Gene Profiling After Knocking-down Expression Of Nucleostemin In HL-60Cells Using DNA Microarray

Objective:Construct a lentiviral expression vector for RNA interference(RNAi) of nucleostemin(NS) and detect its interference efficiency in HL-60leukemia cell line. After NS expression was inhibited, DNA microarray and subsequent bioinformatic analysis were proceeded to identify the changes of the gene expression profiles, which made the foundation for further exploring the specific mechanism of NS p53-independent signal pathway.Methods:(1) Effective RNAi target sequence was designed and screened towards NS gene sequence. Single-stranded DNA oligo containing RNAi sequence, Loop circle, Age I/EcoR I enzyme cutting site, and termination signal sequence was synthesized and annealed to double-stranded DNA, which was subsequently connected to the Age I/EcoR I-digested GV248lentiviral vector with GFP and puromycin resistance marker to form a recombinant lentiviral vector. Then it was transformed into competent E.coli cells, and the positive clones were confirmed by colony PCR and sequencing. The recombinant vector and two lentivirus packing plasmids (pHelperl.Oand pHelper2.0) were co-transfected into293T cells to obtain packaged lentivirus particles. Viral titer was then determined. Subsequently, the recombinant lentivirus were transfected into human leukemia HL-60cell line, then the transfection efficiency was observed under inverted fluorescence microscope, and the inhibition rate was detected by real-time PCR. (2) HL-60cells transfected with NS-siRNA lentiviral vector were set as the experimental group, while the cells transfected with the negative control lentiviral vector were set as the control group.Total RNA from each sample was amplified and transcribed into Cy3-labeled cRNA. The labeled cRNAs were hybridized onto the Whole Human Genome Oligo Microarray. After having washed the slides, the arrays were scanned and the data were extracted. After raw data normalization and filtering, differentially expressed genes with statistical significance were identified through Fold Change≥2or≤0.5. Real-time PCR was then proceeded to demonstrate the reliability of the gene chip data. GO analysis and Pathway analysis were applied to determine the roles of these differentially expressed genes played in these or GO terms or biological pathways..Results:(1) DNA sequencing showed that RNAi sequence was inserted into the GV248vector and the recombinant lentiviral vector was constructed successfully. The recombinant lentivirus harvested from293T cells had a titer of4×108TU/ml. Observation under inverted fluorescence microscope showed the lentiviral transfection efficiency was higher than80%in HL-60cells. Real-time PCR showed NS mRNA expression level of experimental group was significantly lower than negative control group(P<0.05). The interference efficiency52.3%.(2) The purity, integrity and quality of RNA were met the requirements for microarray hybridization.1953differentialy expressed genes were identified through Fold Change≥2or≤0.5, in which943genes were up-regulated and1010genes were down-regulated. Real-time PCR demonstrated the reliability of the data.These differentially expressed genes may involve in great kinds of functions and signal pathways through GO analysis and Pathway analysis, in which the association with protein processing in endoplasmic reticulum(ER) is the most significant.Conclusion:(1) The lentiviral vector for RNAi of NS has been successfully constructed with high titer, and it could inhibit NS mRNA expression effectively in three human leukemia cell lines HL-60, NB4and K562, which laid the foundation for follow-up studies.(2) After NS was knocked down, the mechanism of biological characteristics changes in HL-60cells was complex, it may involve in many kinds of functional proteins, metabolism and signal pathways. Pathway analysis of the up-regulated genes indicated that the association with protein processing in endoplasmic reticulum is the most significant, and pathway anaysis of the down-regulated genes showed that the change of metallothioneins is the most significant. This indicated that HL-60cells apoptosis after knocking-down NS expression may associated with ER stress induced apoptosis, and the weak proliferation capacity tumorigenic ability may associated with down-regulation of metallothioneins. But it still needs follow-up experiments to confirm. This study made the foundation for further explore the specific mechanism of NS p53-independent pathway in HL-60cells.