| Objective: To investigate the effects of Livin targeted microRNA on hunman ovarian cancer cell SKOV3.Two pairs of microRNA sequence were designed and synthesized, and then transfected to SKOV3 cells. Detect the expression of Livin mRNA, the generation, apoptosis and cycle of cells. Also to explore the possible mechanisms of Livin targeted microRNA on the human ovarian cancer cell SKOV3.Methods:1. human ovarian cancer cell SKOV3 resuscitative and culture, cells morphological were observed by inverted microscope.2. Designed and synthesised the Livin targeted microRNA and linked to plasmid vector. Transfect the SKOV3 cells beforehand by the liposome. 24h and 48h after that, the green fluorescence cells and the transfection efficiency were observed by fluorescence microscope.3. The expression of Livin mRNA were analyzed by reverse transcriptase polymerase chain reaction(RT-PCR).4. The anti-proliferative effect of Livin targeted microRNA on SKOV3 were measured by MTT colorimetric method.5. The cell cycle and apoptosis were assessed by flow cytometry(FCM).Results:1. The observation by inverted microscope and fluorescence microscope: 24h after transfect, SKOV3 cells changed significantly in morphology observed by microscope. The culture solution was clear, control group cells growth were concentrated, glossiness was good. The transfected group cells density is lower than blank group, glossiness comes down, and part of the cells were expression green fluorescnce ;48h after transfect the control group cells already has overlapping growth, The changes were more obvious in the transfect group. The cells which expression green fluorescnce were 70-80 percent, the transfection was successful.2. RT-PCR showed that 48h after Livin targeted microRNA transfect, Livin mRNA of different groups compare with reference. The ratio of Livinα: the control and negative group were 0.58±0.21%,0.53±0.06%. but the transfect group was 0.36±0.06%, the differences among control,negative and transfect group were significant(P<0.05). And the Livinβ: the control and negative group were 0.75±0.10%, 0.73±0.15%. but the transfect group was 0.48±0.20%, The differences were significant(P<0.05),showed that the Livin mRNA expression weakened, the RNA interference for ovarian cancer was significant. The micRNA interference for SKOV3 is feasible.3. MTT showed that 24h after Livin targeted microRNA transfect, the inhibitory ratio of negative group was 2.44±0.78%.But the transfect group was 30.37±2.17%, difference among them was significant(P<0.05).48h after transfected the inhibitory ratio of negative group was 2.54±0.30%,but the transfect group was 46.49±3.36%,difference among them was significant(P<0.05).72h after transfected the inhibitory ratio of negative group was 3.33±0.20%,but the transfect group was 55.26±2.69%,difference among them was significant(P<0.05).The inhibitory ratio were showed increasing tendency in certain time range.4. FCM showed that 48h after Livin targeted microRNA transfect, there was an apoptosis peak in the transfect group SKOV3 cells and the ratio of S phase decline, cell mainly arrested in G0/G1 phase by flow cytometry,and the apoptosis percentage raised up. The apoptosis index of the control and negative group were 1.26±0.19%,5.57±0.21%,but the transfect group was 17.44±1.41%, differences among control, negative and transfect group were significant(P<0.05).The proliferation index of the control and negative group were 76.97±2.80%,69.20±2.26%, but the transfect group was 53.47±2.51%, differences among them were significant (P<0.05). It showed that the proliferation inhibit is significant. Conclusion:1. Designed and synthesised the Livin targeted microRNA and linked to plasmid vector.Transfected into the SKOV3 cells by liposome successfully.2. Livin targeted microRNA transfect can weaken the expression of Livin mRNA. lay a foundation for the application of microRNA to RNAi and gene therapy.3. Livin targeted microRNA transfect human ovarian cancer cell SKOV3,can inhibit the proliferation of human ovarian cancer cell in a time dependent manner.4. Livin targeted microRNA transfect can arrested SKOV3 cell cycle G0/G1 phase, effected cancer cell DNA composition, and induced the apoptosisThe experment is only preliminary research, which definite mechanism but only, pend for studying in a deepgoing way further.
|
PDF Full Text Request