Down-regulation of midkine gene expression by small interfering RNA affects melanoma cell adhesion and invasion

Objective To study the effects of midkine (MK) gene-targeting small interfering RNA (siRNA)on the invasion of melanoma cells.Methods Three MK gene-targeting siRNAs (S1,S2 and S3)were designed,constructed,and transfected into human A375 melanoma cells.Real-time PCR was performed to measure the expression of MK gene and to screen the siRNA with best efficacy.Then,A375 cells were transfected with the optimal siRNA of various doses (3.125,6.25 and 12.5 nmol/L)followed by additional culture of various durations(24,48,72 hours).Some A375 cells remaining untreated served as the blank control group,and some transfected only with liposomes served as the vector control group.Reverse transcription (RT) -PCR and Western blot were conducted to detect the mRNA and protein expression of MK,respectively,MTT assay to observe the adhesion of A375 cells,and Boyden chamber was used to evaluate cell invasion.Results The expression of MK mRNA was downregulated by all the three siRNAs,especially by the siRNA S3,which was used in the following transfection experiment.Real-time quantitative PCR revealed that the MK mRNA expression was reduced by the siRNA in a dose- (r24hours=-0.906,r4Bhours=-0.922,r72hours=-0.939,all P<0.01)and time-dependent(r3.125nmol/L=-0.889,r625nmol/L=-0.935,r125nmol/L=-0.928,all P<0.01)manner.MTT assay showed that the percentage of adhesing cells was 73.66%±2.25%,49.36%±2.16%and 28.35%±1.68%in A375 cells transfected with the siRNA of 3.125,6.25 and 12.5 nmol/L,respectively.The number of cells migrating across the chamber filter was 23.9±1.6,12.1±1.5,5.6±1.2 among A375 cells transfected with the siRNA of 3.125,6.25 and 12.5 nmol/L,respectively,significantly lower than that in the blank control group(36.8±1.5).The percentage of adhesing cells and number of migrating cells decreased with the dose of siRNA(r=-0.936,-0.915,P<0.01,0.05,respectively).Conclusions MK gene might play an important role in the adhesion and invasion of melanonla cells.To down-regulate the expression of MK gene by siRNA may suppress the adhesion and invasion of melanoma cells. Key words: Melanoma,experimental; Cell line,tumor; Midkine; RNA,small interfering