microRNA-25 targets PKCζ and protects osteoblastic cells from dexamethasone via activating AMPK signaling

AMP-activated protein kinase (AMPK) activation could protect osteoblasts from dexamethasone (Dex). This study aims to provoke AMPK activation via microRNA downregulation of its negative regulator protein kinase C ζ (PKCζ). Results show that microRNA-25-5p (miR-25-5p) targets PKCζ's 3’ untranslated regions (UTRs). Forced-expression of miR-25 downregulated PKCζ and activated AMPK in human osteoblastic cells (OB-6 and hFOB1.19 lines), which thereafter protected cells from Dex. Reversely, expression of antagomiR-25, the miR-25 inhibitor, upregulated PKCζ and inhibited AMPK activation, exacerbating Dex damages. Notably, PKCζ shRNA knockdown similarly activated AMPK and protected osteoblastic cells from Dex. AMPK activation was required for miR-25-induced osteoblastic cell protection. AMPKα shRNA or dominant negative mutation almost completely blocked miR-25-induced cytoprotection against Dex. Further studies showed that miR-25 expression increased NADPH activity and suppressed Dex-induced oxidative stress in osteoblastic cells. Such effects by miR-25 were abolished with AMPKα knockdown or mutation. Significantly, miR-25-5p level was increased in patients’ necrotic femoral head tissues, which was correlated with PKCζ downregulation and AMPK hyper-activation. These results suggest that miR-25-5p targets PKCζ and protects osteoblastic cells from Dex possibly via activating AMPK signaling.


Research Paper
induced osteoblast cell death [23].The results of these studies imply that AMPK activation exerts pro-survival functions in osteoblasts/osteoblastic cells.

AMPK activation is required for miR-25induced cytoprotection in osteoblastic cells
Thus, miR-25 expression activated AMPK and protected osteoblastic cells from Dex.The link between the two was then explored.Genetic strategies were utilized to block AMPK activation.AMPKα shRNA or dominant negative AMPKα ("dn-AMPKα", T172A) was introduced to the miR-25-expressing OB-6 cells.Western blot results in Figure 5A demonstrated that    miR-25-induced AMPK activation was almost blocked by AMPKα shRNA or mutation.Importantly, miR-25induced cytoprotection against Dex was also abolished in AMPKα-silenced or -mutated cells (Figure 5B and 5C).Therefore, miR-25 was no longer cytoprotective when AMPK was silenced or mutated (Figure 5B and 5C).It should be noted that AMPKα shRNA or mutation augmented Dex-induced damages in OB-6 cells (no miR-25, Figure 5A-5C).These results are consistent with the antigomir-25 results (Figure 3), and further confirmed that AMPK inhibition would potentiate Dex-induced cytotoxicity in osteoblastic cells.Therefore, AMPK activation is required for miR-25-induced cytoprotection in osteoblastic cells.

miR-25-5p upregulation correlates with PKCζ downregulation and AMPK activation in human osteonecrosis tissues
It has been shown that AMPK activation was upregulated in patients' necrotic femoral head tissues [23].Here, miR-25-5p expression level in human necrotic femoral head tissues was tested, and its level was compared with that in the surrounding normal femoral head tissues (Figure 6A).In the necrotic tissues, the level of miR-25-5p was upregulated (Figure 6A), yet PKCζ (protein and mRNA) level was decreased (Figure 6B-6D).AMPK activity, indicated by p-ACC, was also increased in the necrotic femoral head tissues (Figure 6C).Therefore, miR-25 upregulation and PKCζ depletion could be at least one reason of AMPK activation in patients' necrotic femoral head tissues [23].
Although miR-25-5p is a predicted target of PKCζ, it appears to be not conserved.As a matter of fact, the miRNA database (TargetScan v7.1) fails to find a single conserved miRNA for PKCζ.This could be due to the fact that the entire 3'UTR of PKCζ is poorly conserved among different cell types.Fortunately, here we show that miR-25-5p targets and downregulates PKCζ in osteoblastic cells, which activates AMPK signaling and protects cells against Dex.It will be interesting to test the potential effect of miR-25-5p on PKCζ and AMPK signaling in other cells.
Excessive Dex usage could lead to osteoporosis [1,2] and/or osteonecrosis [3].In the Dex-taking patients' bones, decreased number of osteoblasts and increased number of apoptotic osteoblast cells are often detected [1,2].Here, the in vitro results showed that miR-25-5p downregulated PKCζ and activated AMPK signaling to protect osteoblastic cell from Dex. Intriguingly, in human necrotic femoral head tissues, miR-25-5p expression was significantly increased, which was correlated with PKCζ downregulation and AMPK activation.Thus, in the following studies, it will be very interesting to further test the possible effect of miR-25-5p in animal model of Dex-induced bone damages.
In summary, these results conclude that miR-25 targets PKCζ and protects osteoblastic cells from Dex via activating AMPK signaling.

Chemicals and reagents
Dex was obtained from Sigma Aldrich (Shanghai, China).All cell culture reagents were obtained from Gibco (Shanghai, China).All the antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Cell culture
The OB-6 [4] and hFOB1.19[32] human osteoblastic cells were obtained from the Cell Bank of Shanghai Institute of Biological Science (Shanghai, China).Osteoblastic cells were cultured as described [4,32].

Quantitative real-time polymerase chain reaction (qRT-PCR) assay
As described in our previous studies [9,11], total RNA was extracted by the SV total RNA purification system (Promega, Shanghai, China).Extracted RNA was reverse-transcribed through the reverse transcriptase (Promega).cDNA derived from 500 ng of RNA was amplified by quantitative real-time polymerase chain reaction ("qRT-PCR").The SYBR Green PCR kit (Applied Biosystems, San Diego, CA) was applied to detect expression of targeted mRNAs.GAPDH primers (F-5′-AAG GTG AAG GTC GGA GTC-3′ and R-5′-TGT AGT TGA GGT CAA TGA AGG-3′) and PKCζ primers (F-5′-GCG TAC TGC GGC CAG TGC-3′ and R-5′-CTT GGC ATA GCT TCC ACG-3′) were described [33].PCR was performed in triplicate and was conducted using a Real-Time PCR Detection System (7500; ABI, Shanghai, China).mRNA expression was quantified using the ΔΔ Ct method using GAPDH as the internal control.Mature hsa-miR-25-5p expression was assessed using TaqMan microRNA assay using the primer described [34].Mature hsa-miR-25-3p primer was described previously [35].Five ng of total RNA was reverse-transcribed using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystem) and the looped primer provided by the specific TaqMan microRNA assay [36].All the primers and sequences were synthesized by Genepharm (Shanghai, China).

Western blot assay
As described [9,11], cell lysates were extracted via RIPA lysis buffer (Bio-sky, Nanjing, China).Aliquots of 30 µg lysates per sample were electro-transferred on 10% SDS-PAGE gel, following by transfer to PVDF membranes.The blots were then incubated with designated primary and secondary antibodies.The antigen-antibody binding was detected via enhanced chemiluminescence (ECL) reagents.ImageJ software was applied to quantify protein band.

Cell death detection
Cell death was tested by counting cells using a cytometer after addition of trypan blue, which stained the cytoplasm of dead cells.Cell death percentage (%) = the number of trypan blue stained cells/the number of total cells (×100%) [11].

Apoptosis assay by enzyme-linked immunosorbent assay (ELISA)
As described [39], the Histone-DNA Apoptosis ELISA Detection Kit (Roche, Palo Alto, CA) was applied to quantify cell apoptosis with indicated treatment.

shRNA knock and stable cell selection
The two lentiviral shRNAs (GV248-puromycin vector) against human PKCζ were designed, synthesized and verified by Genepharm Co. (Shanghai, China).The AMPKα shRNA was described in our previous study [39].Osteoblastic cells were seeded onto 6-well plates with 50% of confluence.The lentiviral shRNA (10 μL/ mL) were added to cultured cells for 24 hours.Afterwards, cells were cultured in puromycin (1 μg/mL)-containing complete medium, until resistant colonies can be identified (10-14 days).The expression of target protein (PKCζ or AMPKα) in the stable cells was tested by Western blot assay.The scramble lentiviral shRNA (Santa Cruz) was added to the control osteoblastic cells.

AMPK dominant negative mutation
The dominant-negative mutant of AMPKα (dn-AMPKα, T172A) construct was a gift from Dr. Lu's group [40], which was transfected to osteoblastic cells via Lipofectamine 2000 [40], and stable cells were selected via neomycin (1 μg/mL, Sigma).Transfection efficiency was always verified via Western blot assay in the stable cells.

NADPH activity assay
NADPH activity assay was described in previous studies [23,41].Briefly, after treatment of cells, the lysates were incubated with NADP-cycling buffer plus glucose-6-phosphate dehydrogenase (G6PD, Sigma) at 60 °C for 30 min [23].Afterwards, glucose 6-phosphate (G6P, Sigma) was added to the mixture, and the change in absorbance at 570 nm was measured every 30 s for 4 min at 30 °C.The concentration of NADP+ was calculated by subtracting [NADPH] from [total NADP].NADPH activity was then calculated through NADPH/ NADP+ [41].

Reactive oxygen species (ROS) assay
ROS production was measured via a DCFH-DA fluorescent dye (Invitrogen).After treatment, cells were incubated with 1 μM of DCFH-DA (Invitrogen) for 30 min.Cells were then washed and analyzed for fluorescence using the flow cytometer (BD, Shanghai, China).The ROS intensity in the treatment group was normalized to that of control group.

Human tissue specimens
Surgery-isolated fresh necrotic femoral head tissues and their surrounding normal femoral head tissues were collected from Dex-taking patients.Fresh tissue specimens were dissolved in tissue lysis buffer (BiYunTian Biotechnology Research Institute, Nantong, China) and were subjected to qRT-PCR assay and Western blot assay.The experiment protocols requiring human samples were approved by the institutional review board and ethics committee of all authors' institutions, and written informed consent was obtained from each patient.A total of 9 patients were included.All studies were conducted according to the principles expressed in the Declaration of Helsinki.

Statistics
The data presented were mean ± standard deviation (SD).Statistical differences were analyzed by one-way ANOVA followed by multiple comparisons performed with post hoc Bonferroni test (SPSS version 18.0).Values of p < 0.05 were considered statistically significant.

Figure 1 :
Figure 1: microRNA-25 downregulates PKCζ and activates AMPK signaling in human osteoblastic cells.A. microRNA-25-5p ("miR-25-5p") targets the 3' untranslated regions of human PKCζ.Human osteoblastic OB-6 cells B-D. or hFOB1.19 cells E-G. were transfected with has-pre-miR-25 or the control microRNA ("miR-C"), and stable cells were established; Expressions of miR-25-5p (B and E) and PKCζ mRNA (C and F) were tested by quantitative real-time PCR ("qRT-PCR") assay; Expression of listed proteins in these cells was tested by Western blot assay, and phosphorylations of AMPKα and ACCα were quantified (D and G).Experiments in this figure were repeated three times, and similar results were always obtained."Trans" stands for transfection reagents only (B-G).*p<0.05 vs. group "miR-C" (B-G).

Figure 2 :
Figure 2: microRNA-25 protects human osteoblastic cells from Dex. Stable osteoblastic OB-6 cells A-C. or hFOB1.19 cells D-F. expressing microRNA-25 ("miR-25") or control microRNA ("miR-C") were treated with or without Dex (1 μM) for 24 hours, cell viability (MTT assay, A and D), cell apoptosis (Histone DNA ELISA assay, B and E) and cell death (trypan blue assay, C and F) were tested.Experiments in this figure were repeated three times, and similar results were obtained."Ctrl" stands for untreated control group."Trans" stands for transfection reagents only.*p<0.05 vs. "miR-C" cells with Dex treatment.

Figure 4 :
Figure 4: PKCζ knockdown activates AMPK and protects human osteoblastic cells from Dex. OB-6 cells were infected with lentiviral PKCζ shRNA ("-1 or -2") or non-sense control shRNA ("scr shRNA"), and stable cells were established; miR-25-5p and PKCζ mRNA expression levels in these cells were tested A., PKCζ protein expression and AMPK activation were also tested B. Above cells were treated with or without Dex (1 μM) for 24 hours, cell viability (C, MTT assay) and apoptosis (D, Histone DNA ELISA assay) were shown.PKCζ shRNA ("-1")-expressing OB-6 cells were transfected with miR-25, expressions of miR-25-5p E. and PKCζ mRNA F. were shown; These cells were also treated with or without Dex (1 μM) for 24 hours, cell viability G. and apoptosis H. were tested."Ctrl" stands for untreated control group.Experiments in this figure were repeated three times, and similar results were obtained.*p<0.05 vs. "scr shRNA" cells (A, C-H).

Figure 5 :
Figure 5: AMPK activation is required for miR-25-induced cytoprotection in osteoblastic cells.Stable OB-6 cells with miR-25 were constructed with dominant negative AMPKα ("dn-AMPKα", T172A), AMPKα shRNA, or the scramble control shRNA ("scr shRNA"), expressions of listed proteins in these cells were tested by Western blots A. Above cells were treated with or without Dex (1 μM), cell viability (MTT assay, 24 hours, B., apoptosis intensity (Histone DNA ELISA assay, 24 hours, C. and ROS content (DCFH-DA fluorescent dye assay, 6 hours, D. were tested; NADPH activity in above cells was also shown (4 hours, E).Experiments in this figure were repeated three times, and similar results were obtained."Ctrl" stands for untreated control group.*p<0.05 vs. "scr shRNA" cells.

Figure 6 :
Figure 6: miR-25-5p upregulation correlates with PKCζ downregulation and AMPK activation in human osteonecrosis tissues.Expressions of miR-25-5p A. and PKCζ mRNA B. in surgery-isolated femoral head tissues (both normal or "S" and necrotic or "N") from GC-taking patients (n=9) were tested by qRT-PCR assay; Expression of listed protein was tested by Western blots (C, and PKCζ expression was quantified in D). *p<0.05 vs. "S" tissues.