Under hypoxia,Glut-1 mRNA expression in Patu8988 cells treated with 200 nM of LV-HIF-1αRNAi for 24,48,and 72 hr,respectively,was positively correlated with the inhibition rate of cell proliferation(r=0.728,P<0.05),as well as the inhibition rate of 18F-FDG uptake(r=0.746,P<0.05),while the latter two displayed a positive correlation with each other too(r=0.620,P<0.05).Conclusion Under hypoxia,RNAi targeting HIF-1α significantly inhibited the expression of Glut-1 mRNA in Patu8988 pancreatic cancer cells and their uptake of 18F-FDG Our results suggest that LV-HIF-1αRNAi may form a new treatment for pancreatic cancer,and the effectiveness of the treatment can be early assessed with 18F-FDG imaging.Part Ⅱ The target therapeutic effect of functionalized graphene oxide nanoparticles graphene oxide-polyethylene glycol-folic Acid-1-pyrenemethylamine hydrochloride-mediated RNA interference of HIF-1α gene on the uptake of 18F-FDG and other biological behaviors in human pancreatic cancer cellsObjective To explore the effect of polyethylene glycol(PEG)-functionalized graphene oxide-folic acid-1-pyrenemethylamine hydrochloride(GO-PEG-FA-PyNH2)-mediated RNA interference of Hypoxia-inducible factor 1-alpha(HIF-1α)on the effect of the biological behavior of human pancreatic cancer cell Patu8988.Methods The RNA interference(RNAi)functionalized graphene oxide particles GO-PEG-FA-PyNH2-HIF-1αRNAi that specifically target the HIF-1α with gene transfection was constructed,and the protein expression of HIF-1α in Patu8988 cells was determined by western blot and fluorescent quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)was used to detect the transfected GO-PEG-FA-PyNH2-HIF-1αRNAi.The gene expression level of glucose transporter-1(Glut-1)after RNAi knockdown of HIF-la in Patu8988 cells was determined by using real-time RT-PCR and western blotting.The transwell invasion chamber method was used to detect the invasive ability of Patu8988 cells,and MTT assay was used to detect the proliferation of Patu8988 cells before and after transfection.The cell cycle of Patu8988 was detected by flow cytometry,and the effect of HIF-1α knockdown on uptake of 18F-FDG in Patu8988 cells was detected by gamma counter,and the inhibitory effect on nude mice bearing pancreatic cancer was detected too.Results The mRNA and protein levels of HIF-1α in cells transfected with functionalized particles GO-PEG-FA-PyNH2-HIF-1αRNAi were decreased under the hypoxia or normoxia state.The MTT assay showed that the absorbance of GO-PEG-FA-PyNH2-HIF-1αRNAi experimental group was significantly lower than that of the negative control group(P<0.05)and blank control group(P<0.05).Moreover,the proportion of G0/G1 cells in GO-PEG-FA-PyNH2-HIF-1αRNAi experimental group was higher than that of negative control group(P<0.05)and blank control group(P<0.05).The number of migrated cells in the GO-PEG-FA-PyNH2-HIF-laRNAi experimental group was much lower than that in the blank control group(P<0.05)and the negative control group(P<0.05).Furthermore,the expression of Glut-1 mRNA in GO-PEG-FA-PyNH2-HIF-1αRNAi group was lower than that in negative control group(P<0.05)and blank control group(P<0.05).Consistently,the uptake of 18F-FDG in the GO-PEG-FA-PyNH2-HIF-1αRNAi group was also lower than that in the negative control group(P<0.05)and the blank group(P<0.05),the GO-PEG-FA-PyNH2-HIF-1αRNAi group can inhibit tumor growth better than other groups,and prolong the survival time of animal models(P<0.05).Conclusion Under normoxic/hypoxic conditions,RNA interference for HIF-1αsignificantly decreased Glut-1 mRNA expression and uptake of 18F-FDG in pancreatic cancer cells and have obvious tumor suppression effects.It was possible to use 18F-FDG imaging for early observing the therapeutic effect of GO-PEG-FA-PyNH2-HIF-laRNAi on pancreatic cancer.