The Study Of Imaging Of Pre-mRNA Splicing With A Luciferase Reporter Gene

Objective: In most eukaryotic genes,protein coding sequences(exons)are interrupted by non-coding sequences(introns)and the introns in the nucleus need to be removed before the nuclear export is translated into protein.This show that pre-m RNA splicing is an important step in the expression of most eukaryotic genes,and has the important implication for the physiological functions and pathological states of cells.In recent studies,gene expression at the m RNA level has generally been monitored using traditional biochemical methods.However,these methods can generally only be detected in vitro or influenced by the specificity of the molecule.And they cannot accurately monitor the pre-m RNA splicing process in real time,nor can they fully realize the pre-m RNA splicing imaging in vivo.Therefore,it is necessary to develop an ideal molecular probe to monitor the pre-m RNA splicing process in real time.Materials and Methods: In this study,we first assembled an artificial intron in the luciferase gene reading frame to construct a luciferase reporter gene(Luc-intron).At the same time,we set up a control luciferase reporter gene(Luc-control)without introns.We used the lentivirus system to construct two stable cell lines,A549-luc-intron and A549-luc-control.Afterwards,we used a Dual-Luciferase Reporter System method to measure the luciferase activity at the dependence of dose and time under the action of the exogenous drug,isoginkgetin(ISO).RT-PCR was used to verify the splicing condition.Then,we used bioluminescence imaging technology to monitor the pre-m RNA splicing process at the animal level.Finally,we used this luciferase reporter gene for preliminary screening and validation of drugs.Results: We found that the bioluminescent signal increased with the number of cells and had a high degree of correlation to prove the stability of the two cell lines.At the cellular level,luciferase activity decreased with increasing of the concentration or time of the ISO.It showed drug concentration and time dependence.RT-PCR experiments also verified the luciferase assay results.In addition,we found that ISO could inhibit cell growth and splicing,and this inhibition was reversible.Then,we used the bioluminescent imaging technology to monitor that the bioluminescent signal was significantly reduced after the effect of ISO at the animal level.And it indicated that the splicing process was inhibited.The above results indicated that the luciferase reporter system could monitor the pre-m RNA splicing process in real time.Finally,based on this luciferase reporter system,some small molecule drugs were initially screened.The experimental results showed that diosmetin could activate the luciferase fluorescence signal and the luciferase signal was concentration gradient dependent.We tentatively concluded that diosmetin could promote pre-m RNA splicing.We found that diosmetin could inhibit cell growth and induce apoptosis,which were detected by the CCK-8 cell activity assay and the Annexin V-FITC/PI double staining assay.Conclusion: The luciferase reporter developed in this study can effectively monitor the pre-m RNA splicing process in real time.At the same time,we can use this reporter system to screen small molecule drugs and provide new tools for drug development and treatment of splicing abnormalities.