The Analysis Of Expression And Correlation Of STAT3,CyclinD1,Bcl-2,BAX And Caspase-9 Protein In RNA Interference Silencing STAT3 Gene Lung Adenocarcinoma A549 Cells

Objective:To investigate the expression of STAT3,CyclinD1,Bcl-2,BAX and Caspase-9 in RNA interference silencing STAT3 gene lung adenocarcinoma A549 cells. Methods : (1)RNA interference in lung adenocarcinoma A549 cells silence STAT3 gene expression: Search STAT3mRNA on the whole genome sequence from CNKI, using BLAST homology analysis software to the entire gene sequence STAT3mRNA as a template, gene-coding region from the initiation codon looking for with the design characteristics of the downstream target sequence in accordance with the principle of siRNA design, three design STAT3-specific shRNA expression above the gene sequence, synthesized by chemical synthesis of three double-stranded siRNA were si-h-STAT3-1, si-h-STAT3-2, si-h-STAT3-3, and the establishment of si-h-GAPDH positive control group, NControl negative control group, NControl fluorescence in the control group and blank control group, the use of liposome-mediated transfection method of A549 cells transfected with 16h in the fluorescence microscope to observe the situation transfection. (2)conventional PCR detection of the efficiency of RNA interference: 24h in the collection of cells transfected with TRIZOL extraction of RNA, reverse transcription into cDNA, in order to target genes for the purpose of STAT3 gene, housekeeping genesβ-actin as reference gene, amplification primers were designed to PCR products by agarose gel electrophoresis, the purpose of testing the efficiency of bands and interference. (3)Cell disruption and protein extration :300ul of cells lysate was added in each bottle of cells.Then bottles were put on the ice to oscillate thoroughly for 30minutes and cells were centrifuged to obtain supernatant protein by the high speed refrigerated centrifuge.(4)The protein expression of STAT3,CyclinD1,Bcl-2,BAX and Caspase-9 was detected by Western blot. (5)Image analyxis:The Western-blot results were scaned and transmitted into computer,and Image–pro Plus image analysis software system was used to analyze the protein band,the band IOD was used to represent protein expression quantity.So the difference of protein level in two group cells and the correlation between STAT3 and CyclinD1, or Bcl-2, or BAX ,or Caspase-9 in lung adenocarinoma cell A549 was analyzed. Results:(1)Successful design and chemical synthesis of siRNA, transfected into A549 cells successfully;(2)The expression of STAT3,CyclinD1, Bcl-2 decreased significantly(P<0.05)and increased significantly. (3)In lung adenocarinoma cell A549,it was also found that the expression of CyclinD1, Bcl-2 was in a positive correlation with STAT3 (P<0.05); BAX in a negative correlation with STAT3;the expression of Caspase-9 doesn't have any correlation with STAT3 Conclusions: (1) Successful design and chemical synthesis of siRNA, transfected into A549 cells successfully.RNAi of STAT3 can effectively inhibit STAT3 expression,suppress the proliferation of cancer cells,and promote apoptosis.The RNA interfering technique targeting of STAT3 maybe can provide a new pathway for the gene therapy of lung cancer. (2) STAT3-targeting siRNA can significantiy inhibit cyclinD1,Bcl-2 expression.the expression of CyclinD1, Bcl-2 was in a positive correlation with STAT3. CyclinD1, Bcl-2 maybe the downstream gene of STAT3,they have synergistic effect on suppressing the growth of A549 cells and promoting apoptosis.(3) STAT3-targeting siRNA can significantly increase BAX,Caspase-9 expression. the expression of BAX was in a negative correlation with STAT3. The protein level of Caspase-9 doesn't have any correlations with STAT3 in lung adenocarinoma cell A549.