RNA Interference Silencing STAT3 Gene In Lung Adenocarcinoma A549 Cell Growth Inhibition Study

Objective: To investigate the RNAi technology in human lung cancer cell lines A549 blocking STAT3 gene effect, the application of fluorescence quantitative PCR and Western-blot (Western blot method) before and after the detection of interference of the expression of STAT3 to examine STAT3 in the pathogenesis of lung cancer role in the treatment of lung cancer to find a new target. Methods: (1) chemical synthesis of siRNA and transfected A549 cells:①A549 lung cancer cells as target cells, STAT3 gene as a target gene, from STAT3mRNA search CNKI gene sequence, BLAST homology analysis software to STAT3mRNA gene sequence as a template, gene-coding region from start codon downstream to find with the design characteristics of the target sequence, in accordance with the principle of siRNA design, the design of three STAT3-specific shRNA expression of the gene sequence, synthesized by chemical synthesis of three double-stranded siRNA, named for sihSTAT3-1, sihSTAT3-2, sihSTAT3-3. SiRNA1 group will be divided into experimental, siRNA2 Group, siRNA3 Group, sihGAPDH positive control group, NControl negative A549 cells in the control group and blank control group.②the use of liposome-mediated method, synthetic siRNA transfected cells in each group, 16 hours after transfection fluorescence microscope, the observed transfection efficiency. (2) fluorescence quantitative PCR assay after RNA interference inhibition rate STAT3mRNA:①to target genes for the purpose of STAT3 gene, housekeeping genesβ-actin as reference gene.Transfected cells were collected after 24h, using TRIZOL extraction RNA, reverse transcription into cDNA, agarose gel electrophoresis to detect the purpose of strip.②in transfected 24h, 48h, 72h, respectively, the collection of cells, by reverse transcription polymerase chain reaction (RT-PCR) and STAT3 target gene was the housekeeper geneβ-actin product of the cDNA and as a standard gradient dilution, the use of SYBR Green I quantitative PCR, come standard curve, curve and amplification curves of dissolution.③Comparison of the relative quantitative method STAT3mRNA domain relative value of the expression of multiple inhibitory rate reached to carry out statistical analysis. (3) the application of Western-blot assay interference STAT3 protein expression after the inhibition rate:①to select a more efficient siRNA interference in each group of Stat3 protein in Western-blot detection kit using BCA total protein concentration of samples, polypropylene gel electrophoresis (SDS-PAGE), Coomassie brilliant blue staining of protein bands and electricity transfer closed PVDF membrane, immune response (by adding a second anti-1and anti-2), chemiluminescence bands display purposes.②Western-blot results will be sent to the computer scanning, using Image-Proplus image analysis software system for analysis of protein bands to protein bands of gray-scale value represents the amount of protein expression, usingβ-actin band gray-scale value correction, the band reached the relative gray value. The results of statistical analysis. Results: (1) the success of Design and Synthesis of siRNA, and successfully transfected into A549 lung cancer cells. (2) fluorescence quantitative PCR results showed that transfection 24h, 48h, 72h after the interference group and blank group STAT3 gene expression was significantly reduced (P<0.05), siRNA- STAT3 to the STAT3 gene expression were inhibited, which 0-48 hours showed a marked increase in inhibition rate trends, within 48 hours to 72 hours inhibited the rate of change was not obvious.Corresponding to three different sites of siRNA-STAT3 expression inhibited STAT3mRNA was no significant difference (P>0.05); negative control and blank control the expression of STAT3 was no significant difference (P>0.05). (2) Western blot showed that: before and after transfection the level of Stat3 protein expression were inhibited, inhibition rate of 76.92%, with significant differences (P<0.05), reference gene in each group no significant differences in expression. Conclusion: (1) of the experimental use of chemical synthesis successfully synthesized STAT3 gene for siRNA, the success of the establishment of STAT3 gene RNA interference technology.(2) the synthesis of siRNA transfected into A549 cells by fluorescence quantitative PCR detection of gene expression levels of STAT3, and STAT3 through the Western blot detection of protein expression showed that: STAT3mRNA after transfection and protein expression were inhibited in both, of reference gene expression have little effect on the synthesis of siRNA of STAT3 has an efficient and specific silencing effect. (3) The experimental use of RNAi technology, effectively inhibited lung adenocarcinoma A549 cells, the expression of STAT3 gene, induced apoptosis. To further explore the STAT3 gene in lung cancer occurrence and development of the important role of the foundation, as well as in the treatment of lung cancer to find the target more effectively provide the right direction.