The Construction And Photoacoustic Imaging Of Tyrosinase Reporter Gene Regulated By MicroRNA-9

Micro RNAs(mi RNAs),a class of small RNAs including 22~24 nucleotides,are significant regulators of many cellular functions.One mi RNA can have many target genes,also different mi RNAs can target a same gene.Recent studies have identified micro RNA-9(mi R-9)as one such mi RNA plays an important role in cell growth,differentiation,neurogenesis,immunity and apoptosis.Mi R-9 is decreased in many types of cancer,such as many breast cancer cell lines.Conventional detection methods,such as northern blot,Real-time PCR or microarray,have been used to assess mi RNA expression.However,these techniques are labor-intensive and time-consuming,and require the fixation or lysis of cells.Recent remarkable advances in molecular imaging techniques have provided the capability of noninvasive repeated quantitative imaging of mi RNAs in living animals.Photoacoustic imaging(PAI),a novel nonionizing and noninvasive modality on account of optical absorption excitation and ultrasonic detection,provides high-resolution optical contrast information in deep tissues and breaking through resolution restrictions of pure optical imaging techniques,which is gaining attention in the field of medical imaging.It is highly sensitive to melanin,one of the principal absorbers in skin,and tyrosinase is the key enzyme in charge of generation of melanin.Melanin is a very suitable contrast agent for PAI,which is non-toxic,stable and has good biological compatibility.Using tyrosinase report gene and photoacoustic imaging as a system to detect the expression of mi R-9,is of great significance of cancer treatment and its clinical application.The goal of this research is to monitor the change of mi R-9 expression noninvasively in vivo using photoacoustic imaging and new molecular probes.5-aza-2'-deoxycytidine can influence the expression of mi R-9 in A549 cells.As the product of tyrosinase,melanin has strong absorption ability and good biocompatibility properties,the tyrosinase gene and mi R-9 target sequence were synthesized into a new photoacoustic molecular probes which expression is regulated by mi R-9.Combine the probes with light sound equipment to monitor the change of mi R-9 expression.First,build tyrosinase report gene,detect melanin expression in vitro and validate report gene targeting to mi R-9: using gene synthesis plasmid TYR,TYR-3×PT;Two plasmids were transfected into 293 T cells at a certain concentration gradient,respectively,then detect tyrosinase activity and melanin content;mi R-9 and TYR-3×PT were simultaneously transfected into 293 T cells at a certain concentration gradient,then detect tyrosinase activity and melanin content.Second,photoacoustic imaging is performed with EP tubes,phantom and mice,respectively: 293 T cells transfected with different concentrations of plasmid,293 T cells transfected with the same concentration of plasmid and different concentrations of mi R-9,and A549 cells transfected by plasmid before and after dosing were executed 3D photoacoustic handheld scanning with EP tube and photoacoustic tomography imaging with phantom;The mices which thighs were rapidly injected with A549 cells transfected with plasmid before and after dosing implemented 3D photoacoustic handheld scan and photoacoustic tomography imaging.Successful build the tyrosinase report gene targeting mi R-9,mi R-9 can inhibit tyrosinase report gene expression.Within a certain range,the report gene expression increases with the increased concentration of its transfection,mi R-9 inhibition also along with the increase of its transfection concentration increased.To methylation drug 5-Aza-2'-deoxycytidine promoted the expression of A549 cells mi R-9.Photoacoustic imaging to detect the plasmid and mi R-9 different transfection concentration signal caused by the differences,and drugs before,during and after the stimulus signal differences in mice in vivo and in vitro.We proved that tyrosinase report gene is suitable for photoacoustic imaging.Using tyrosinase report gene targeted mi R-9 and photoacoustic equipment,change of mi R-9 expression can be detected in A549 cells before and after 5-Aza-2'-deoxycytidine stimulus.