SiRNA Targeting mTOR Effectively Prevents the Proliferation and Migration of Human Lens Epithelial Cells.

Chunmei Zhang,Hongling Liu,Yahui Xi,Na Jin,Jingjing Liu,Guiming Zhang,Ram Nagaraj

doi:10.1371/journal.pone.0167349

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.

Highlights

Posterior capsule opacification (PCO), known as after-cataract, is the most common complication and the primary reason for decreased visual acuity after extracapsular cataract surgery [1]
We demonstrated a decrease in the total mammalian target of rapamycin (mTOR) mRNA in human lens epithelial B3 cells (HLE B3) cells treated with mTOR-small interfering RNA (siRNA) relative to controls at 24, 48 and 72 hours after transfection, respectively (14.6%, 49.3% and 62.8% decrease, respectively) (Fig 1A and 1B)
The main findings in this study are: 1) mTOR-siRNA effectively reduced mTOR levels in human lens epithelial (HLE) B3 cells; 2) Attenuating mTOR dramatically inhibited HLE B3 proliferation; 3) The activities of p70S6K and AKT, downstream effectors of mTORC1 and mTORC2, were reduced by mTOR interference; 4) transforming growth factor (TGF)-β-induced epithelial- mesenchymal transition (EMT) was blocked by mTOR inhibition

Summary

Introduction

Posterior capsule opacification (PCO), known as after-cataract, is the most common complication and the primary reason for decreased visual acuity after extracapsular cataract surgery [1]. The primary cause of PCO formation is the proliferation of the residual lens epithelial cells (LECs). Several lines of evidence indicated that the phosphatidylinositol 3-kinase (PI3K)/the mammalian target of rapamycin (mTOR) signalling pathway may be involved in the LECs proliferation and migration. Intracellular mTOR forms two distinct protein complexes (mTORC): mTORC1 and mTORC2 [5] Both mTORC1 and mTORC2 are able to modulate proliferation and migration, they exert their functions via distinct signalling pathways. MTORC1 activates ribosomal S6 kinases (S6K1 and S6K2) and eukaryotic initiation factor 4E (eIF4E) to regulate cellcycle progression and protein synthesis [6,7], whereas mTORC2 phosphorylates protein kinase B (PKB, AKT) at serine 473 to modulate cell differentiation, proliferation, invasion, and glucose metabolism [8,9,10]

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