Down Regulation Of MTOR By Lentivirals Contributes To Apoptosis And Inhibits Cell Proliferation In Lung Cancer

ObjectiveLung cancer is an aggressive malignancy with the higher incidence in men than that in women. Laryngal squamous cell carcinoma(LSCC) is the main kind of lung cancer. Gene deregulation in patiens associates with lung caner risk. mTOR as one kind of protein kinase has been confirmed to control cancer progression by regulating cancer relative genes. Early studies also shown that mTOR is crucial to the development of cancer and also provides a new, potential therapeutic target for treating cancer.RNA interference(RNAi) is a popular technique that can down-regulate gene expression specifically. In this study, RNAi was performed to regulate mTOR expression in A549cells. After mTOR down-regulation, MTT assay, colony formation assay, Transwell and Murine xenograft model were performed to test the influence of mTOR on A549cell progression. In addition, to further understand the mechanism of mTOR in lung cancer regulation, the expression of P70S6K, MT1-MMP, HIF-1a, VEGFA and cyclinD1were analysised by western blot.Methods1According to coding region of mTOR, mTOR specific shRN A expression plasmids were constructed and transfected into A549cells, western blot was performed to detect the expression of mTOR. After the most efficiency shRNA selected, lentivirus with GFP (lenti-GFP-mTOR) or Puro (lenti-Puro-mTOR) resistance marker were packaged.2After treated with lenti-GFP-mTOR, MTT assay, colony formation assay, flow assay and Transwell were performed to analysis the proliferation and invasion ability of A549cells.3In vivo, stable cell lines were established by treated A549cells with lenti-Puro-mTOR and control lentivirus.1×107cells were injected into athymic nude mice. The mice were monitored and tumor size was measured, and tumor volumes were calculated as width (mm)×width (mm) X length (mm)×0.5.23days after injection, the mice were killed.4Statistical analysis:Data are expressed as means±SEM. Statistical analysis was performed using the SPSS software version16.0. All the data were analyzed by ANOVA. P<0.05was considered statistically significant.Results1Four mTOR specific shRNA lentivirus expression plasmids were constructed, Sh-mTOR-6506, Sh-mTOR-3266, Sh-mTOR-4995, Sh-mTOR-6216. After trasfection, Sh-mTOR-6216was selected to be the most inhibition rate of mTOR in A549cells by western blot. Then lentivirus with GFP (lenti-GFP-mTOR) or Puro (lenti-Puro-mTOR) resistance marker containing mTOR-6216shRNA were packaged.2In vitro, MTT assay was performed to detect A549cell vability after being treated with lenti-GFP-mTOR and control lentivirus. The absorbance at570nm was as followed, Mock,0.687+0.103, control group,0.667±0.037, lenti-GFP-mTOR,0.356±0.059. Compared with control, lenti-GFP-mTOR had significant inhibition on A549cell(P<0.05). There is no significant difference between mock group and control group.3Colony formation assay was performed to detect A549cell proliferation after treated with lenti-GFP-mTOR and control lentivirus. The clone number were culculated, Mock,63±10.536, control group,59.333±9.713, lenti-GFP-mTOR,16±4.583. lenti-GFP-mTOR had greater influence on A549cell colony formation ability compared with control group. Clone numbers between lenti-GFP-mTOR and control group had significant difference(.P<0.05).4Transwell was performed to detect A549cell invasion24h after being treated with lenti-GFP-mTOR and control lentivirus. The results were shown that after treated with lenti-GFP-mTOR, A549cell invasion ability was decreased compared with control goup, and there is significant difference (P<0.05), but no significant difference between Mock and control group. The passed cells were shown, Mock,124.333±6.110, control group,109.333±11.504, lenti-GFP-mTOR,18±5.568.5The results of flow cytometry analysis shown that down-regulation of mTOR expression by lenti-GFP-mTOR leaded to G1phase arrest and contributed to A549cell apoptosis compared with control group. The proportion of apoptosis cell was as followed, Mock,0.337±0.103, control group,2.473±0.634, lenti-GFP-mTOR,54.973±6.377. 6The expressions of P70S6K, MT1-MMP, HIF-la, VEGFA and cyclinDl were analyzed by western blot after mTOR down-regulated. Data shown that, mTOR is the positate regulator of these genes, down-regulation of mTOR decreased the expression of P70S6K, MT1-MMP, HIF-la, VEGFA and cyclinD1.7In vivo, tumor volume was recorded after mice were killed. Tumor volume in the group of lenti-Puro-mTOR was28.308+3.521mm3, which was significantly less than that in control group (P<0.05). Tumor volume of Mock group and control group were,72.767±14.677mm3and62.625±6.358mm3respectivly, and there is no significant difference between them.Conclusions1mTOR specific silencing lentivirus with GFP of Puro selecting marker was successfully packaged,2In vitro, down-regulation of mTOR expression by lenti-GFP-mTOR inhibited A549cell proliferation, invasion ability, and contributed to cell apoptosis. Western blot analysis shown that the expression of P70S6K, HIF-la, VEGFA and cyclinDl were decreased after mTOR silenced.3In vivo, compared with control group, down-regulation of mTOR in A549cells inhibited tumor genesis, the tumor volume in lenti-Puro-mTOR group was significantly less than that in control group.