The Effection Of C-fes ShRNA On The Proliferation,differentiation And Apoptosis Of HL-60 Cells

The leukemia is one of the most common heterogeneity hematopoietic malignancies, and the research has been into molecular biology from colonia medicine and cell biology as the development of the technology of molecular biology. Now cell differentiation can be seen as a much more predominant theory. It presumed that genetic mutation could prevent terminal differentiation of cells and cause cumulate of the immature cells to induce canceration.c-fes belongs to protein-tyrosine kinase family which implicated in the regulation of the generation and fuction of the common hematopoietic cells, c-fes mRNA protein levels and kinase activity increase upon induction of granulocyte cell differentiation. During the process of the cell growth, proto-oncogene is activated into oncogene through gene translocation,gene amplification,insertion and point mutation, then cell will have immortality and malignancy proliferation .Recent research has shown that c-fes expressed in many kinds of neoplastic disease, the c-fes proto-oncogene is expressed at high levels in the terminal stages of granulocytic differentiation. In addition, c-fes has been implicated in apoptosis pathways. It was observed that inhibition c-fes by incubating the HL-60 cells with a specific c-fes antisense oligodeoxynucleotide, the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.RNA interference (RNAi) is the most effective gene silencing technology, which can specifically inhibit the transcription of target genes, and in turn reduce the expression level and function of the corresponding protein. Therefore, using RNAi technology, we choose c-fes as the target gene. We transiently transfected c-fes shRNA into HL-60 cells by lipofectamine reagent. After observing the proliferation,differentiation and apoptosis, the experiment advanced to investigate the effect of combination use arsenious oxide with siRNA.Methods:1 Using in vitro cell culture technique, we transfected c-fes shRNA into HL-60 cells by lipofectamine reagent.The cells were divided into four groups:①blank control group;②transfection reagent group of lipofectamineTM2000;③plasmid KB group;③pGenesil1.1c-fes shRNA group. In order to detect the interference effect, we observe the expression of c-fes protein in HL-60 cells using FCM.2 The four groups of HL-60 cells were cocultured with different concentrations of As2O3 (0.1μmol/L,0.5μmol/L,1.0μmol/L,2.0μmol/L). By using the RQ-PCR technology,we detected the changes of c-fes mRNA level in different time interval(0h,24h,48,72h). The following detection regarding the proliferation,differentiation and apoptosis was observed as above-mentioned. Cells were stained with Wright-Giemsa for general morphology. The inhibition rate of HL-60 cells was examined by methyl-thiazolyl-tetrazolium(MTT) test . The differentiation of HL-60 cells were examined by NBT reduction reaction. Annexin-V/FITC staining was used to evaluated the apoptosis rate.Results:1 c-fes shRNA inhibited c-fes protein expression: FCM results showed that c-fes protein could be significantly reduced by c-fes shRNA. The reduction appeared from 12h after interference, and the maximum reduction rate was 76.36%, which appeared at 48h. However, transient transfection of c-fes shRNA could only present for a limited period, and the c-fes protein had gone up at 72h after transfection.2 The cell morphology appeared to apoptosis earlier after treated with low concentrations of As2O3(0.1μmol/L,0.5μmol/L,1.0μmol/L,2.0μmol/L)in c-fes shRNA group, it is peaked up 72 hours after treated with 2.0μmol/L As2O3.The nucleolus almost disappearance, karyoplasmic ratio diminished, karyomorphism irregular, more vacuole in intracytoplasm, the effect was in a time-dependent manner.3 With the growing of As2O3 concentration and time, RQ-PCR analysis of CON group, LIP group and KB Group showed that there was a gradually increased tendency of c-fes mRNA expression levels ,but no significant differences were found among the three groups (P>0.05). The c-fes mRNA expression levels of c-fes shRNA group did not elevated after the treatment with low concentration (0.1μmol/L, 0.5μmol/L) As2O3 in different time interval (0h, 24h, 48h, 72h). The expression level of c-fes mRNA increased slightly after 72h, but still lower than the CON group, LIP group and KB of the same period.4 There were no significant difference among the CON group, LIP group and KB group regarding the proliferation,differentiation and apoptosis(P>0.05). 5 Treated with the different concentrations of As2O3(0.1μmol/L,0.5μmol/L,1.0μmol/L,2.0μmol/L), the inhibition rate increased as the concentration raised in the CON group, LIP group and KB group. The inhibition rate in c-fes shRNA group was higher than the concurrent group as does and time extend(P<0.05).6 Treated with the concentrations of As2O3(0.1μmol/L,0.5μmol/L), HL-60 showed significantly differentiated and in dose-dependent and time-dependent manner in the CON group, LIP group and KB group. The differentiation rate of HL-60 cells in c-fes group had no significant difference with the untreated group 12 hours after cocultured with As2O3 by 0.1μmol/L,0.5μmol/L(P>0.05), 24 hours and 48 hours descended, and were lowest at 48 hours(P<0.05). With the concentrations of As2O3(1.0μmol/L,2.0μmol/L), the differentiation rate of HL-60 cells kept in a low level, with a maximum reduction at 24 hours.7 Flow cytometry (FCM) with the Annexin-V/FITC staining displayed a promoted apoptosis with concentrations of As2O3(0.1μmol/L,0.5μmol/L,1.0μmol/L,2.0μmol/L)after interference The apoptosis rate in c-fes shRNA group was higher than the concurrent group as does and time extend(P<0.05),with 41.36% at 24 hours by 2.0μmol/L.Conclusion:1 RNA interference targeting c-fes shRNA reduced the c-fes expression successfully. In the meantime, the growth status and differentiation of HL-60 cells were significantly inhibited, and HL-60 cells were induced apoptosis.2 Cocultured with different concentrations of As2O3 after RNA interference, the growth status and differentiation of HL-60 cells were more severe inhibited, and HL-60 cells were induced apoptosis more effective, what's more, the combination use can decrease the dose of As2O3.