| BackgroundMicroRNAs, one of the small endogenous non-coding RNAs, play major roles in cell proliferation and apoptosis by inhibiting the translation of their target mRNAs or mediating their degradation. The aberrational level of microRNAs expression in numerous cancers indicate that they may be involved in tumor developement and progress as either tumor suppressors or oncogenes.It has been reported that microRNAs are involved in the functional role and regulation of mTOR, as mTORC1 promotes myocytes fusion by regulating expression of miR-1 and miR-125b, while rictor, one of the specific constituents of mTORC2, is regulated by miR-218. However, the role of microRNAs in triggering tumor cell survival remains to be elucidated. We have identified more than fifty microRNAs regulated by mTOR by a preliminary screen using an expression profile approach and herein hypothesize that mTOR may modulate cell survival through some microRNAs.Methods and Results1. Expression level of miR-9* was up-regulated by PP242, a general mTOR inhibitor, but not by rapamycin, a specific mTORC1 inhibitor.MCF-7 cells were treated with DMSO, rapamycin (100nM) or PP242 (200nM) for twenty-four hours and the expression profile of microRNAs were analyzed. Amongst the microRNAs responsive to PP242 or rapamycin, fifteen microRNAs were solely regulated by PP242, but not by rapamycin, suggests these microRNAs may be specially modulated by mTORC2.As our previous study indicate that mTORC2, but not mTORC1, is closely related to cell survival, we analyzed the possible role of mTORC2-specific microRNAs in it. When mimics of these microRNAs were transfected into MCF-7 cells, only miR-9* mimics could significantly promote cell apoptosis. Consistently, qRT-PCR (quantitative real-time PCR) results further confirmed that miR-9* was specifically regulated by mTORC2. While mRNA expression of miR-9* was amplified by 2.55 fold (P<0.01) when MCF-7 cells were treated with PP242 (200nM) for 24h, no significant change of it were detected when cells were treated with rapamycin (100nM).2. miR-9* promote cell apoptosis and suppress cell proliferation.Possible role of miR-9* in apoptosis was further substantiated in various cell lines across different histological origins. An 18.3 fold higher expression level of miR-9* (P<0.05) was observed after 40nM of mimics of it was transfected into MCF-7 cells, as shown by qRT-PCR, suggests the mimics are effective.5nM,20nM and 40nM mimics of miR-9* was transfected into MCF-7 cells for 24h, respectively. Cell death rate increased by 14%(P<0.05), and cell numbers decreased by 13.8%(P <0.01) when MCF-7 cells were transfected with miR-9* mimics (40nM). Furthermore, after serum-starvation, cell death rate of MCF-7 increased by 9%(P< 0.05) in response to transfection with miR-9* mimics (40nM). Putative role of miR-9* in 5-FU mediated apoptosis was further analyzed. MCF-7 cells were treated with OμM,75μM,150μM,300μM or 450μM of 5-FU for 24h, and significant increase in cell death was observed in cells treated with 5-FU above 300μM (including 300μM). After transfected with miR-9* mimics for 48h, MCF-7 cells were treated with OμM,75μM,150μM or 300μM of 5-FU for further 24h, and cell death rate increased by 22%, respectively. Moreover, after transfected with miR-9* mimics for 72h and treated with 75μM of 5-FU for further 24h, cell death rate increased by 23.5%(P<0.01). Furthermore, apoptosis of MCF-7 induced by miR-9* were confirmed by monitoring the cleaved form of PARP, one of the molecular marker of early apoptosis. In addition, the facts that miR-9* could induce apoptosis and inhibit proliferation were also observed in A549 and PC-3M cells. All in all, these results suggest that miR-9* plays pivotal roles in apoptosis.3. miR-9* targeted E2F1 in breast cancer cells.We further investigated the mechanism by which miR-9* promotes apoptosis. E2F1, E2F3 and MDM2 were identified as putative targets of miR-9* by bioinformatic analysis, which could play important roles in miR-9* induced apoptosis.3’-UTR of these genes were inserted into a luciferase reporter, pMir-report and the resulting reporters were pMir-3’UTR-E2F1, pMir-3’UTR-E2F3 and pMir-3’UTR-MDM2, respectively. Interestingly, relative luciferase unit (RLU) was reduced by 38.2%(P<0.01) when miR-9* was co-transfected with pMir-3’UTR-E2F1. Furthermore, expression of E2F1 protein decreased in a concentration-dependent manner when different concentrations of miR-9* mimics were transfected into MCF7. Reversely, increased expression of E2F1 protein was monitored when inhibitor of miR-9* was conducted into these cells. Likewise, similar results were obtained in MDA-MB-231 cells. Collectively, these results suggest that miR-9* may suppress the expression of E2F1 in breast cancer cells.4. miR-9* mediated pp242-induced apoptosis by targeting E2F1We further corroborated that it was mTORC2, but not mTORCl that mediated cell apoptosis. The death rate of MCF-7 cells remained insignificantly affected when treated with rapamycin (100nM) for 48h, but increased by 13.3%(P<0.01) when stimulated with pp242 (200nM) for the same time. Meanwhile, expression level of E2F1 protein was insignificantly changed when treated with rapamycin, but was significantly lowered when treated with pp242. Consistently, E2F1 protein expression was dramatically down-regulated when either mTOR or rictor was depleted using two siRNAs targeting different mRNA regions, which is the main constituent of both mTORCl and mTORC2 or solely mTORC2, respectively, but remained insignificantly changed when Raptor was silenced, which is the main component of mTORC1. In addition, when serum-starved for further 24h, the death rate of MCF-7 cells increased by 21%(P<0.001) or 17.1%(P<0.001), after they were transfected with siRNAs of rictor or mTOR for 48h.We then investigated whether inhibition of miR-9* will protect cells from pp242-induced apoptosis. In the presence of pretreatment with inhibitor of miR-9* for 24h, death rate of MCF-7 cells remained insignificantly changed after they were treated with pp242 (100nM) for 24h, while in the absence of pretreatment, it increased by 13.3% after pp242 stimulation. These results indicate apoptosis induced by pp242 could be reversed by inhibitor of miR-9*. Furthermore, it was shown by western blot that PARP cleavage increased drastically after pp242 treatment in the absence of pretreatment with inhibitor of miR-9*, but remained unchanged in the presence of that pretreatment.Finally, a MDA-MB-231 xenograft tumor model was established by injection of MDA-MB-231 cells into nude mice subcutaneously, while feeding nude mice with pp242 resulted in a significant increase in death rate in cells within the MDA-MB-231 xenograft tumor, feeding them with rapamycin had no significant effect. Moreover, as shown by RT-PCR, expression of miR-9* in the tumor tissue was significantly up-regulated when the mice were fed with pp242. And shown by western blot, expression level of E2F1 in the tumor tissue was lowered when the mice were fed with pp242, but insignificantly changed when they were fed with rapamycin.ConclusionCollectively, our study revealed a novel pathway by which mTORC2 modulate cell survival, namely, the mTORC2-Akt-miR-9*-E2F1 pathway and provided us new insights into mTORC2’s role in anti-apoptosis. Inhibition of mTORC2 reduces expression of E2F1 by decreasing phosphorylation of Thr473 of Akt and increasing miR-9* expression, thus promoting cell apoptosis. Whether miR-9* may serve as a potential target for cancer therapy and how Akt regulates miR-9* expression will be investigated further. |
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