The Effect Of SiRNA Targeting EGFR On Human Lens Epithelial Cells

Objective:We designed the siRNA of epidermal growth factor receptor(EGFR)and transfected it into the Human lens epithelial cells(HLEs)to knockdown the expression of EGFR to evaluate the effect of targeting si-EGFR on the proliferation of HLEs.Next,we selected the most adaptable concentration of recombinant EGF for stimulating the HLEs treated by si-EGFR to further test the suppressive effect of small interfering RNA(siRNA)and study the inhibition mechanisms of si-EGFR on HLEs.Methods:1.Design and chemically synthesize siRNA targeting EGFR according to the principle of design of siRNA,and constructe the expression vector of si-EGFR and name as pSilencer2.1-shRNA-EGFR.Then prepare to make pSilencer2.1-U6neo free-running vector as negative control,transfect respectively the two plasmid into HLEs by Lipofeetamine 2000.Locate EGFR by immunofluorescence technique.The expression levels of EGFR mRNA and protein were detected respectively by real time PCR and Western blot.Cell proliferation was determined by MTS and cell growth curve,and cell growth state was observed by flow cytometry.2.Use different concentration recombinant EGF(0.1?1?10ng/mL)to stimulate HLEs to observe the morphologic change and growth condition of HLEs,at the moment of adding recombinant EGF,measure OD490 and OD570 continuous 4d by MTS and count the number of cells to draw the cell growth curve to detect cell proliferation activity.3.Selecte the most adaptable concentration of recombinant EGF for stimulating the HLEs transfected with the two plasmids respectively,measure OD490 and OD570 for 4d by MTS and draw the cell growth curve by counting the number of cells for 7d to detect cell proliferation activity.Results:1.The expressions of EGFR in both mRNA and protein in HLEs were effectively decreased by the siRNA targeting human EGFR gene.The EGFR mRNA level in the si-EGFR was 0.450625 at 48h after transfection,and was significantly lower than that of the negative control group(1).The EGFR protein levels in the si-EGFR was 0.085±0.013,and was significantly lower than that of the negative control group(0.712±0.054)and blank control group(0.740±0.056).2.Flow cytometric analysis show:in the group treated by si-EGFR,the percentage of cells in G1 phase increased to 41.6%,as compared to negative control group,which had only 29.5%of cells in G1 phase.The percentage of si-EGFR cells in G2 phase decreased to 27.5%,as compared to 43.7%in the negative control group The proliferation index of si-EGFR cells was 140.4%as compared to 239%in negative controls.MTS and cell growth curve results showed that the growth rate of HLEs was decreased markedly at 24 to 72 hours(P<0.01).Moreover,the cell growth activity was decreased obviously.These results indicate that EGFR plays an important role in the transition of the cell cycle and then affect the growth of HLE cells.3.In a certain range,the proliferation ability of cells is rising following the increase of EGF concentration,but if the range was exceeded,it may exert an opposing effect.The result of MTS is the same to the cell growth curve,and the most adaptable concentration of recombinant EGF to simulate cell proliferation activity is 1 ng/mL.4.Compared to the control group,it can be said that si-EGFR group was almost unswayed when co-supplied with EGF.Even spurred by EGF,siRNA of EGFR can obviously and stably inhibit the growth of HLE cells in vitro.The result of MTS is the same to the cell growth curve.Conclusion:1.pSilencer2.1-shRNA-EGFR expression vector by chemical synthesis in vitro can degrade the expression of EGFR in HLEs;accordingly inhibit the proliferation activity of cells.Thus,siRNA targeting EGFR may provide a totally new way for preventing Posterior capsule opacification(PCO).2.In a certain range,the proliferation ability of cells is rising following the increase of EGF concentration,but if the range was exceeded,it may exert an opposing effect.The most adaptable concentration of recombinant EGF to simulate cell proliferation is 1ng/ml.3.Even spurred by the most adaptable concentration of recombinant EGF,siRNA of EGFR can obviously and stably inhibit the growth of HLE cells in vitro and the inhibition come true through EGF-EGFR signal pathway blockage.