The Study Of Alternative Polysdenylation Detection Of Proto-oncogene Based On Ultrasensitive Electrochemical Biosensor

Alternative polyadenylation?APA?is a pervasive mechanism in eukaryotes and plays an important role in expressional regulation of genes.Genes can choose different polyadenylation sites to produce various mRNA isoforms with different lengths of 3'untranslation region?3'UTR?.Current researches show that proto-oncogenes tend to use the isoforms with short 3'UTR in different cancer cells and cancer tissues.These isoforms are also closely related to the diagnostic typing and prognosis of tumors,so it may be used as a potential novel cancer biomarker with good clinical value and application prospect.Although traditional biological methods?qPCR,Northern blot,high-throughput sequencing?are widely used in the field of APA detetion,there are some shortcomings to limit their application,such as high cost,complicated operation,time-consuming,and so on.Recently,electrochemical detection technniques have attracted considerable atttion.Compared with traditional methods,the electrochemical biosensors have many outstanding advantages,including controllable linear range,low cost,miniaturization,simplicity,good selectivity,and high sensitivity.However,untill now no research has been reported on APA quantification using electrochemical detection.Therefore,we have effectively combined supramolecular materials,nanoparticles,and biological probes to highly improve the sensitivity and specificity of biosensors.These biosensors greatly reduced the detection limit and realized the rapid detection of APA.In this paper,using electrochemical technology we have accomplished the ultrasensitivive APA detections of two pro-oncogenes?Dicer1 and CCND2?which have been reported to be biomarkers in breast cancer and lung cancer,respectively.The two studies represent as following;Part I:SCX6-RGO composites were prepared by hydrothermal method.Au nanomaterials were introduced to increase the sensing capability.All constructed materials were characterized by Fourier transform infrared spectroscopy?FTIR?,thermogravimetric analysis?TGA?,Zeta potential,and atomic force microscope?AFM?.Methylene blue?MB?was selected as an electrical signal and detected by differential pulse voltammetry?DPV?.The total RNAs were extracted from human breast cancer cell lines BT474 and SKBR3 and reverse-transcribed to cDNA as detection samples.Subsequently,the expression of Dicer1isoforms regulated by APA was determined using electrochemical biosensors.The results showed that the expression of the Dicer1isoform with shorter 3'UTR?Dicer1-S?was obviously higher than that of the isoform with longer 3'UTR?Dicer1-L?both in BT474 and SKBR3 cell lines.The detection ranges of Dicer1-S and Dicer1-L were 10^-1410^-9M and 10^-1510^-10M,respectively.The LODs of Dicer1-S and Dicer1-L were 3.5 fM and 0.53 fM,respectively.These agreed well with the results analyzed by the RT-qPCR method.Thus,the proposed biosensor manifested high specificityand sensitivity,implying a potential application value.Part II:SCX8-RGO composites were prepared by one-step method.Simultaneously,Fe₃O₄ nanomaterials were synthesized and Au nanoparticles were added to synthesize Au@SCX8-RGO and Au@Fe₃O₄ composite materials.The materials were characterized by IR,TEM and XPS.The DPV detection was performed by using the screen printing electrode and using toluidine blue?TB?as an electrical signal.The total RNAs extracted from human lung cancer cell lines H292,and normal lung epithelial cell Base-2B were used to directly detect CCND2 APA.The results of electrochemical detection showed that the expression of the CCND2 isoform with shorter 3'UTR?CCND2-S?was obviously higher than that of the isoform with longer 3'UTR?CCND2-L?by the constructed biosensor in H292 cell lines.The detection ranges of CCND2-S and CCND2-L were 10^-1810^-11M and 10^-1710^-11M,respectively.The LODs of CCND2-S and CCND2-L were 0.176 and 9.5 aM,respectively.These results were same as the results analyzed by RT-qPCR method.To our knowledge,this is the first quantitation of different isoforms regulated by APA mechanims using electrochemical detection.The detection results suggested that the proposed electrochemical sensing strategy is thus expected to provide a new method for determination of novel biomarkers and a novel method for the diagnosis and prognosis of cancer.