Construction Of Eukaryotic Expression Vector Of SiRNA Specific For STAT1 Gene And Its Influence On Expression Of STAT1 Protein In 16HBE Cells

Objective: Signal transducer and activator of transcription1 (STAT1) is a transcription factor that founded firstly in STAT family,which Participate in signal transduction of Interferon(IFN) and regulate innate immunity.A number of cytokines could activate STAT1 through JAK-STAT path like gamma-Interferon (IFN-γ) et al,which induced target gene to express and participated the signal transduction process mediated by cytokines, thereby led to inflammation and hyperreactivity of airway.The expresstion of STAT1 was up-regulated in bronchus epithelial cells of asthma.we constructed eukaryotic expression vector of shRNA specific for STAT1 gene and transfected it into human airway epithelial cells (16HBE) stimulated by IFN-γand detected its interference activity, which will establish a favourable foundation for further study of gene therapy of the inflammation disease like asthma et al. Method: 1. Construction of Eukaryotic Expression Vector of siRNA Specific for STAT1 Gene: We retrieved gene sequences of two kinds variant for STAT1 gene from genebank and selected their common sequences.Small interfering RNA ( siRNA) was designed according to the Tuschl's principle,two fragments were chosen as STAT1 interference fragment finally,another fragment was designed by array random of any fragment as negative control,then they were converted into cDNA coding small hairpin RNA (shRNA) of STAT1 gene.The cDNA was synthesized into double strands and inserted into pGenesil-1.1 eukaryotic expression vector with kanamycin resistance and enhanced green fluorescent protein, and then the recombinant plasmid was transformed into strain DH5α, amplificated and extracted.Eventually, the plasmid identified by restriction enzyme SacI and Gel electrophoresis was used for sequencing. Synthetized recombinant plasmid was named respectively pGSTAT1-1,pGSTAT1-2, and negative control pG-HK. 2. The influence of recombinant plasmid on expression of STAT1 protein in 16HBE cell: We adopted recombinant plasmid and Lipofectamine 2000 with the proportion of 1﹕1 to 1﹕5 to transfect into 16HBE cells. In addition to the blank control group, there were six groups in total with three wells in each.Then,observated the expression of fluorescence in 24, 48, 72 hours after transfection and identified the transfection efficiency in different time and ratio; 16HBE cells that had been dealed by IFN-γwas divided into four groups with three wells in each,the total protein of cells was extracted at 0 min,30 min,12 hours,24 hours after Stimulation,and the relative amount of STAT1 protein was detected through Western blotting method.We selected the proportion that plasmid and Lipofectamine 2000 were 1μg:2μl and divided 16HBE cells which had been stimulated by IFN-γfor 30 minutes into blank grup,pGSTAT1-1 group,pGSTAT1-2 group,and pG-HK group.Then, recombinant plasmid were transfected, the total protein of cells was extracted arter 48 hours,we adopted Western blotting method to detect relative amount of STAT1 protein in every group to study the influence of recombinant plasmid on expression of STAT1 protein in 16HBE cell.We did statistical analysis by One-way ANOVA and randomized block design in SPSS13.0 software, When P value was under 0.05,the difference had statistical significance. Result: All the results of digestion and sequencing showed that two siRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into 16HBE cell successfully.That the proportions of recombinant plasmid and Lipofectamine 2000 were 1μg:2μl and the time of 48 hours after transfection were the more effective,which amounted to 58.21%(P < 0.01); Detected by Western blotting, the expression levels of STAT1 protein after stimulation of IFN-γin 30min group were higher than 24h group (P=0.01), so were 30minutes and another three groups (P=0.000). But there wasn't statistic difference between 30minutes group and 12 hours group after Stimulation of IFN-γ(P=0.531);Through Western blotting detection, the transfection of recombinant plasmid could reduce the expression level of STAT1 protein. The relative content of STAT1 protein in pGSTAT1-2 group was significantly lower than the blank group and pG-HK group at 48 hours after transfection(P=0.01), and so was pGSTAT1-1 group(P<0.05).The rate of inhibition of pGSTAT1-2 was 57.62% and the pGSTAT1-1 was 30.64%. At the same time, There was statistic difference between pGSTAT1-2 group and pGSTAT1-1 group(P =0.04). Conclusion: 1. Two siRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into human airway epithelial cells successfully. 2. to stimulate airway epithelial cells with IFN-γcould make expression of STAT1 protein increased and reached the peak in 30 minutes and last until 24 hours. 3. STAT1-specific siRNA eukaryotic expression vector could inhibit expression of STAT1 protein in 16HBE cells,however, the difference of target site caused different efficiency for constructed recombinants. 4. This method was convenient and trustworthy, making STAT1 as a target in the RNA interference technology is expected to become a new method of gene therapy for most of diseases with airway inflammation.