ABSTRACT
Objective To investigate the effects of miR-10a expression on migration and invasion of human pancreatic cancer cells AsPC-1.Methods Small interfering RNA targeting at miR-10a (miR-10a-siRNA) was constructed,then it was transfected into pancreatic cancer AsPC-1 cells,and nonsense siRNA (Nc-siRNA) group and blank control group was established.Real time PCR assay was used to detect the expression of miR-10a in the 3 groups,and wound healing assay and Transwell assay were used to determine the migration and invasion abilities of cancer cells.The amount of matrix metalloproteinase-13 (MMP-13) in supernatant of cancer cell culture of each group was examined by ELISA assay.Results The miR-10a levels in control group,NC-siRNA group and miR-10a-siRNA group were 1.05 ±0.08,1.03 ±0.06,0.02 ±0.01 ; and the number of transmembrane cell were (150 ± 2.6),(145 ± 2.2),(62 ± 1.8),the levels of MMP-13 in the supernatant were (108.5 ± 2.8),(107.8 ± 2.5),(35.8 ± 1.5) pg/ml.The values were significantly lower in miR-10a-siRNA group than those in control group and NC-siRNA group (P ＜ 0.01).The distance of cultured clone in miR-10a treated cancer cells (736± 18 μm) was significantly longer than those in the controls (385 ±5 μm) and NC-siRNA group (395± 13 μm,P＜0.01).Conclusions Down-regulation of miR-10a by siRNA may inhibit migration and invasion of pancreatic cancer AsPC-1 cells,and the downregulated expression of MMP-13 may be one of the important mechanisms.
ABSTRACT
Objective To study the effects of Forkhead box protein M1 (FoxM1) down regulation by small interfering RNA(siRNA) on chemosensitivity and mechanism of human pancreatic cancer cell and its mechanism.Methods Three FoxM1 siRNAs were designed and constructed.All cancer cells were divided into different groups,after transfected with FoxM1 siRNA for different time,the cultured cells were harvested to carry on the next tests.Expression of FoxM1 were determined by red-time PCR and Western blot,and prolifearion and chemosensitivity were evaluated by MTT assay,and the phosphorylation of Akt protein was examined by Western blot.Results FoxM1 siRNA could down-regulate the FoxM1 expression in a dose-and time-dependent manner.The MTF results showed that the inhibit rates was 17.78％,17.56％,35.39％,52.81％,70.98％ indifferentgroups [Con-A + Gemcitabine,Con-B + Gemcitabine,siRNA (3.125nM) + Gemcitabine,siRNA (6.25nM) + Gemcitabine and siRNA(12.5nM) + Gemcitabine,respectively.The phosphorylation of Akt protein was inhibited in a dose-dependent manner.Conclusions FoxM1 siRNA could sensitize human pancreaticr cancer cells chemotherapy sensitivity,it is the one of the important mechanisms through down-regulate Akt phosphorylated levels,but the molecular mechanism need to be explored further.
ABSTRACT
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.