Preliminary Study On Antisense Imaging Targeting LncRNA HOTAIR

Objective Long-non-coding RNA（long noncoding RNA,LncRNA）HOTAIR（HOX transcript antisense RNA）is amplified and overexpressed in many human tumors and can be used as an effective target for tumor therapy.^99mTc-labeled antisense oligodeoxynucleotide（antisense oligonucleotides,ASON）can show the expression of HOTAIR,which is of value in the diagnosis and treatment of malignant tumors.It provides important information for the occurrence and prognosis of malignant tumors.The aim of this study was to synthesize a new antisense oligonucleotide probe ^99mTc-HYNIC-ASON targeting LncRNA HOTAIR with radionuclide ^99mTc,identify the physicochemical properties of the probe in vivo and in vitro,and the effect of liposome transfection on the proliferation and migration of human glioma U87 cells.The in vivo biodistribution and imaging of malignant glioma xenografts were also investigated.Methods The antisense oligonucleotide（ASON）probe sequence for lnc RNA HOTAIR,5’-AATTCTTAAATTGGGCTGG-3’,was designed and synthesized.The sequence of the mismatched antisense oligonucleotide（ASONM）probe was 5’-AATACTTAGATTAGGCAGG-3’.Two sequences were methylated and thio-modified to improve the stability and anti-degradation ability.NH₂C₆was used as the linker,that is,the synthetic formula was 5’-NH₂C_6-ASON-3’,and ^99mTc was coupled with bifunctional chelating agent HYNIC and Tricine buffer was used as the buffer solution.The ASONs and ASONMs were labeled with the same method and purified by Sephadex G25.The radiolabeling yield,radiochemical purity and stability of the peak tube probe in fresh human serum were detected by ITLC paper chromatography with salt water as the developer,and the anti-degradation ability of the probe was detected by agarose gel electrophoresis.The malignant glioma U87 cell line was resuscitated and the radioactive labeled probe was transfected with liposome 2000.The uptake rate of the probe was measured byγ radioimmunoassay,and the changes of migration and proliferation of tumor cells were detected by cell scratch test and Cell Counting Kit-8（CCK-8）test,respectively.In addition,U87 cells were amplified and cultured,and 1×10¹⁰/1.5ml each mouse was inoculated into the right foreaxilla of BALb/c nude mice.When the diameter of the tumor was about 1.0-1.5cm,the tumor-bearing nude mice with regular shape and no significant difference in tumor size were selected for the follow-up experiments.In the in vivo biodistribution experiment,20 xenografts were randomly and averagely divided into five groups and each one was injected with liposome-encapsulated antisense probe ^99mTc-HYNIC-ASON 1μg and 2.59MBq（100μl）through tail vein respectively.Each group was sacrificed at 1 h,2 h,3 h,4 h and 6 h after injection.The organ or tissue of heart,liver,spleen,kidney,stomach,small intestine,bladder,muscle,bone and tumor were weighed,and their radioactivity counts were measured.In the imaging experiment,4 tumor-bearing nude mice were randomly divided into antisense imaging group,mismatch group,blocking group and ^99mTc control group.The xenografts of antisense group and mismatch group were injected with 4μg,14.8MBq（150μl）Lip-^99mTc-HYNIC-ASON and ^99mTc-HYNIC-ASONM respectively;the blocking group was injected with 10μg liposome-transfected unlabeled probe 2 hours before Lip-^99mTc-HYNIC-ASON injection;the ^99mTc control group was injected with ^99mTc solution with the same radioactivity as that of the experimental group.SPECT imaging of the four groups was performed at 1 h,2 h,4 h,6 h and 8 h after injection,respectively,and the radioactive uptake of the tumor site was observed.All data were processed by SPSS22.0 statistical software,and the measurement data were expressed by mean±standard deviation（±s）.T test was used for comparison between the two groups,and analysis of variance was used for multi-group comparison.The criterion of significance:when P<0.05,it showed that the difference between the data of the examined groups was statistically significant.Results（1）The radiolabeling yeild of antisense probe ^99mTc-HYNIC-ASON and mismatched probe ^99mTc-HYNIC-ASONM determined by ITLC were（91±1.5）%and（90±0.6）%,respectively,and the radiochemical purity was more than 89%.Gel electrophoresis experiments confirmed that ^99mTc and the probe were successfully radiolabeled and no obvious degradation;the probe showed good stability in incubation with normal saline and human fresh serum in 37 degree Celsius,and the radiochemical purity stayed higher than 80%within 12 hours.（2）The results of cell uptake assay showed that 6 hours after liposome transfection,the maximum uptake rate of probe Lip-^99mTc-HYNIC-ASON in human glioma U87 cells was 3.0%,which was significantly higher than that in the non-transfection group（0.6%）（t=15.6～21.56,P<0.01）.The results of CCK-8 test and cell scratch test showed that Lip-^99mTc-HYNIC-ASON could inhibit the proliferation and migration of tumor cells,and there was significant difference between the two groups（t=2.336～30.23,P<0.05;t=51.54,P<0.01,F=331.8,P<0.01）.（3）The study of biodistribution showed that the tumor uptake of antisense probe Lip-^99mTc-HYNIC-ASON encapsulated by liposome was highly concentrated and significantly higher than that of mismatch group,and the ratio of tumor-to-muscle was also significantly higher than that of mismatch group.At the same time,the tumor could be clearly demonstrated by SPECT imaging 1 hour after injection of liposome-encapsulated antisense probe Lip-^99mTc-HYNIC-ASON,and the best acquisition time was at 2 hours post-injection.On the contrary,no tumor was found in mismatch group and blocking group at any time point post-injection.Conclusion^99mTc-HYNIC-ASON antisense oligonucleotide probe targeting HOTAIR malignant glioma was successfully synthesized.The probe showed a higher radiolabeling yeild,good stability and strong targeting binding ability,and can inhibit the proliferation and migration of glioma U87 cells.The liposome encapsulated oligonucleotide probe can be used for real-time imaging of lnc RNA HOTAIR in malignant gliomas in vivo to observe the expression of HOTAIR.It is a new type of non-invasive targeting probe and expected to be used in the early and specific diagnosis of malignant glioma.