BACKGROUND AND AIM: miRNA is an important factor for tumorigenesis which could act as a potential molecular target for tumor diagnosis. The goal of this study was to explore a new method for visualizing the expression of let-7 in lung adenocarcinoma A549 cells by Cerenkov luminescence imaging (CLI) and gamma camera imaging. METHODS: The human sodium/iodine symporter (hNIS) and 3'-UTR sequence of the ras gene (RU) that complementarily binds to let-7 were cloned with hNIS serving as the reporter gene. The expression of hNIS regulated by let-7 in the fusion gene hNIS-RU was constructed; the let-7 primer (pri-let-7), which could specifically bind to RU and the mir-143 primer (pri-mir143) not binding with RU, was cloned. A549 cells were transfected with hNIS or hNIS-RU, and additional cells were cotransfected with hNIS-RU and different concentrations of pri-let-7 or pri-mir143. The cells were incubated with 740kBq (131)I-containing media for 1h, 24h after transfection. CLI, gamma camera imaging, and γ counting were subsequently conducted, and the correlation among CLI, gamma camera imaging, and γ counting was compared when cotransfected with pri-let-7. RESULTS: CLI, gamma camera imaging, and radioactive counting showed that hNIS-transfected A549 cells had significantly higher uptake of (131)I compared to non-transfected cells. The uptake of (131)I in hNIS-RU transfected A549 cells decreased to approximately 70% compared to hNIS-transfected cells, since hNIS-RU expression was suppressed by intracellular let-7. After cotransfection with hNIS-RU and various concentrations of pri-let-7, (131)I uptake gradually decreased with increasing pri-let-7, while (131)I uptake remained roughly unchanged in the presence of hNIS-RU cotransfected with different amounts of pri-mir143. CLI was highly correlated with gamma camera imaging (r(2)=0.9893) and radioactivity counting (0.9779). CONCLUSIONS: Based upon miRNA-regulated reporter genes which mediate the uptake of the radionuclide, both CLI and gamma camera imaging can noninvasively detect miRNA expression in cells, which may provide a new way for the visualization of miRNA expression.
BACKGROUND AND AIM: miRNA is an important factor for tumorigenesis which could act as a potential molecular target for tumor diagnosis. The goal of this study was to explore a new method for visualizing the expression of let-7 in lung adenocarcinoma A549 cells by Cerenkov luminescence imaging (CLI) and gamma camera imaging. METHODS: The humansodium/iodine symporter (hNIS) and 3'-UTR sequence of the ras gene (RU) that complementarily binds to let-7 were cloned with hNIS serving as the reporter gene. The expression of hNIS regulated by let-7 in the fusion gene hNIS-RU was constructed; the let-7 primer (pri-let-7), which could specifically bind to RU and the mir-143 primer (pri-mir143) not binding with RU, was cloned. A549 cells were transfected with hNIS or hNIS-RU, and additional cells were cotransfected with hNIS-RU and different concentrations of pri-let-7 or pri-mir143. The cells were incubated with 740kBq (131)I-containing media for 1h, 24h after transfection. CLI, gamma camera imaging, and γ counting were subsequently conducted, and the correlation among CLI, gamma camera imaging, and γ counting was compared when cotransfected with pri-let-7. RESULTS: CLI, gamma camera imaging, and radioactive counting showed that hNIS-transfected A549 cells had significantly higher uptake of (131)I compared to non-transfected cells. The uptake of (131)I in hNIS-RU transfected A549 cells decreased to approximately 70% compared to hNIS-transfected cells, since hNIS-RU expression was suppressed by intracellular let-7. After cotransfection with hNIS-RU and various concentrations of pri-let-7, (131)I uptake gradually decreased with increasing pri-let-7, while (131)I uptake remained roughly unchanged in the presence of hNIS-RU cotransfected with different amounts of pri-mir143. CLI was highly correlated with gamma camera imaging (r(2)=0.9893) and radioactivity counting (0.9779). CONCLUSIONS: Based upon miRNA-regulated reporter genes which mediate the uptake of the radionuclide, both CLI and gamma camera imaging can noninvasively detect miRNA expression in cells, which may provide a new way for the visualization of miRNA expression.
Authors: Nikunj B Bhatt; Darpan N Pandya; William A Dezarn; Frank C Marini; Dawen Zhao; William H Gmeiner; Pierre L Triozzi; Thaddeus J Wadas Journal: Methods Mol Biol Date: 2018
Authors: Thomas C Wilson; Stephen A Jannetti; Navjot Guru; Nagavarakishore Pillarsetty; Thomas Reiner; Giacomo Pirovano Journal: Int J Radiat Biol Date: 2020-07-02 Impact factor: 2.694