Investigation Of The Relationship Between Dicer Activity And Lung Cancer Using Electrochemical Techniques

Objective：Dicer, a key enzyme in RNA silence pathway, is responsible to cut duplex RNAinto19-21bp short RNA. Recent researches have demonstrated that the level of Diceris closely associated with the cancer prognosis, which the low expression of Diceralways means poor survival.However, the detailed relationship between Dicer activityand lung cancer has not been clarified clearly.So, we have fabricated a novelelectrochemical biosensor for detecting dicer activity, which can furthercontributetoelucidate the potential relations between Dicer activity and clinical pathology ofnon small cell lung cancer (NSCLC).Methods:Based on the intrinsic slicer activity of dicer, we have constructed anelectrochemical method to detect its activity. Firstly, we designed a double-strandedRNA, one of which contained a thiol group at3’ end that could be covalently linkedto a pre-treated gold electrode. After the incubation with dicer, the double-strandedRNA on the electrode surface was cleaved into two fragments. Due to the lowermelting temperature, the formed duplex would deform and left the shorter strandattached on the electrode at room temperature (22°C). Finally, the remainedfragmentson the electrode were hybridized with a methylene blue modified single-strandedDNA, which makes the electrochemical signal proportionate to the activity of dicer.Thus, an assay method for detecting the activity of dicer has been established.Results：1.Optimization of the experimental conditions: (1)30minutes and37°C are the optimal time and temperature conditions forincubating RNA duplex modified electrode with dicer, respective.(2) The molar ratio between RNA1: RNA2of0.2μM:0.5μM is sufficient for theformation of RNA duplex.(3)30°C and2hours are the optimal conditions for hybridization of methyleneblue modified DNA.2. In this method, a linear range between the activity and concentration of theenzyme can be obtained in a certain detection range.3. The activity of dicer activity is lower in the tissue from NSCLC than innormal tissue. Furthermore, the activity of Dicer decreases in the cancer with higherclinical stages.Conclusion：1. With a constant amount of substrate, the activity of dicer increases with theconcentration until a saturation.2. The detectable activity of dicer was as low as0.01U/mL based on thesignal-to-noise (S/N) level. Owing to its high sensitivity and inexpensive instruments,the proposed method may be used for the detection of other malignant tumors.3. The mutagenesis of Dicer gene might be responsible for the staging of lungcancer. The activity of Dicer decreases with a higher clinical stage.