microRNA-19a protects osteoblasts from dexamethasone via targeting TSC1

Activation of mTOR complex 1 (mTORC1) could protect human osteoblasts from dexamethasone. Tuberous sclerosis complex 1 (TSC1) is mTORC1 upstream inhibitory protein. We demonstrate here that microRNA-19a (“miR-19a”, -3p) targets the 3' untranslated regions of TSC1 mRNA. Expression of miR-19a downregulated TSC1 in OB-6 osteoblastic cells and primary human osteoblasts. miR-19a activated mTORC1 and protected human osteoblasts from dexamethasone. mTORC1 inhibition, by RAD001 or Raptor shRNA, almost completely abolished miR-19a-induced osteoblast cytoprotection against dexamethasone. Knockdown of TSC1 by targeted shRNA similarly induced mTORC1 activation and protected osteoblasts. Moreover, miR-19a activated mTORC1-dependent NF-E2-related factor 2 (Nrf2) signaling and inhibited dexamethasone-induced reactive oxygen species production in osteoblasts. Together, miR-19a protects human osteoblasts from dexamethasone possibly via targeting TSC1-mTORC1 signaling.

To further confirm that TSC1 downregulation is the direct cause of mTORC1 activation in human osteoblasts, a TSC1-expression construct (see Method) was introduced to miR-19a-expressing OB-6 cells. The construct restored TSC1 expression ( Figure 2E). Importantly, exogenous TSC1 expression in OB-6 cells almost completely blocked miR-19a-induced mTORC1 activation (tested by p-S6K1, Figure 2E). In miR-19a-expressing primary human osteoblasts, the TSC1 construct similarly restored TSC1 expression and abolished mTORC1 activation ( Figure  2F). As expected, the TSC1 construct didn't affect miR-19a expression by LV-miR-19a in the osteoblasts (Data not shown). These results imply that TSC1 silence should be the direct and primary cause of mTORC1 activation by miR-19a in human osteoblasts.
Dex. To test this hypothesis, shRNA method was again utilized to knockdown TSC1. As demonstrated, the applied lentiviral TSC1 shRNA (purchased from Santa Cruz Biotech) potently downregulated TSC1 in OB-6 cells ( Figure 5A). TSC2 protein level was also downregulated ( Figure 5A). Correspondingly, mTORC1 activation, or p-S6K1, was boosted ( Figure 5A). In the TSC1-shRNAexpressing OB-6 cells, introduction of LV-miR-19a failed to further change TSC1 expression and mTOR activation ( Figure 5A). Importantly, OB-6 cells with TSC1 shRNA were also protected from Dex, showing reduced viability reduction ( Figure 5B) and cell death ( Figure 5C). the lentiviral miR-19a expression vector ("LV-miR-19a", two lines, "Line1/2"), non-sense scramble control microRNA ("miRC"), or the parental control OB-6 cells ("Ctrl"), were subjected to Western blotting assay of listed proteins (A and B). Primary human osteoblasts were infected with LV-miR-19a or miRC for 48 hours, expressions of listed proteins were shown (C and D). Stable OB-6 cells with LV-miR-19a ("Line1") (E), as well as the primary human osteoblasts with LV-miR-19a (F), were further transfected with TSC1 cDNA construct ("wt-TSC1", 0.20 μg/mL, for 48 hours) or the empty vector (pSuper-puro-EGFP, "Vec"), total cell lysates were subjected to Western blotting assay of listed proteins. The indicated protein band was quantified (total gray), and its value was normalized to the loading control. Experiments in this figure were repeated four times, and similar results were obtained.
Intriguingly, expression of miR-19a (by LV-miR-19a) in the TSC1 shRNA-expressing OB-6 cells was unable to further protect cells from Dex ( Figure 5B and 5C). These results together indicate that TSC1 is the primary target of miR-19a in mediating its osteoblast cytoprotection.

DISCUSSION
In the current study, we propose that miR-19a is a TSC1-targeting miRNA in human osteoblasts. Expression of miR-19a downregulated TSC1 and activated mTORC1 signaling in both OB-6 osteoblastic cells and primary human osteoblasts. Significantly, miR-19a protected with miR-19a expression vector ("LV-miR-19a") or the non-sense scramble control microRNA ("miRC"), as well as the parental control cells ("Ctrl"), were treated with/out Dex (1 μM) for 48 hours, cell viability (CCK-8 assay, A and D), cell death (LDH release assay, B and E) and apoptosis (Histone DNA ELISA assay, C) were examined. Data were expressed as mean ± SD (n = 5). "C" stands for untreated control group. * p < 0.05 vs. "C". # p < 0.05 vs. Dex treatment of "miRC" cells. Experiments in this figure were repeated three times, and similar results were obtained. human osteoblasts from Dex, and activation of mTORC1 is required for the process. mTORC1 inhibition, by RAD001 or Raptor shRNA, almost completely abolished miR-19a-induced osteoblast cytoprotection. Similarly, TSC1 shRNA activated mTORC1 and protected osteoblasts from Dex. Notably, miR-19a was in valid in TSC1-silenced OB-6 cells. These results imply that TSC1 should be the primary and direct target protein of miR-19a in mediating  osteoblastic cells were infected with lentiviral scramble control shRNA ("SCR-sh"), TSC1 shRNA ("TSC1-sh") or plus lentiviral miR-19a expression vector ("+LV-miR-19a"), cells were further selected by puromycin, expressions of listed proteins were shown (A). The abovementioned cells were also treated with/out Dex (1 μM) for 48 hours, cell survival (B) and death (C) were tested. The indicated protein band wan quantified, and its value was normalized to the loading control (A). Data were expressed as mean ± SD (n = 5). "Ctrl" stands for parental control cells. "C" stands for untreated control group. * p < 0.05 vs. "C". # p < 0.05 vs. Dex treatment of "SCR-sh" cells. Experiments in this figure were repeated three times, and similar results were obtained. its cytoprotective activity in osteoblasts. Indeed, we show that TSC1 mRNA's 3′-UTR luciferase assay was also largely attenuated after miR-19a expression.
The transcription factor Nrf2 signaling is one key cellular defense mechanism against oxidative stress [40,45]. Recent studies have proposed that mTORC1 could be a upstream signaling for Nrf2 activation [38,39]. Activated mTORC1 could induce Nrf2 phosphorylation at Ser-40, causing its departure from Keap1. This will lead to Nrf2 stabilization, its nuclear translocation and activation [38,39]. Here, expression of miR-19a induced Nrf2 stabilization and expression of Nrf2-regulated genes (HO-1, NQO-1 and GCL-C). Inhibition of mTORC1, by RAD001 or Raptor shRNA, almost completely blocked miR-19a-induced mRNA expression of Nrf2regulated genes. These results indicate that miR-19ainduced mTORC1 activation could possibly activate Nrf2 signaling, which likely inhibits Dex-induced oxidative stress. The detailed mechanism may warrant further characterizations.

Chemicals and reagents
Dexamethasone (Dex), puromycin and RAD001 were provided by Sigma Aldrich (Nantong, China). Cell culture reagents were purchased from Gibco Co. (Nantong, China). All the antibodies utilized in this study were obtained from Cell Signaling Technology (Nanjing, China). Lipofectamine 2000 reagent was provided by Invitrogen (Suzhou, China). All the primers were from Genepharm (Shanghai, China) unless otherwise mentioned.

OB-6 cell culture
The adherent OB-6 osteoblastic cells were seeded at a density of 5000 cells/cm 2 and were cultured in α-minimal essential medium (MEM) with 10% fetal bovine serum (FBS), plus 1% each of penicillin, streptomycin, and glutamine.

Culture of primary human osteoblasts
The redundant trabecular bone fragments were obtained from the written-informed consent healthy donors at Huai'an First People's Hospital (Huai'an, Jiangsu, China). The trabecular bone fragments were minced and washed 2-3 times with warm PBS, which were then digested using DNase and collagenase type II. After 2 h digestion at 37°C, bone debris was separated by using a 70-μm nylon mesh (Falcon). Cells were precipitated by centrifugation, washed several times to remove excess collagenase and DNase enzymes. The primary osteoblasts were then resuspended in α-MEM supplemented with 10% FBS, which were plated at 2 × 10 4 cells/cm 2 . After confluence, cells were trypsinized, resuspended, and seeded at 1.5 × 10 4 /cm 2 . The primary osteoblasts at passage 3-5 were utilized for further experiments. The protocols were approved by Ethics Review Board of Nanjing Medical University.

TSC1 3′-UTR luciferase assay
TSC1 mRNA's 3′-UTR was amplified via the primers as described [50], which was sub-cloned into pMIR-Report plasmid [50]. The reporter plasmid was then utilized as template to generate a miR-19a response element. The plasmid with the perfect match contain the complementary sequences of the mature miR-19a-3p behind the firefly luciferase gene [49]. OB-6 cells were transfected with luciferase reporter plasmid by Lipofectamine 2000. The luciferase activity was measured 24 hours after transfection using the Dual-Luciferase reporter assay system (Promega, Shanghai, China).

Western blotting assay
The detailed protocols for Western blotting assay were described in detail in our previous studies [6,7,11,12]. The total gray of the protein band was quantified via ImageJ software, and the value was normalized to the loading control.

shRNA
The lentiviral TSC1 shRNA (catalog no. sc-37437-V), the lentiviral Raptor shRNA (catalog no. sc-44069-V), and the lentiviral scramble control shRNA (catalog no.sc-108060) were all purchased from the Santa Cruz Biotech (Shanghai, China). The lentiviral shRNA (10 μL virus/1 mL medium, per well) was added directly to the cultured osteoblasts for 24 hours. Afterwards, puromycin (0.5 μg/mL, Sigma) was added to select stable cells. Knockdown of the targeted-protein in the stable cells was verified via Western blotting assay.

Exogenous TSC1 expression
The full length human TSC1 cDNA was synthesized, sequence-verified and provided by Genepharm (Shanghai, China), which was inserted into the pSuper-puro-EGFP vector (Addgene, Shanghai, China). The construct was transfected to miR-19a-expressing osteoblasts via Lipofectamine 2000 transfection. Expression of TSC1 in the resulting cells was verified by the Western blotting assay.

Statistical analysis
All values were expressed as means ± standard deviation (SD). The statistical significance of differences among groups were determined by one-way analysis of variance (ANOVA) followed by the Tukey's post hoc multiple comparison tests. p < 0.05 was considered significant.

Author contributions
All authors carried out the experiments, participated in the design of the study and performed the statistical analysis, participated in its design and coordination and helped to draft the manuscript.