Suppression of lentiviral vector-mediated mTOR gene silencing on growth of human lens epithelial cell in vitro

Background

Mammalian target of raparrmycin (mTOR) acts as a critical molecular link between growth signals and the processes underlying cell growth. The overexpression of mTOR in human lens epithelial cells (HLECs) is associated with posterior capsular opacification (PCO). Applying the mTOR shRNA transfection technique to silence mTOR in HLECs is significant for the targeting treatment of PCO.

Objective

This study was to investigate the function and morphology of HLECs infected by mTOR shRNA lentiviral vector.

Methods

293T cells were transfected by recombinant plasmid containing mTOR shRNA, blank plasmid and PBS to prepare different lentiviral vectors. HLECs were routinely cultured and were divided into the mTOR shRNA transfected 1, 2, 3 groups, blank plasmid group and PBS group, and different lentiviral vectors were added in the medium to infect the cells. The expression of mTOR protein in HLECs was detected using Western blot to determine mTOR gene silence, and the group with the highest efficiency of silence was screened as the sequent experiment group. The proportion of HLECs in the G₁ phase was analyzed by flow cotymetry. The expressions of α-smooth muscle actin (α-SMA) and E-Cadherin proteins in the cells were assayed by Western blot. The viability of HLECs was assessed by cell counting kit-8 (CCK8) test.

Results

Successfully packaged lentivirus were found in the supernatant after infection of mTOR shRNA in the mTOR shRNA transfected 1, 2, 3 groups with the green fluorescence in >80% cells, while green fluorescence protein (GFP) response was absent in the blank plasmid group and the PBS group. The HLECs of mTOR shRNA transfected groups and blank plasmid group grew well due to the resistance for puromycin of plasmid, however, more dead cells were found in the PBS group. Western blot revealed the weakest expression of mTOR mRNA in the cells in the mTOR shRNA transfected 3 group. The expressing bland of α-SMA in the HLECs was weaker in the mTOR shRNA transfectd 3 group than that in the blank plasmid group or PBS group, and the expression of E-Cadherin was stronger in in the mTOR shRNA transfectd 3 group than that in the blank plasmid group or PBS group. The absorbance of the mTOR shRNA transfectd 3 group was significantly declined in comparison with the blank plasmid group and the PBS group, with significant difference among the three groups in both 1 hour and 2 hours after experiment (all at P<0.05). The proportion of cells in G₁ phase was (60.00±1.78)% in the mTOR shRNA transfected 3 group, which was significantly increased in comparison with (53.48±1.86)% in the blank plasmid group and (53.02±1.49)% in the PBS group (F=18.910, P=0.002).

Conclusions

mTOR gene is silenced after mTOR shRNA plasmid transfected into HLECs. This procedure can invert the epithelial-mesenchymal transition (EMT), inhibit cell growth and put off cellular cycle in HLECs in vitro.

Lentiviral vector; Short hairpin RNA; Posterior capsule opacification; Mammalian target of raparrmycin; Epithelial-mesenchymal transition; Lens, crystal; Epithelial cells; Gene silence