Construction Of Eukaryotic Expression Vector Of SiRNA Specific For HnRNP B₁ Gene And Its Effect Of HnRNPB₁ SiRNA On Lung Cancer Cell A549

ObjectiveAll studies have shown that HnRNP B₁ is over-expression in lung cancer,however,it is unclear how HnRNP B₁ plays a role in pathogenesis. To construct eukaryotic expression vector of siRNA specificfor HnRNP B₁ as a molecular target tool to cleave HnRNP B₁ mRNA in lung cancer cell, to initially investigate the effect of the recombinant plasmid on HnRNP B₁ mRNA expression of A549cells, to provide the experimental evidence and a new tool to further explore the function of HnRNP B₁ gene and the feasibility of its gene therapy.Methods1. Genome sequences of HnRNP B₁ gene was retrieved from Genbank, small interfering RNA (siRNA) was designed according to the Tuschl's principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs (shRNAs) of siRNA for HnRNP B₁ gene. The cDNA was synthesized and inserted into plasmid psiPNA. The recombinant plasmid being eukaryotic expression vector was controlled by the Hl promoter of RNA polymerase III, and identified by the PCR and the sequence analysis. 2. The plasmid was transiently transfected into A549 cells for 24 by Lipofectamine 2000. Expression of HnRNP B₁ mRNA was assayed RT-PCR. The recombinant plasmid had the screening resisting antibiotics.After steadily transfection, their positive mono-cell clones being resistant to G418 are isolated. The vectors were transfected into A549 cells by liposome 2000.The expression level of HnRNP B₁ mRNA and protein were detected by real-time RT-PCR and western blot analysis at different times after transfection.3. The cell proliferations of A549 cells were measured with MTT method after transfection. The changes of biological behavior were monitored by cell growth curves.4. The cell cycles and cell apoptosis of A549 cell were measured with flow cytometry method after transfection.Results1. It was confirmed that the HnRNP B₁-specific siRNA expression vectors were constructed successfully by PCR analysis and DNA sequencing.2. After the recombinant plasmids were transiently transfected into A549 cells by Lipofectamine 2000, the mRNA level of HnRNP B₁ decreased 46%,65%, 58% and 73% on A549 cells.The control groups have no the effects on A549 cells.3. HnRNP B₁ mRNA expression was inhibited 77.69%, 74.37%, 75.65% and 83.04% by A,B,C and D vector at 1 week after the formation of A549 cellular clone.The HnRNP B₁ protein was reduced.While vector E is transfected, the expression level of HnRNPB₁ mRNA and protein were not varied significantly at 1w.4. HnRNPB1 mRNA expression was inhibited 65.8% and 70.3% by vector A, D into A549 cells at the fourth week after the formation of cellular clone, and the HnRNP B₁ protein was decreased. While vector E is transfected, the expression level of HnRNPB₁ mRNA and protein were not varied significantly at 4w.5. HnRNP B₁ mRNA and protein expression couldnot be inhibited by vector A, D into A549 cells at the sixth week after the formation of cellular clone. While vector E is transfected, the expression level of HnRNPB₁ mRNA and protein were not varied significantly at 6w.6. the cell proliferations of A549 cell were inhibited by the A, B, C and D recombinant plasmid after the formation of cellular clone. The cell proliferation and apoptosis were significantly not changed.7. After transfected by vectors, the ratios of Gl phase of A549 cell was improved and the ratios of S phase was descended significantly at 1 week after the formation of cellular clone, and the apoptotic rates induced byA, B,C and D were respectively 17.8±0.45%,19.4±1.35%,16.6±2.91% and 28.5±1.59% after the formation of A549 cellular positive colonies. The ratios of G1 phase A549 cell was reduced and the ratios of S phase was increased gradually at the 4w and the 6w after transfected by vector A, D and the apoptoic rates by A, D were respectively 11.2±1.43%,15.9±2.13% and 6.7%,4.9%.Apoptosis effects were decreased gradually as time longed. Inhibition effects were sustained for at least four weeks after the formation cellular clone.Conclusions(1) The expression of HnRNPB₁ gene in A549 cells can specifically inhibited efficiently and was sustained for at least four weeks after the formation of cellular clone by HnRNPB₁-specific siRNA expression vector.(2) HnRNP B₁ -specific siRNA expression vector could adjust the cell cycle and inhibit the cancer cell proliferation and promote apoptosis. So it might be one of the ideal strategies for gene therapy of lung cancer.