Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation

Biochemical and Biophysical Research Communications xxx (xxxx) xxx

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation Cong-ya Zhu a, b, 1, Chen Yao c, 1, Lun-qing Zhu d, 1, Chang She a, *, Xiao-zhong Zhou a, ** a

Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, China Department of Orthopaedics, Yancheng No.1 People's Hospital, The Fourth Affiliated Hospital of Nantong University, Yancheng, China c Department of Orthopedics, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China d The Center of Diagnosis and Treatment for Children's Bone Diseases, The Children's Hospital Affiliated to Soochow University, Suzhou, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 June 2019 Accepted 15 June 2019 Available online xxx

Dexamethasone (DEX) exerts potent cytotoxicity against cultured human osteoblasts. The current study examined the role of the circular RNA HIPK3 (circHIPK3) in the mechanism of cell death. We found that circHIPK3 expression was downregulated in DEX-treated human osteoblasts and circHIPK3 levels decreased in human necrotic femoral head tissues. In OB-6 osteoblastic cells and primary human osteoblasts ectopic overexpression of circHIPK3 potently suppressed DEX-induced apoptosis and programmed necrosis. Conversely, knockdown of circHIPK3by targeted siRNAs enhanced DEX-induced cytotoxicity in human osteoblasts. We further observed that microRNA-124 (miR-124), a key miRNA sponged by circHIPK3, accumulated following DEX treatment in OB-6 cells and primary osteoblasts. Confirming the role of miR-124 in DEX-induced cytotoxicity, miR-124 inhibitor attenuated cell death in human osteoblasts. Conversely, forced overexpression of miR-124 mimicked DEX-induced actions and induced cytotoxicity in human osteoblasts. We conclude that DEX-induced cytotoxicity in human osteoblasts is associated with circHIPK3 downregulation. © 2019 Elsevier Inc. All rights reserved.

Keywords: Dexamethasone Osteoblasts circHIPK3 and miR-124

1. Introduction Excessive and sustained dexamethasone (DEX) treatment is the leading cause secondary osteoporosis [1,2] and osteonecrosis [3] in human patients. Osteoblasts, which are essential for bone formation and remodeling [4,5], are the primary target cells of DEX [1,2]. Patients receiving DEX consistently show osteoblast depletion, increased osteoblast cell apoptosis/necrosis, as well as decreased osteoblastogenesis [1,2]. Treatment of cultured osteoblasts with DEX induces significant cell apoptosis and programmed necrosis [6,7]. Our group has been examining the pathophysiology of DEXinduced osteoblast injury and mechanisms to protect human osteoblasts from DEX-induced injury [8e12]. Circular RNAs (circRNAs) have characteristic covalently-closed loop structures, but

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X.-z. Zhou). 1 Co-first authors.

without 50 -30 polarity and polyadenylated tails [13,14]. These highly-stable non-coding RNAs can alter gene expression and function by sponging microRNAs (miRNAs) [13,14]. In this study, we tested the potential role of circRNAs in human osteoblasts, which are remain largely unknown. Circular RNA HIPK3 (circHIPK3, circ_0000284) is derived from exon2 of human HIPK3 [15], with its mature sequence close to 1100 bp [15]. Recent studies have demonstrated that circHIPK3 exerts diverse functions in different human tissues and cells. Shan et al., reported that upregulated circHIPK3 in diabetes mellitus (DM) is involved in retinal vascular dysfunction [16]. Furthermore, circHIPK3 upregulation has been shown to promote human cancer cell progression via sponging tumor-suppressive miRNAs [15,17e19]. Results of the present study demonstrate that circHIPIK3 downregulation mediates DEX-induced cytotoxicity in human osteoblasts. 2. Materials and methods

(C.

She),

[email protected]

2.1. Chemicals and reagents Dexamethasone (DEX), puromycin, MTT, TUNEL, DAPI and JC-1

https://doi.org/10.1016/j.bbrc.2019.06.073 0006-291X/© 2019 Elsevier Inc. All rights reserved.

Please cite this article as: C.-y. Zhu et al., Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.073

2

C.-y. Zhu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

dyes were provided by Sigma-Aldrich (St. Louis, MO). Cyclophilin-D (CyPD), adenine nucleotide translocator 1 (ANT-1), sphingosine kinase (SphK) types 1 (SphK1), signal transducer and activator of transcription 3 (STAT3) and other antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). Cell culture reagents were obtained from Gibco Co. (Carlsbad, CA). All the primers, sequences and viral constructs were purchased from Shanghai Genechem Co. (Shanghai, China), unless otherwise mentioned.

circHIPK3 knockdown was confirmed by qPCR. 2.7. Cell viability assay At 3  103 cells per well OB-6 cells or primary human osteoblasts were seeded into 96-well plates. Following the applied treatments, cell viability was measured performing a MTT assay. MTT optical density (OD) was recorded at the test wavelength of 550 nm.

2.2. Cell culture 2.8. Cell death assay Using the previously-described protocols [12,20,21], human OB6 osteoblastic cells, purchased from the Cell Bank of Shanghai Institute of Biological Science (Shanghai, China), were cultured and differentiated [20]. The primary human osteoblasts, provided by Dr. Ji [22], were cultured using a previously-described protocol [22,23], with approval by the Ethics Committee of Soochow University. Written-informed consent was obtained from the donor.

At a density of 1.5  104 cells per well OB-6 cells or primary human osteoblasts were seeded into 24-well plates and treated with DEX. Lactate dehydrogenase (LDH) levels were measured by a LDH kit (Roche, Shanghai, China). Medium LDH percentage (vs. total LDH), indicating cell death intensity, was calculated [12,26]. 2.9. Apoptosis assays

2.3. Human femoral head tissues As described early [12,24], surgery-isolated necrotic femoral head tissues, as well as the paired surrounding normal femoral head tissues, were from twelve (12) written-informed consent DEX-taking patients with femoral head resection. The protocols were approved by the ethics committee of Soochow University, and recommendations according to the Declaration of Helsinki. 2.4. The quantitative real-time PCR (qPCR) analysis qPCR protocols are described in detail in our previous studies [25,26], using an ABI Prism 7700 Fast qPCR system (Shanghai, China) [25], and a DDCt method for quantification. U6 RNA was amplified to normalize expression levels of circHIPK3 and listed miRNAs. All the primers for qPCR assay of this study are listed in Table-1. 2.5. circHIPK3 overexpression The lentiviral pGLV3-U6-GFP-Puro vector encoding circHIPK3 (“LV-circHIPK3”) used to transduce human osteoblasts was provided as a gift from Dr. Cao [27]. Stable cells were selected by puromycin (2 mg/mL) for 96 h. circHIPK3 overexpression was verified by qPCR. Control cells were transduced with the pLenti6-puro-GFP vector control (“LV-Vec”). 2.6. circHIPK3 siRNA Two circHIPK3 siRNAs, with non-overlapping and sequences (”-1/-2”, represents “S1/S2” [27]), as well as the scramble nonsense control siRNA (“si-C00 ), were provided by Dr. Cao [27]. The siRNA transfection (500 nM each) protocol using Lipofectamine 2000 (Invitrogen, Shanghai, China) was previously described [12]. Table 1 Primers of the qPCR assay. miR-124-F miR-124-R miR-152-F miR-152-R miR-338-F miR-338-R U6 eF U6-R circHIPK3-F circHIPK3-R

50 - GGACTTTCTTCATTCACACCG-30 50 -GACCACTGAGGTTAGAGCCA-30 50 -TCAGTGCATGACAGAACT-30 50 -GAACATGTCTGCGTATCTC-30 50 -ATATCCTGGTGCTGAGTG-30 50 -GAACATGTCTGCGTATCTC-30 50 -CTCGCTTCGGCAGCACATATACT-30 50 -ACGCTTCACGAATTTGCGTGTC-30 50 -TATGTTGGTGGATCCTGTTCGGCA-30 50 -TGGTGGGTAGACCAAGACTTGTGA-30

Apoptosis assays, including the caspase-3 activity assay, the Histone DNA ELISA assay, and Annexin V FACS, were described in detail in our previous studies [12,28,29]. 2.10. Mitochondrial depolarization With mitochondrial depolarization JC-1 dye aggregates to form the green monomers [30]. Following applied treatments OB-6 cells or primary human osteoblasts were stained with JC-1 (5.0 mg/mL), washed and analyzed immediately with a fluorescence spectrofluorometer at emission of 527 nm. 2.11. Mitochondrial isolation, Western blotting and mitochondrial immunoprecipitation (Mito-IP) The protocols of the protein-related assays, including Western blotting, Mito-IP (testing mitochondrial CyPD-ANT-1 immunocomplex) and data quantification were described in detail in previous studies [11,12,25,26,31,32]. 2.12. Ectopic miR-124 overexpression OB-6 cells were seeded into the six-well plates at 50% confluence. The lentiviral pri-miR-124 construct, provided by Dr. Wu [19], was transduced into OB-6 cells. After selection by puromycin (2 mg/ mL, for 96 h) stable cells were established. miR-124 overexpression was confirmed by qPCR. 2.13. Transfection of miR inhibitors OB-6 cells and primary human osteoblasts were seeded into sixwell plates at 50% confluence. Cells were transfected with 500 nM of the applied miR inhibitor and control miR inhibitor using Lipofectamine 2000 for 24 h, and repeated another round (total 48 h). miR inhibitors were from Shanghai Genechem Co. (Shanghai, China). 2.14. Statistics Data were mean ± standard deviation (SD). Statistical analyses were performed by ANOVA through multiple comparisons with post hoc Bonferroni test (SPSS version 20.0, Chicago, IL). To test significance between two treatment groups we applied the twotailed unpaired T test. Values of P < 0.05 were statistically significant.

Please cite this article as: C.-y. Zhu et al., Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.073

C.-y. Zhu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

including miR-379, miR-584 and miR-654 [15], were not detected in OB-6 cells (Data not shown). In primary human osteoblasts DEX treatment (1 mM, 12 h) similarly induced circHIPK3 downregulation (35.3 ± 5.1% of control level, P < 0.05) (Fig. 1C), whereas levels of miR-124, miR-152 and miR-338 increased to 5.5 ± 0.6, 5.5 ± 0.7, and 3.8 ± 0.2 folds (Fig. 1D). These results show that DEX downregulates circHIPK3, while upregulating miR-124, miR-152 and miR-338 in human osteoblasts. We next measured circHIPK3 expression in human necrotic femoral head tissues and found that circHIPK3 was downregulated. As described [12], a set of twelve (12) necrotic femoral head tissues isolated from DEX-treated patients were analyzed. When compared to expression in surrounding healthy femoral head tissue (“S”), circHIPK3 levels were significantly downregulated in the necrotic femoral head tissues (“N”, 32.5 ± 8.3% of “S” tissues, P < 0.05)

3. Results 3.1. circHIPK3 is downregulated in DEX-treated human osteoblasts and its level decreased in human necrotic femoral head tissues To test the potential effect of DEX on circHIPK3 expression in osteoblasts, qPCR was performed. OB-6 human osteoblastic cells were treated with DEX at 1 mM, as previously described [11,12,26]. DEX time-dependently decreased circHIPK3 expression in OB6 cells (Fig. 1A), with levels reduced to 99.5 ± 3.7%, 80.6 ± 4.5%, 38.0 ± 2.1% and 30.7 ± 2.3% compared to control after 3 h, 6 h, 9 h and 12 h (Fig. 1A). In contrast, the levels of circHIPK3-sponged miRNAs, including miR-124, miR-152 and miR-338 [15,27,33], were significantly increased in OB-6 cells following 6e12 h of DEX (1 mM) treatment (Fig. 1B). Other known circHIPK3 target miRNAs,

A

OB-6 cells

B

OB-6 cells 9

0.06

* *

0.03

0

Ctrl

3h

6h

9h

DEX (1 μM)

C

Primary osteoblasts

6h

9h

12h

miR-124

** *

miR-338

2

Ctrl DEX (1 μM), 12h

0

F

0.08

*

Ctrl

DEX (1 μM), 12h

8

Relative expression (Folds vs. “S”)

0.12

0

3h

miR-152

4

0.16

0.04

Ctrl

Primary osteoblasts

Relative expression (Folds vs. “Ctrl”)

circHIPK3 (vs. U6) circHIPK3 (vs. U6)

E

** * * * DEX (1 μM)

6

*

*

*

miR-338

3

D

0.08

0

miR-152 6

*

*

miR-124

0

12h

0.12

0.04

Relative expression (Folds vs. “Ctrl”)

circHIPK3 (vs. U6)

0.09

*

3

n=12

N S S. Surrounding normal femoral head tissues N. Necrotic femoral head tissues

6

miR-124 miR-152 miR-338

4

* *

*

2

0

n=12

S N S. Surrounding normal femoral head tissues N. Necrotic femoral head tissues

Fig. 1. circHIPK3 is downregulated in DEX-treated human osteoblasts and levels decreased in human necrotic femoral head tissues. OB-6 cells (A and B) or the primary human osteoblasts (C and D) were left untreated (“Ctrl”, same for all figures) or treated with dexamethasone (“DEX”, 1 mM) for applied time periods; Expression of circHIPK3 and listed miRNAs (miR-124, miR-152 and miR-338) was tested by qPCR, their levels normalized. Expression ofcircHIPK3 (E) and listed miRNAs (F) in surgery-isolated femoral head tissues (both normal and necrotic) from twelve (12) DEX-taking patients was shown. Data presented were mean ± standard deviation (SD) (Same for all figures). For each assay, n ¼ 5 (AeD). *P < 0.05 vs. “Ctrl” group (AeD). *P < 0.05 vs. surrounding normal (“S”) femoral head tissues (E and F). Experiments in this figure were repeated three times to ensure consistency of results.

Please cite this article as: C.-y. Zhu et al., Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.073

4

C.-y. Zhu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

(Fig. 1E). Conversely, levels of miR-124, miR-152 and miR-338 in “N” tissues increased to 4.6 ± 1.0, 5.8 ± 1.2, 3.9 ± 0.8 folds respectively (Fig. 1F), when compared to those in “S” tissues (P < 0.05). 3.2. Ectopic circHIPK3 overexpression inhibits DEX-induced apoptosis and programmed necrosis in OB-6 cells To study the role of circHIPK3 downregulation in DEX-induced cytotoxicity in human osteoblasts, OB-6 osteoblastic cells were transduced with a lentiviral circHIPK3-expressing construct (“LVcircHIPK3”, from Dr. Cao [27]), and two stable cell lines established: LV-circHIPK3 (L1/L2), with puromycin selection. As shown in Fig. 2A, qPCR results demonstrated that circHIPK3 levels increased 4e5 folds in the stable cells (P < 0.05 vs. control cells), even after DEX treatment (Fig. 2A). Significantly, in LV-circHIPK3-expressing OB-6 cells, DEX (1 mM)-induced reduction in viability (MTT OD) (Fig. 2B) and cell death (LDH release, Fig. 2C) were significantly attenuated. As previously observed [12,21,26], in control OB-6 cells DEX treatment induced apoptosis activation, causing caspase-3 activation (Fig. 2D), caspase-3-PARP [poly (ADP-ribose) polymerase] cleavage (Fig. 2E) and increased Annexin V staining (Fig. 2F and G). Significantly, ectopic overexpression of circHIPK3, by LVcircHIPK3, potently inhibited DEX-induced apoptosis activation in OB-6 cells. As we previously reported [11,26], DEX induces mitochondrial association of CyPD and ANT-1, two key mitochondrial permeability transition pore (mPTP) components, leading to mitochondrial depolarization, cell apoptosis and programmed necrosis. JC-1 dye assay results, Fig. 2H, showed that DEX-induced mitochondrial depolarization (JC-1 intensity increase) was largely attenuated by LV-circHIPK3 in OB-6 cells (Fig. 2H). Furthermore, LV-circHIPK3 prevented DEX-induced mitochondrial CyPD-ANT-1 association tested by mito-IP assay [11] (Fig. 2I), and reduced cytochrome C (“Cyto-C00 ) cytosol release (Fig. 2J). These results demonstrate that forced circHIPK3 overexpression inhibited DEX-induced programmed necrosis in OB-6 cells. LV-circHIPK3 alone had no significant effect on OB-6 cell viability (Fig. 2AeJ). 3.3. circHIPK3 downregulation mediates DEX-induced cytotoxicity in human osteoblasts Next we tested whether forced circHIPK3 overexpression can inhibit DEX-induced apoptosis and programmed necrosis in primary human osteoblasts. Primary human osteoblasts transduced with LV-circHIPK3 showed significantly increased circHIPK3 expression even after DEX treatment (Fig. 3A). DEX-induced viability reduction (Fig. 3B) and cell death (Fig. 3C) of primary osteoblasts were attenuated by LV-circHIPK3. Annexin V FACS assay results demonstrated that circHIPK3 overexpression attenuated DEX-induced apoptosis activation in human osteoblasts (Fig. 3D). Additionally, DEX-induced mitochondrial depolarization (Fig. 3E) and cytosol cytochrome C release (Fig. 3F) were also inhibited by LV-circHIPK3. If circHIPK3 downregulation is essential for DEX-induced death of human osteoblasts, further circHIPK3 depletion would be anticipated to enhance DEX cytotoxicity. To test this hypothesis, two circHIPK3 siRNAs (”-1/-2”, gifts from Dr. Cao [27]), with nonoverlapping sequences, were individually transfected into OB6 cells. qPCR results in Fig. 3G demonstrated that each applied circHIPK3 siRNA further downregulated circHIPK3 expression in DEX-treated OB-6 cells. Consequently, DEX-induced viability reduction (Fig. 3H) and cell death (Fig. 3I) were significantly increased. Similar results were obtained in primary human osteoblasts, where circHIPK3 silencing by targeted siRNAs (Fig. 3J) potentiated DEX-induced cytotoxicity (Fig. 3K and L).

3.4. miR-124inhibition protects human osteoblasts from DEXinduced cytotoxicity Previous studies suggest that circHIPK3 physically binds to miRNAs to cause their degradation [15,18]. In this study, we found that DEX induced accumulation of three miRNAs (miR-124, miR152 and miR-338) in osteoblasts (Fig. 1). To test the link between the accumulation of these miRNAs and DEX-induced cytotoxicity, OB-6 osteoblastic cells were pretreated with the inhibitors of these miRNAs. As demonstrated, transfection of the miR-124 inhibitor (“miR-124i”) attenuated the DEX-induced viability (MTT OD) reduction (Fig. 4A), cell death (medium LDH release, Fig. 4B) and apoptosis (Histone-bound single strand DNA accumulation, Fig. 4C) in OB-6 cells. In contrast, the miR-152 inhibitor (“miR-152i”) and the miR-338 inhibitor (“miR-338i”) failed to affect DEX-induced cytotoxicity (Fig. 4AeC). Further experimental results showed that miR-124 levels were significantly reduced by LV-circHIPK3 (“L1”, see Fig. 2), but increased with transfection of circHIPK3 siRNA (”-1”) (Fig. 4D). Additionally, expression of SphK1 and STAT3, two pro-survival targets of miR-124 [34e36], were downregulated in DEX-treated OB-6 cells (Fig. 4E), where miR-124 levels were increased (see Fig. 1). The above results suggest that miR-124 accumulation, caused by circHIPK3 downregulation, mediates DEX-induced cytotoxicity in human osteoblasts. Therefore, ectopic overexpression of miR-124 would be predicted to mimic the action of DEX. To test this hypothesis, a lentiviral pri-miR-124 construct (LV-miR-124, from Dr. Wu [19]) was transduced into OB-6 cells, resulting in over 25 folds’ increase of mature miR-124 expression (Fig. 4F). As shown, LV-miR124 induced a reduction in viability (Fig. 4G) and cell death (Fig. 4G) in OB-6 cells. Importantly, DEX treatment of LV-miR-124expressing OB-6 cells failed to further enhance miR-124 expression (Fig. 4F) or induce additional cell death and apoptosis (Fig. 4G). These results confirm that miR-124 accumulation is a key mechanism of DEX-induced osteoblast cytotoxicity. In the primary human osteoblasts, miR-124i similarly attenuated DEX-induced viability reduction and cell death (Fig. 4H). Furthermore, miR-124 levels were inhibited by LV-circHIPK3 (Fig. 4I), but increased after circHIPK3 silencing (Fig. 4I). The miR-124 targets SphK1 and STAT3 were also downregulated by DEX in primary osteoblasts (Fig. 4J). 4. Discussion Our recent studies have suggested that targeting non-coding RNAs could be a novel and useful strategy to protect human osteoblasts from DEX. For example inhibition of microRNA-29a induced Gab1 [growth factor receptor bound 2 (Grb2)-associated binder-1] upregulation and Akt activation, to protect OB-6 cells from hydrogen peroxide (H2O2) [21]. Long non-coding RNA (LncRNA) EPIC1 (Lnc-EPIC1) can also protect human osteoblasts from DEX possibly via interacting with Myc [12]. However the expression and potential function of circRNAs in human osteoblasts are still largely unknown. circHIPK3 has been reported to promote cell survival by sponging and suppressing several functional miRNAs [15,17,18]. Our results show that circHIPIK3 downregulation is vital in mediating DEX-induced cytotoxicity in human osteoblasts. In both OB-6 osteoblastic cells and primary human osteoblasts DEX treatment significantly downregulated circHIPIK3 expression, and circHIPIK3 depletion by targeted siRNAs further increased the cytotoxicity of DEX in human osteoblasts. Significantly, we found that circHIPIK3 levels are reduced in human necrotic femoral head tissues. Conversely lentivirus-mediated ectopic overexpression of circHIPIK3 attenuated DEX-induced apoptosis and programmed necrosis

Please cite this article as: C.-y. Zhu et al., Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.073

C.-y. Zhu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

# #

0

DEX (1 μM), 12h

Ctrl

G

*

CleCaspase-3

19kD-

# #

1.15

**

0.26

0.07 (vs. Tubulin)

ClePARP

89kD0.27

0.06

Ctrl

Propidium iodide (PI)

20.91%

1.49%

7.83%

* #

* 0.4

Ctrl

#

*

DEX (1 μM), 6h

10

#

*

Ctrl

DEX (1 μM), 20h

LV-circHIPK3 (L2) 0.88%

6.38%

40 kDa-

-CyPD

33 kDa-

-ANT-1 0.90

Input (Mito-)

2.71%

J

Annexin V

DEX (1 μM), 12h

LV

0.8

0

Annexin V

DEX (1 μM), 6h Mito-IP: CyPD

-c irc LV HI PK -c 3 irc (L H 1) IP K 3 (L 2)

I

-V ec

LV-Vec LV-circHIPK3 (L1) LV-circHIPK3 (L2)

*

3.56%

LV

1.2

#

Tubulin

5.19% Annexin V

*

20

DEX (1 μM), 20h LV-circHIPK3 (L1)

LV-Vec

H

LV-Vec LV-circHIPK3 (L1) LV-circHIPK3 (L2)

0

DEX (1 μM), 16h

5.08%

30

0.01 (vs. Tubulin)

55kD-

0

DEX (1 μM), 24h

0.14

0.11 (vs. CyPD)

40 kDa-

-CyPD

33 kDa-

-ANT-1

35 kDa-

-VDAC-1

rc L V HI PK -c 3 irc (L H 1) IP K 3 (L 2)

2

Ctrl

ec

4

10

-V

6

# #

**

DEX (1 μM), 16h

8

*

20

0

DEX (1 μM), 24h

E LV-Vec LV-circHIPK3 (L1) LV-circHIPK3 (L2)

F

Mito depolarization (JC-1 OD)

0.2

Annexin V (%)

10

*

LV -

Caspase-3 activity (folds vs. “Ctrl”)

12

0.4

Ve c LV -c irc H LV IP K -c 3 irc (L H 1) IP K 3 (L 2)

D

Ctrl

**

0.6

LV-Vec LV-circHIPK3 (L1) LV-circHIPK3 (L2)

-c i

*

0

# #

30

LV

0.2

C

LV

**

0.4

LV-Vec LV-circHIPK3 (L1) LV-circHIPK3 (L2)

0.8

Viability (MTT OD)

circHIPK3 (vs. U6)

B

LV-Vec LV-circHIPK3 (L1) LV-circHIPK3 (L2)

Cell death (Medium LDH %)

OB-6 cells

A

5

0.35

0.14

-Cyto C

15 kDa-

0.11 (vs. Erk1/2)

-Erk1/2

42 kDa-

Cyto-

Fig. 2. Ectopic circHIPK3 overexpression inhibits DEX-induced apoptosis and programmed necrosis in OB-6 cells. OB-6 cells, transduced with the lentiviral circHIPK3 expression construct (pLenti6-puro-GFP-circHIPK3,“LV-circHIPK3”) or the control empty vector (“LV-Vec”), were selected by puromycin to achieve stable cell lines; Cells were further treated with dexamethasone (“DEX”, 1 mM) for indicated time periods; circHIPK3 expression was detected by qPCR (A); Cell viability and death were tested by MTT assay (B) and LDH release assay (C), respectively; Cell apoptosis was tested listed assays (DeG). Mitochondrial depolarization (JC-1 dye assay, H), CyPD-ANT-1 mitochondrial association (“Mito-IP” assay, I) and cytochrome C (“Cyto-C”) cytosol release (J) were tested as well. Expression of listed proteins were quantified (E, I and J). For each assay, n ¼ 5. *P < 0.05vs. “Ctrl” treatment in “LV-Vec” cells. #P < 0.05vs. vs. “DEX” treatment in “LV-Vec” cells. Experiments in this figure were repeated four times to ensure consistency of results.

in human osteoblasts. At least nine miRNAs, including miR-124, miR-152, miR-193a, miR-29a, miR-29b, miR-338, miR-379, miR-584 and miR-654, are potential targets of circHIPK3 [15]. miR-124 was shown to inhibit

osteoblast differentiation [37,38]. In the current study, we found that DEX treatment in human osteoblasts induced circHIPK3 reduction and miR-124 accumulation, accompanied with downregulation of miR-124 pro-survival targets: SphK1 and STAT3.

Please cite this article as: C.-y. Zhu et al., Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.073

C.-y. Zhu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

0.8

circHIPK3 (vs. U6)

Viability (MTT OD)

*

0.6

0.4

0.2

Ctrl

0.5

*

0.4 0.3

*

0

DEX (1 μM), 12h

D

#

0.2

Ctrl

LV-Vec

12.11%

30 25 20

#

15

*

10 5 0

Ctrl

*

30

LV-circHIPK3

0.60%

DEX (1 μM), 24h

E

LV-Vec LV-circHIPK3

4.17%

Annexin V (%)

Propidium iodide (PI)

7.45%

9.69%

20

#

*

10

5.76% 0

H

OB-6 cells

Ctrl

OB-6 cells

0.8

0.06

0.03

0

* Ctrl si-C

#

*

-1

#

Viability (MTT OD)

circHIPK3 (vs. U6)

0.09 0.6

0.2

0

-2

*

0.4

Ctrl

si-C

-1

-2

Primary osteoblasts

circHIPK3 (vs. U6)

0.12

0.08

0.04

0

*

#

#

-1

-2

* * Ctrl si-C

si-circHIPK3 DEX (1 μM), 12h

Viability (MTT OD)

0.6

0.4

0.2

0

* #

#

-1

-2

* * Ctrl si-C

si-circHIPK3 DEX (1 μM), 24h

#

30

0.6

F

LV-Vec LV-circHIPK3

Primary osteoblasts

*

#

0.4

*

-Cyto C 0.31

0.2

0.10 (vs. Erk1/2)

-Erk1/2 DEX (1 μM), 12h 0

Ctrl

DEX (1 μM), 6h

#

* *

*

20

10

0

Ctrl

si-C

-1

-2

si-circHIPK3

DEX (1 μM), 24h

K

OB-6 cells

40

si-circHIPK3

DEX (1 μM), 12h

Primary osteoblasts

#

* *

si-circHIPK3

J

#

Primary osteoblasts

DEX (1 μM), 20h

I

DEX (1 μM), 24h

L Cell death (Medium LDH %)

G

Annexin V

Cell death (Medium LDH %)

Annexin V

*

LV-Vec LV-circHIPK3

35

DEX (1 μM), 24h

Primary osteoblasts DEX (1 μM), 20h

Primary osteoblasts 40

0.1

*

0

LV-Vec LV-circHIPK3

0.6

#

C

Primary osteoblasts

LV -V ec LV -c irc H IP K 3

B

Primary osteoblasts

Mito depolarization (JC-1 OD)

A

Cell death (Medium LDH %)

6

Primary osteoblasts # 60

#

*

-1

-2

* 40

*

20

0

Ctrl si-C

si-circHIPK3 DEX (1 μM), 24h

Fig. 3. circHIPK3 downregulation mediates DEX-induced cytotoxicity in human osteoblasts. The primary human osteoblasts, transduced with the lentiviral circHIPK3 expression construct (pLenti6-puro-GFP-circHIPK3,“LV-circHIPK3”) or the control empty vector (“LV-Vec”), were further treated with dexamethasone (“DEX”, 1 mM) for indicated time periods; circHIPK3 expression was detected by qPCR (A); Cell viability and death were tested by MTT assay (B) and LDH release assay (C), respectively; Cell apoptosis was tested Annexin V FACS assay (D). Mitochondrial depolarization (JC-1 dye assay, E) and cytochrome C (“Cyto-C”) cytosol release (F) were tested as well. OB-6 cells (GeI) or the primary human osteoblasts (JeL) were transfected with the applied circHIPK3 siRNA ([“si-circHIPK3 (1/-2)”], 500 nM, for 48 h) or the scramble control siRNA (“si-C00 , 500 nM, for 48 h), treated with dexamethasone (“DEX”, 1 mM) for applied time periods, circHIPK3 expression (G and J), cell viability (H and K) and death (I and L) were tested. Expression of listed proteins were quantified (F). For each assay, n ¼ 5. *P < 0.05vs. “Ctrl” treatment. #P < 0.05 vs. vs. “DEX” treatment in “LV-Vec”/“si-C” cells. Experiments in this figure were repeated four times to ensure consistency of results.

Conversely, miR-124 inhibition protected human osteoblasts from DEX-induced cell death. Mimicking the action of DEX by forced overexpression of miR-124 induced significant cytotoxicity in OB-

6 cells, and DEX treatment failed to induce further OB-6 cell death. Taken together, miR-124 accumulation following circHIPK3 downregulation appears to be the primary mechanism of DEX-

Please cite this article as: C.-y. Zhu et al., Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.073

C.-y. Zhu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

***

0.2

DEX (1 μM), 24h

0

E

45kD-

SphK1 0.78

1

STAT3 0.25 (vs. Tubulin)

Tubulin

20 15 10 5 0

H

IP

miR-C +DEX LV-miR-124

*

0.2

Ctrl

miRCi miR-124i

DEX (1 μM), 24h

Cell death (Medium LDH %)

*

40

*

30 20 10 0

DEX (1 μM), 24h

P > 0.05

#

* Ctrl miRCi miR-124i

* *

0.3

0

miR-C +DEX LV-miR-124

DEX (1 μM), 24h

0.20 (vs. Tubulin)

* 2

0.78

*

0

*

*

20 10 0

miR-C +DEX LV-miR-124

Dexamethasone (DEX) circHIPK3 downregulation

STAT3

86kD-

55kD-

30

P > 0.05

SphK1

45kD-

1

K

0.76

3

40

miR-124 accmulation

0.21 (vs. Tubulin)

Tubulin

Osteoblast death

Ve LV c -c irc H IP K3 si -c irc H IP K3

-c

#

0.4

0.6

J miR-124 expression (vs. “LV-Vec”)

K3

* *

25

si

-c LV

Viability (MTT OD)

0.6

Ctrl

0.9

I

Primary osteoblasts

0

30

K3

55kD-

0

G

irc

H

IP

-V

irc

LV

0.80

*

0

H

0.20 (vs. Tubulin)

86kD-

*

0.4

DEX (1 μM), 24h

Viability (MTT OD)

Ctrl

2

#

P > 0.05

DEX (1 μM), 12h

*

3

Ctrl

F

4

ec

miR-124 expression (vs. “LV-Vec”)

D

*

10

0.8

LV -

Ctrl

#

Relative expression (vs. Ctrl)

0

20

***

miRCi miR-152i miR-338i miR-124i

Cell death (Medium LDH %)

P > 0.05

0.4

***

miRCi miR-152i miR-338i miR-124i

P > 0.05

1.2

DEX (1 μM), 12h

*

C

P > 0.05 30

Ctrl

#

Cell death (Medium LDH %)

0.6

Viability (MTT OD)

B

miRCi miR-152i miR-338i miR-124i

Histone DNA ELISA OD

OB-6 cells

A

7

Fig. 4. miR-124 inhibition protects human osteoblasts from DEX-induced cytotoxicity. OB-6 cells (AeC) or the primary human osteoblasts (H) were transfected with 500 nM of miR-152 inhibitor (“miR-152i”), miR-338 inhibitor (“miR-338i”), miR-124 inhibitor (“miR-124i”) or the non-sense control miR inhibitor control (“miRCi”) for 48 h, followed by dexamethasone (“DEX”, 1 mM) treatment for 24 h, cell viability, death and apoptosis and were tested by MTT (A and H), LDH (B and H) and Histone DNA ELISA (C) assays, respectively. Expression of miR-124 in OB-6 cells (D) and primary human osteoblasts (I) with LV-circHIPK3, the control empty vector (“LV-Vec”), or circHIPK3 siRNA ([“si-circHIPK3 (1)”], 500 nM, 48 h) was shown. OB-6 cells or the primary human osteoblasts were treated with dexamethasone (“DEX”, 1 mM) for 12 h, expression of listed proteins in total cell lysates was shown (E and J). Stable OB-6 cells, with the lentiviral pri-miR-124 construct (“LV-miR-124”) or control miR construct (“miR-C00 ), were treated with dexamethasone (“DEX”, 1 mM) for 24 h, miR-124 expression (F), cell viability (G) and death (G) were tested similarly. The proposed signaling pathway of this study (K). Expression of listed proteins were quantified (E and J). For each assay, n ¼ 5. *P < 0.05vs. “Ctrl” treatment (A-C and H). #P < 0.05vs. “DEX” treatment in “miRCi” cells (A-C and H).*P < 0.05vs.“LV-Vec”/“miR-C” cells (D, F, G and I). Experiments in this figure were repeated three times to ensure consistency of results.

induced cytotoxicity in human osteoblasts (see the proposed signaling pathway in Fig. 4K). This is further supported by the fact that miR-124 levels are upregulated in necrotic femoral head tissues. Although the underlying mechanism warrants further characterization, our results highlight that the circHIPK3-miR-124 cascade is a novel target for the protection of human osteoblasts from DEX.

Fundings The study was supported by the National Natural Science Foundation of China (81171712, 81602359 and 81402475). Grants from Natural Science Foundation of Jiangsu Province (BK20151213) and Innovation Project of Jiangsu Province (BK 201423 and BK20160340).The authors have no conflict of interests, and by Second Affiliated Hospital of Soochow University Preponderant

Please cite this article as: C.-y. Zhu et al., Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.073

8

C.-y. Zhu et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

Clinic Discipline Group Project (XKQ2015003). Conflict of interests Authors have no conflict of interests. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.06.073. References [1] D. den Uyl, I.E. Bultink, W.F. Lems, Advances in glucocorticoid-induced osteoporosis, Curr. Rheumatol. Rep. 13 (2011) 233e240. [2] R.S. Weinstein, Clinical practice. Glucocorticoid-induced bone disease, N. Engl. J. Med. 365 (2011) 62e70. [3] M.A. Kerachian, C. Seguin, E.J. Harvey, Glucocorticoids in osteonecrosis of the femoral head: a new understanding of the mechanisms of action, J. Steroid Biochem. Mol. Biol. 114 (2009) 121e128. [4] B. Souttou, D. Raulais, M. Vigny, Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways, J. Cell. Physiol. 187 (2001) 59e64. [5] H.A. Himburg, G.G. Muramoto, P. Daher, S.K. Meadows, J.L. Russell, P. Doan, J.T. Chi, A.B. Salter, W.E. Lento, T. Reya, N.J. Chao, J.P. Chute, Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells, Nat. Med. 16 (2010) 475e482. [6] M. Almeida, L. Han, E. Ambrogini, R.S. Weinstein, S.C. Manolagas, Glucocorticoids and tumor necrosis factor alpha increase oxidative stress and suppress Wnt protein signaling in osteoblasts, J. Biol. Chem. 286 (2011) 44326e44335. [7] S.I. Yun, H.Y. Yoon, S.Y. Jeong, Y.S. Chung, Glucocorticoid induces apoptosis of osteoblast cells through the activation of glycogen synthase kinase 3beta, J. Bone Miner. Metab. 27 (2009) 140e148. [8] S. Guo, Y. Xie, J.B. Fan, F. Ji, S. Wang, H. Fei, alpha-Melanocyte stimulating hormone attenuates dexamethasone-induced osteoblast damages through activating melanocortin receptor 4-SphK1 signaling, Biochem. Biophys. Res. Commun. 469 (2016) 281e287. [9] S. Guo, L. Mao, F. Ji, S. Wang, Y. Xie, H. Fei, X.D. Wang, Activating AMPactivated protein kinase by an alpha1 selective activator Compound 13 attenuates dexamethasone-induced osteoblast cell death, Biochem. Biophys. Res. Commun. 471 (2016) 545e552. [10] F. Ji, L. Mao, Y. Liu, X. Cao, Y. Xie, S. Wang, H. Fei, K6PC-5, a novel sphingosine kinase 1 (SphK1) activator, alleviates dexamethasone-induced damages to osteoblasts through activating SphK1-Akt signaling, Biochem. Biophys. Res. Commun. 458 (2015) 568e575. [11] Y.F. Zhen, G.D. Wang, L.Q. Zhu, S.P. Tan, F.Y. Zhang, X.Z. Zhou, X.D. Wang, P53 dependent mitochondrial permeability transition pore opening is required for dexamethasone-induced death of osteoblasts, J. Cell. Physiol. 229 (2014) 1475e1483. [12] X.Y. Zhang, H.J. Shan, P. Zhang, C. She, X.Z. Zhou, LncRNA EPIC1 protects human osteoblasts from dexamethasone-induced cell death, Biochem. Biophys. Res. Commun. 503 (2018) 2255e2262. [13] K.K. Ebbesen, T.B. Hansen, J. Kjems, Insights into circular RNA biology, RNA Biol. 14 (2017) 1035e1045. [14] X. Meng, X. Li, P. Zhang, J. Wang, Y. Zhou, M. Chen, Circular RNA: an emerging key player in RNA world, Briefings Bioinf. 18 (2017) 547e557. [15] Q. Zheng, C. Bao, W. Guo, S. Li, J. Chen, B. Chen, Y. Luo, D. Lyu, Y. Li, G. Shi, L. Liang, J. Gu, X. He, S. Huang, Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs, Nat. Commun. 7 (2016) 11215. [16] K. Shan, C. Liu, B.H. Liu, X. Chen, R. Dong, X. Liu, Y.Y. Zhang, B. Liu, S.J. Zhang, J.J. Wang, S.H. Zhang, J.H. Wu, C. Zhao, B. Yan, Circular noncoding RNA HIPK3 mediates retinal vascular dysfunction in diabetes mellitus, Circulation 136 (2017) 1629e1642. [17] K. Zeng, X. Chen, M. Xu, X. Liu, X. Hu, T. Xu, H. Sun, Y. Pan, B. He, S. Wang, CircHIPK3 promotes colorectal cancer growth and metastasis by sponging miR-7, Cell Death Dis. 9 (2018) 417.

[18] G. Chen, Y. Shi, M. Liu, J. Sun, circHIPK3 regulates cell proliferation and migration by sponging miR-124 and regulating AQP3 expression in hepatocellular carcinoma, Cell Death Dis. 9 (2018) 175. [19] D. Kai, L. Yannian, C. Yitian, G. Dinghao, Z. Xin, J. Wu, Circular RNA HIPK3 promotes gallbladder cancer cell growth by sponging microRNA-124, Biochem. Biophys. Res. Commun. 503 (2018) 863e869. [20] H. Ding, T. Wang, D. Xu, B. Cha, J. Liu, Y. Li, Dexamethasone-induced apoptosis of osteocytic and osteoblastic cells is mediated by TAK1 activation, Biochem. Biophys. Res. Commun. 460 (2015) 157e163. [21] J.W. Ruan, C. Yao, J.Y. Bai, X.Z. Zhou, microRNA-29a inhibition induces Gab1 upregulation to protect OB-6 human osteoblasts from hydrogen peroxide, Biochem. Biophys. Res. Commun. 503 (2018) 607e614. [22] S. Zhao, L. Mao, S.G. Wang, F.L. Chen, F. Ji, H.D. Fei, MicroRNA-200a activates Nrf2 signaling to protect osteoblasts from dexamethasone, Oncotarget 8 (2017) 104867e104876. [23] H.D. Fei, Q. Yuan, L. Mao, F.L. Chen, Z.H. Cui, S. Tao, F. Ji, Assessment of GSK1904529A as a promising anti-osteosarcoma agent, Oncotarget 8 (2017) 49646e49654. [24] J.B. Fan, J.W. Ruan, W. Liu, L.Q. Zhu, X.H. Zhu, H. Yi, S.Y. Cui, J.N. Zhao, Z.M. Cui, miR-135b expression downregulates Ppm1e to activate AMPK signaling and protect osteoblastic cells from dexamethasone, Oncotarget 7 (2016) 70613e70622. [25] Y. Xu, S.J. Fang, L.J. Zhu, L.Q. Zhu, X.Z. Zhou, DNA-PKcs-SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation, Biochem. Biophys. Res. Commun. 453 (2014) 362e367. [26] S.T. Li, N.N. Chen, Y.B. Qiao, W.L. Zhu, J.W. Ruan, X.Z. Zhou, SC79 rescues osteoblasts from dexamethasone though activating Akt-Nrf2 signaling, Biochem. Biophys. Res. Commun. 479 (2016) 54e60. [27] Y. Cao, G. Yuan, Y. Zhang, R. Lu, High glucose-induced circHIPK3 downregulation mediates endothelial cell injury, Biochem. Biophys. Res. Commun. 507 (2018) 362e368. [28] Y.R. Zhu, Y. Xu, J.F. Fang, F. Zhou, X.W. Deng, Y.Q. Zhang, Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation, Biochem. Biophys. Res. Commun. 451 (2014) 112e118. [29] S.S. Wang, Y. Lv, X.C. Xu, Y. Zuo, Y. Song, G.P. Wu, P.H. Lu, Z.Q. Zhang, M.B. Chen, Triptonide inhibits human nasopharyngeal carcinoma cell growth via disrupting Lnc-RNA THOR-IGF2BP1 signaling, Cancer Lett. 443 (2019) 13e24. [30] M.M. Brooks, S. Neelam, R. Fudala, I. Gryczynski, P.R. Cammarata, Lenticular mitoprotection. Part A: monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3beta inhibitor, SB216763, Mol. Vis. 19 (2013) 1406e1412. [31] L.B. Cheng, L. Cheng, H.E. Bi, Z.Q. Zhang, J. Yao, X.Z. Zhou, Q. Jiang, Alphamelanocyte stimulating hormone protects retinal pigment epithelium cells from oxidative stress through activation of melanocortin 1 receptor-AktmTOR signaling, Biochem. Biophys. Res. Commun. 443 (2014) 447e452. [32] J. Marshall, X.Z. Zhou, G. Chen, S.Q. Yang, Y. Li, Y. Wang, Z.Q. Zhang, Q. Jiang, L. Birnbaumer, C. Cao, Antidepression action of BDNF requires and is mimicked by Galphai1/3 expression in the hippocampus, Proc. Natl. Acad. Sci. U. S. A. 115 (2018) E3549eE3558. [33] H. Yu, Y. Chen, P. Jiang, Circular RNA HIPK3 exerts oncogenic properties through suppression of miR-124 in lung cancer, Biochem. Biophys. Res. Commun. 506 (2018) 455e462. [34] Y. Cheng, Y. Li, Y. Nian, D. Liu, F. Dai, J. Zhang, STAT3 is involved in miR-124mediated suppressive effects on esophageal cancer cells, BMC Canc. 15 (2015) 306. [35] M. Wang, B. Meng, Y. Liu, J. Yu, Q. Chen, Y. Liu, MiR-124 inhibits growth and enhances radiation-induced apoptosis in non-small cell lung cancer by inhibiting STAT3, Cell. Physiol. Biochem. 44 (2017) 2017e2028. [36] J. Xia, Z. Wu, C. Yu, W. He, H. Zheng, Y. He, W. Jian, L. Chen, L. Zhang, W. Li, miR-124 inhibits cell proliferation in gastric cancer through down-regulation of SPHK1, J. Pathol. 227 (2012) 470e480. [37] S.L. Tang, Q.H. Huang, L.G. Wu, C. Liu, A.L. Cai, MiR-124 regulates osteoblast differentiation through GSK-3beta in ankylosing spondylitis, Eur. Rev. Med. Pharmacol. Sci. 22 (2018) 6616e6624. [38] A.S. Qadir, S. Um, H. Lee, K. Baek, B.M. Seo, G. Lee, G.S. Kim, K.M. Woo, H.M. Ryoo, J.H. Baek, miR-124 negatively regulates osteogenic differentiation and in vivo bone formation of mesenchymal stem cells, J. Cell. Biochem. 116 (2015) 730e742.

Please cite this article as: C.-y. Zhu et al., Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.06.073