title: SiRNA Targeting mTOR Effectively Prevents the Proliferation and Migration of Human Lens Epithelial Cells.
abstract: Posterior capsule opacification (PCO) is the most common complication that causes visual decrease after extracapsular cataract surgery. The primary cause of PCO formation is the proliferation of the residual lens epithelial cells (LECs). The mammalian target of rapamycin (mTOR) plays an important role in the growth and migration of LECs. In the current study, we used small interfering RNA (siRNA) to specifically attenuate mTOR in human lens epithelial B3 cells (HLE B3). We aimed to examine the effect of mTOR-siRNA on the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of HLE B3 cells and explore the underlying mechanisms. The mTOR-siRNA was transfected into HLE B3 cells using lipofectamine 2000. The mRNA and protein levels of mTOR were examined to confirm the efficiency of mTOR-siRNA. The levels of mRNA and protein as well as the activity of mTOR down-stream effectors p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB, AKT) were examined using real-time PCR or Western blot, respectively. The cell proliferation was determined using cell counting kit (CCK) 8 and cell growth curve assay. The cell migration was examined using Transwell system and Scratch assay. MTOR-siRNA effectively eliminated mTOR mRNA and protein. The proliferation and migration were significantly suppressed by mTOR-siRNA transfection. mTOR-siRNA reduced the mRNA of p70S6K and AKT in a time-dependent manner. Furthermore, the phosphorylation of p70S6K and AKT was decreased by mTOR-siRNA. MTOR-siRNA also eliminated the formation of mTORC1 and mTORC2 protein complex and blocked the transforming growth factor (TGF)-β-induced EMT. Our results suggested that mTOR-siRNA could effectively inhibit the proliferation, migration and EMT of HLE B3 cells through the inhibition of p70S6K and AKT. These results indicated that mTOR-siRNA might be an effective agent inhibiting HLE cells growth and EMT following cataract surgery and provide an alternative therapy for preventing PCO.
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin, P.R. China.
authors.forename: Chunmei
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin, P.R. China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin, P.R. China.
authors.forename: Na
authors.initials: N
authors.lastname: Jin
authors.affiliation: Department of Ophthalmology, No.2 Hospital of Xiamen, Fujian, P.R. China.
authors.forename: Guiming
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin, P.R. China.
authors.forename: Yahui
authors.initials: Y
authors.lastname: Xi
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin, P.R. China.
authors.forename: Hongling
authors.initials: H
authors.lastname: Liu
mesh_terms: D058442:Capsule Opacification / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007903:Lens Capsule, Crystalline / Q000378:metabolism* / Q000473:pathology; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; C546842:MTOR protein, human; D000076222:Mechanistic Target of Rapamycin Complex 1; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases