Biocharacteristic Effects Of TUBB2C Gene Silencing By Small Interfering RNA On SKM-1 Cell Line

All adult patients, presenting at 24 hospitals in Shanghai,who had initial clinical findings of a hematopoietic abnormality and in accordance with the WHO diagnostic criteria of MDS with no history of treatment were candidates of our study.435 cases were concluded in the nested case-control study cohort we established with close follow-up. Until the end of the follow up,52 patients were lost(12%). In the rest of 383 patients we had contact with,41 patients transformed to acute leukemia(case group),the median transformation time was four months, the rate was 9.4%; the other 358 patients did not progress to AML(control group). Three cases were chosen from the case group, examing the gene expression profile of their bone marrow species when diagnosed and progressed to AML, forming the self-control study.At the same time, we established the case-control study by picking cases in a 1:1 ratio from the control group, matched in the transformation risk factors and also examining their bone marrow species. Through a series of analysis, we found six genes, forming a gene group that could distinguish the two groups. Arranged by the degree of expression difference were TUBB, PSMD1, SLC7A5, ATG3, TUBB2C, TIMM10. Among these six genes, only ATG3 were down-regulated. Our group members have already finished the study of TUBB, PSMD1, ATG3. In my study, Lipofetamine(?) RNAi MAX were used to induce TUBB2C specific siRNAs into SKM-1 cell line, then we observe the effects on biocharacteristics of SKM-1 cell line after the gene silencing of TUBB2C.Part Ⅰ. Study on gene silencing of TUBB2C by gene specific small interfering RNAs in SKM-lcell lineObject:To investigate the transfection effects and gene silencing effects of TUBB2C by RNA interference on SKM-1 cell line.Methods:TUBB2C specific siRNAs (TUB2C-siRNAs) were synthesized chemically. Three pairs of TUBB2C-siRNAs were transfected respectively with LipofectamineTM RNAi MAX for the delivery into the SKM-1 cell line, which was established from an AML patient transformed from MDS. Harvest the cells after 24 hours of transfection and observe the fluorescence. Examine the transfection efficiency by flow cytometry and figure out the gene silencing efficiency through Realtime PCR.Results:After the transfection with liposome, cells with fluorescence could be observed and stable transfection efficiency were performed (stable at aroud 93%). Further test were performed by Realtime PCR, observing that the gene expression of TUBB2C were decreased, and by the TUBB2C-siRNAl transfection, the expression level was reduced to 30% of the blank control group (P<0.05).Conclusion:A stable and preferable gene silencing efficiency could be achieved by transfecting TUBB2C specific siRNA1 with Lipofectamine(?) RNAi MAX.Part Ⅱ. Effects on biocharacteristic changes on SKM-1 cell line after the down-regulation of TUBB2CObject:To investigate the biocharacteristic changes after transfecting TUBB2C specific siRNA into SKM-1 cell line with cationic liposome.Methods:Chose the TUBB2C-siRNA1 with high gene silencing efficiency we got from Part Ⅰ for further study. Observe biocharacteristic changes after the RNAi on SKM-1 cell line among the TUBB2C-siRNA1 group、only liposome transfection group and normal SKM-1 cell group through examing their morphology, cell viability、cell cycle、apoptosis and the in vivo tumorigenic ability in Nod/scid mice model.Results:After transfection at the same period, compared with the other two groups, the TUBB2C-siRNA1 group had a lower proportion of mitotic phase cells and more large cells; with the help of electron microscope, it was easy to see the presence of nuclear condensation. The cell line was inhibited in proliferation and viability. Cells in this group had lower proportion of S phase and higher G0/G1 phase proportion, which suggest that these cells were obstucted at G1/S restriction piont. The cell apoptosis analysis by the FCM demonstrated that there was an increase in early apoptosis proportion. When turned to the Nod/scid mice model, the TUBB2C-siRNA1 group had a significantly lower tumor formation rate and it took a longer time for the tumor formation.Conclusion:After the TUBB2C gene silencing, the SKM-1 cell line was inhibited in proliferation and cell cycle was obstructed in G0/G1 phase, with a higher rate of early apoptosis. The observation on Nod/scid model, having a significant lower tumor formation rate and longer tumor formation time suggests the decrease of tumor malignancy after transfection and TUBB2C may play a role in cell proliferation and apoptosis during MDS progressing to AML.