The Effects Of EIF3d Downregulation On Human Non-small Cell Lung Cancer Cells Biological Behavior

Objective Construct eIF3d-shRNA lentivirus vector and Detect the effects of LV-eIF3d-sh RN A on A549 and 95D cell lines.Methods The siRNA sequence and contrast sequence were searched for the target gene,and then the sequences were synthesized by Shanghai Huangli biotech company.Enzyme sites were added to the end of the sequence to synthesize DNA oligo which was a shRNA expression cassette containing interference sequence.Ecor I and Bam HI were used for enzyme digestion to make p GP vector straightening to form asymmetric sticky ends.To construct the framework vector,we inserted shRNA expression cassettes into p GP vector to transform escherichia coli competent cells and then we sequenced the positive clone by PCR.Assembled vrius particles and shRNA expression plasmid were transfected into293T cells to produce assembled lentivirus particles.Two days after transfection,the medium was collected and filtered by 0.45?m screen.Then we centrifuged the collected medium.For cell infection,A549 and 95D cells were seeded in a volume of 2 ml at a density of 5×10~4 cells/well in 6-well plates and transduced with the constructed lentiviruses containing eIF3d shRNA[sheIF3d(S1)/sheIF3d(S2)]and non-silencing shRNA(sh Con)at a multiplicity of infection(MOI)of 20 and 8,respectively.The infection efficiency was observed after 96 h through a fluorescence microscope for the green fluorescence protein expression.Make 2 groups of the cultured A549 and 95D cells as follows:transfecting LV-eIF3d-shRNA plasmid;transfecting empty LV-shRNA plasmid.After transfection,the expression of eeIF3d-m RN A was determined by q PCR and the expression of eeIF3d protein was determined by Western blotting.MTT assay was performed to determine the proliferation of NSCLC cells.Colony formation assay was performed to determine the colonyforming ability of NSCLC cells.Transwell experiment and scratch test were performed to determine the migration ability of NSCLC cells.The cell cycle distribution was analyzed by flow cytometry using propidium iodide(PI)staining.Intracellular signaling molecules were detected using a Path Scan~?intracellular signaling array kit(Cell Signaling Technology,#7323)according to the manufacturer's procedure.Results We identified clone2 of S1 and clone3/4 as positive clones after shRNA sequence was synthesized.Positive clones were sequenced and the results showed that no mutation and insertion happened in recombinant plasmid.Transfection efficiency of recombinant plasmid was examined through the fluorescence GFP as higher than 90%.The tittering results were as follows:sh Con:7.0×10~6Tu/ml;sheIF3d(S1):1.3×10~6Tu/ml;sheIF3d(S2):7.0×10~6Tu/ml.Recombinant plasmid was transfected into all groups of cells successfully.The expression of eIF3d-m RNA and eIF3d protein was decreased in the LV-eIF3d-sh RN A group.The ability of proliferation and colony formation was decreased in LV-eIF3d-shRNA group compared to NC group.The results of Transwell experiment and scratch test indicated that the migration ability of A549 and 95D cells was declined after knockdown of eIF3d.The cell cycle distribution of A549 cells was analyzed using flow cytometer and the results showed that eIF3d knockdown arrested A549 cells at the G2/M phase.To further illuminate the molecular mechanisms by which eIF3d affects NSCLC cell growth,a Path Scan~?intracellular signaling array kit was used to detect the changes of signaling molecules in 95D cells before and after eIF3d knockdown.The data indicated that eIF3d knockdown could significantly inhibit growth of NSCLC cells via blockade of AKT,HSP27 and SAPK/JNK activations.Conclusion The LV-eIF3d-shRNA was constructed successfully and could be used in the following experiments.Down regulation of eIF3d could reduce the proliferation and migration ability of NSCLC cells.Knockdown of eIF3d could arrest A549 cells at the G2/M phase and block the activations of AKT,HSP27 and SAPK/JNK.