The Investigation Of Lentiviral Vector-mediated MTOR Silence’s Affection On Human Lens Epithelial Cell

Objective:After cataract,also known as posterior capsule opacification(PCO),is the mostfrequent complication after cataract surgery which is also the main cause of visual loss.The pathogenesis of PCO is complex and includes lens epithelial cell (LEC)attachment,proliferation, spreading and migration within the capsular bag and on thesurface of an intraocular lens (IOL).The mamalian target of rapamycin(mTOR) is a keyintermediary in multiple mitogenic signaling pathways and plays a central role inmodulating proliferation,angiogenesis and process of epithelial-mesenchymal transit innormal tissues and neoplastic processes.As highly specific and efficient as it is,RNAinterference (RNAi) is a biological process widely used in knocking down target genes.Inour experiment, cells were firstly transfected by specific shRNA targeting mTOR.To testmolecular markers such as E-cadherin, a-SMA and cell function after gaining stable celllines.We aim at finding out an accurate,therapeutic approach to inhibite PCO in geneticlevel.Methods:1. Preparing recombined GV248.mTORshRNAfor the next transfecting experimentsincluding culturing comptent cells and vector construction.2. HLECs are treated with different groups of GV248.mTORshRNA,gaining stable celllines.Western blot are used to test the efficiency of knocking down.3. The successfully inffected cells are selected to continue relative tests.The expression ofa-SMA and E-cadherin is tested by RT-PCR and western blot.Cell counting kitand Flow Cytometer are used to test proliferation and cell cycle.Results:1. GV248.mTORshRNAproves to be matched with Genebank by sequencing.GFP is seen toevenly distribute almost80%area under fluorescence microscopy.2. mTOR-scilence positive strain expression is apparently down regulated campared withcontrol groups by Western blot.3. The expression of a-SMA is declined in mTOR-scilence positive strain,accompaniedwith E-cadherin increasing,indicating that knocking down mTOR can reverse EMT to acertain extent. 4. mTOR knocking down significantly inhibits proliferation of HLEC and this differenceis statistical significant(P<0.05).Cells with low mTOR expression are almost arrestedin G1phase compared with control groups and this difference is statisticalsignificant(P<0.05).Conclusion:1. mTOR plays a central role in modulating proliferation,angiogenesis and process ofepithelial-mesenchymal transit.Inhibition of mTOR may reverse EMT and may be apotential target of treating PCO.2. shRNA application performed well and tend to be a valuable research tool in cellculture by inducing suppression of specific genes of interests.3. Decreased mTOR expression using shRNA resulted in a significant decrease of cellproliferation.Meanwhile cells division is constrained by cell cycle prolonging.Ourresults testifies that restraining mTOR expression can decrease the a-SMA and increaseE-cadherin,regulating the process of EMT in human lens epithelial cells.So we supposethat interfering targeted gene mTOR could be a potential therapy to prevent PCO.