Effect And Mechanism Of RNAi On Expression Of Bmi-1in Human Colorectal Carcinoma LOVO Cells

Objectives1. To select the most effective small interfering RNA (siRNA) so as tointerfere the expression of Bmi-1gene.2. To evaluate the effect of siRNA on the genetic expression of Bmi-1and its possible downstream gene of P16INK4a in human colorectalcarcinoma LOVO cells.3. To observe the effect of siRNA on proliferation and neoplasminvasiveness in human colorectal carcinoma LOVO cells.Methods1. Fluorescein-labeled double-stranded RNA (dsRNA) oligomer wastransfected into the human colorectal carcinoma LOVO cells bylipofectamine2000(invitrogen, USA) according to the manufacture’sinstructions. After6hs, the transfection efficacy was observed by fluorescence microscope,so as to sifted out the optimal conditions forthe siRNA.2. Four siRNA groups with different Bmi-1siRNA sequence namedsiRNA1, siRNA2, siRNA3, siRNA4(the negative control group)were transfected into the cells cultured in the6-well plates by usinglipofectamine2000respectively. After48hs transfection, the RNA wasextracted out. Real-time PCR was used to detect the Bmi-1mRNAexpression and filtered out the strongest Bmi-1siRNA sequence.3. The protein expression of Bmi-1and its downstream gene P16INK4awere detected by Western Blot (Protein Blot). Experiments wererandomly divided into3groups: the Bmi-1siRNA group(transfectedwith specific Bmi-1siRNA sequence, the siRNA1sequence)，thenegative control group (transfected without siRNA target sequence),the blank control group (transfected without siRNA sequence). After24hs, the siRNA was respectively transfected into the cells cultured inthe6-well plates by using lipofectamine2000, and protein wasextracted after60hs. The protein expression of Bmi-1and itsdownstream gene P16INK4a were then detected by Western Blot.4. The proliferation of human colorectal carcinoma LOVO cells wasindirectly revealed by MTT. Experiments were randomly divided into3groups: the Bmi-1siRNA group (transfected with specific Bmi-1siRNA sequence, the siRNA1sequence), the negative control group (transfected without siRNA target sequence), the blank controlgroup(transfected without siRNA sequence). After24hs, the siRNAwas respectively transfected into the cells cultured in the96-wellplates by using lipofectamine2000, then the cells was continued tocultured at37°C in5%CO2incubator, at12hs,24hs,48hs,60hspost-transfection, then the OD value of each well was detected bymicroplate reader at550nm wavelength, the growth curve wasrespectively drawn for the Bmi-1siRNA group，the negative controlgroup and the blank control group.5. The invasiveness of human colorectal carcinoma LOVO cells wasindirectly revealed by transwell invasion assay. the transwell chamberswere put into24-well culture plates,coated with5μl Matrigel BasementMembrane Matrix. Experiments were randomly divided into3groups:the Bmi-1siRNA group(transfected with specific Bmi-1siRNAsequence,the siRNA1sequence)，the negative control group(transfectedwith no siRNA target sequence),the blank control group(transfectedwith no siRNA sequence). After24hs, the siRNA was respectivelytransfected into the cells cultured in the6-well plates by lipofectamine2000. After48hs,the cells was digested with0.25%trypsin. The cellswith the density of5×105cells/ml was inoculated in pre-preparationroom on the transwell chamber, the transwell chambers were removedafter48hs, the cells was stained and counted under a microscope. Results1. Transfection efficiency of fluorescent label observedCells were observed under the inverted microscope,at the same field ofvision, cells with hair weak or bright green fluorescent were consideredto be transfected successfully, and transfection efficiency was about70%.It illuminated that the chemical synthesis of siRNA mediated bylipofectamine2000had the higher transfection efficiency.2. The mRNA expression was calculated by2–ΔΔCt, the results showedthat the Bmi-1mRNA expression in siRNA1group was lower thanother groups. The inhibiting rate of siRNA1group was75%, and therewas no significant difference between the siRNA2, siRNA3,blankcontrol group (P>0.05). It illuminated that the Bmi-1siRNA1caneffectively interfere the mRNA expression of Bmi-1gene.3. The Bmi-1and P16INK4a protein expression was represented bygray-scale ratio. the results showed that Bmi-1/β-actine proteinexpression in siRNA group,negative control group,blank control groupwere0.532±0.066、1.152±0.038、1.137±0.042, respectively.Itilluminated that Bmi-1protein expression in Bmi-1siRNA group waslower than other groups (P <0.05).And there was no significantdifference between the negative control group and the blank controlgroup(P>0.05).And the P16INK4a/β-actine protein expression insiRNA group,negative control group,blank control group were 2.186±0.137,1.772±0.088,1.717±0.099, respectively.The result showedthat P16INK4a protein expression in Bmi-1siRNA group was higherthan other groups (P <0.05). And there was no significant differencebetween the negative control group and the blank control group(P>0.05).It meaned that after the transfection,the Bmi-1protein expressiondecreased,but the P16INK4a protein expression increased.4. After transfection,the OD value detected by microplate reader at550nm wavelength at12hs,24hs,48hs,60hs in the Bmi-1siRNAgroup were0.20±0.019(12hs),0.37±0.036(24hs),0.48±0.027(48hs)and0.67±0.051(60hs), respectively, all of which was significantlylower than those in the control group and the blank group,(p<0.05).From the first24hs after post-transfection, the cells in Bmi-1siRNAgroup grew more slowly than in the control group and the blank group.Compared with the negative control group and blank control group, theproliferation of cells in Bmi-1siRNA group decreased,the differencewas statistically significant (P <0.05).It illustrated that the proliferationof the cells transfected with Bmi-1siRNA was markedlydiminished.While the proliferation of the cells transfected with thenon-specific siRNA and the cells in the blank control group was nosignificant difference (P>0.05). It showed that the RNAi targeting onBmi-1gene could inhibit the proliferation of the human colorectalcarcinoma LOVO cells. 5. The invasiveness of human colorectal carcinoma LOVO cells wasindirectly revealed by transwell invasion assay.At48hspost-transfection, detected the invasiveness in the Bmi-1siRNAgroup，negative control group and blank control group. The neoplasminvasiveness was detected by the transwell invasion assay,the cellswhich went through the martrigel membrane in the Bmi-1siRNAgroup was181.33±8.14, which was significantly fewer than that in thecontrol group（307±12.12）and the blank group（300.33±8.02）,(p<0.05).It showed that the RNAi targeting on Bmi-1gene couldinhibit the invasiveness of the human colorectal carcinoma LOVOcells.ConclusionsChemical synthesis siRNA targeting on Bmi-1gene transfected tocolorectal carcinoma LOVO cells can effectively inhibit the Bmi-1mRNA and protein expression and increase its downstream geneP16INK4a protein expression.It showed that the RNAi targeting onBmi-1gene could inhibit the invasiveness and the proliferation of thehuman colorectal carcinoma LOVO cells by up-regulating theexpression of P16INK4a protein.