Effects Of Novel Nano-nucleic Acid Preparations On Tyrosinase Expression And Proliferation Of Melanoma A375 Cells

Objective:Design and prepare novel nano-nucleic acid preparations that can be used to interfere with melanin synthesis in vivo.And to investigate its effect on the gene expression of tyrosinase and the proliferation,and apoptosis of melanoma A375 cells in vitro.Methods:1.We designed siRNA that targets Tyrosinase(TYR)gene,and selected the siRNA that inhibits the transcription of TYR mRNA as characterized by real-time fluorescent quantitative PCR.The inhibitory effect of TYR siRNA on TYR protein expression was further analyzed using Western blotting.2.According to the expression of TYR-mRNA detected by using real-time PCR,and the rate of cell death and the cell cycle by using flow cytometry,we compared the expression of TYR and the effects of cell death and cycle of A375 cells by using different concentrations of TYR-siRNA and MITF(Microphthalmia-associated transcription factors)-siRNA,respectively.3.To detemine the ideal moleuclar target and the optimal concentration of siRNA nano-nucleic acid preparations,and investigate the uptake of nano-nucleic acid preparation by melanoma cell A375 by laser confocal microscopy.4.Real-time PCR was used in determinging TYR mRNA expression affected by nano-nucleic acid preparations on melanoma cells A375.MTT assay,DAPI staining and flow cytometry were used to analyze the effects on proliferation,morphology and cell death(apoptosis)by nano-nucleic acid preparations on melanoma cells A375.5.One-way ANOVA was conducted in analyzing the results statistically.Results:1.Real-time quantitative PCR was used to screen out siRNA-2 as the best siRNA for interference with TYR mRNA.At the same time,Western blotting confirmed that it also inhibited the expression of TYR at the protein level,and there were significant differences(P<0.05).2.There is a dose-dependent effect on TYR mRNA expression by TYR-siRNA,showing decreased expression of TYR mRNA as TYR-siRNA increase(P<0.05).The rates of cell apoptosis and the TYR-siRNA concentration applied were also dose-dependent,the increased cell death by apoptosis showed positive correlation to the increased concentrations of TYR-siRNA applied(P<0.05).3.TYR-siRNA arrested the cell cycle of A375 in S phase,in a concentration-dependent manner(P<0.05).4.There were concentration-dependent effects on the expressions of MITF mRNA and the TYR mRNA versus TYR-siRNA concentrations,and the expressions of the TYR mRNA and the MITF mRNA decreased as the siRNA concentrations increase(P<0.05).The rates of cell death by apoptosis were also dependent on the concentrations of TYR-siRNA added,the more the siRNA added,the more cell death(P<0.05).5.MITF-siRNA arrested the cell cycle of the A375 cells in G0/G1 phase,in a concentration-dependent fashion(P<0.05).6.Observation using Laser confocal microscopy indicated that nano-nucleic acid preparations can be taken up into A375 cells.7.After the interventions of nano-nucleic acid preparations on A375 cells,the expression of TYR mRNA decreased(P<0.05),the cell proliferation rates decreased(P<0.05),however the cell apoptosis increased(P<0.05).And morphological changes of the nucleus were observed by laser confocal microscopy.Conclusions:1.Both TYR-siRNA and MITF-siRNA designed in this work effectively inhibited the mRNA expression of TYR,a key enzyme in melanin synthesis.Both TYR-siRNA and MITF-siRNA induced cell apoptosis of the melanoma A375 cells in a concentration-dependent manner.2.TYR-siRNA and MITF-siRNA have different effects on cell cycle.When siRNA used by 300 pmol,especially MITF-siRNA,cell proliferation became abnormal by showing up with tetraploid cells.Accordingly,the TYR can be chosen as a target of action,and the nano-nucleic acid preparation was suggested to be administered by a concentration of 200 pmol,due to the dose-dependent functions.3.The nano-nucleic acid preparations made in this study can be taken up into cells.4.Nano-nucleic acid preparations can target TYR mRNA,inhibit cell proliferation,shrink the cell nucleus,produce heterochromatin,and promote cell apoptosis when used with A375.5.The fibrous nano-nucleic acid preparations are more efficient than the spheroidal nano-nucleic acid preparations in the transfection of siRNA molecules,showing more effective cell proliferation inhibitions and increased cell death by apoptosis.