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Upregulation of miR-199 attenuates TNF-α-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5
Biochemical and Biophysical Research Communications xxx (2018) 1e8
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Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5 Wei Wang a, Zhao Guo b, Sidong Yang a, Hui Wang a, Wenyuan Ding a, * a b
Orthopedics Department, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China Orthopedics Department, Affiliated Hospital of Hebei University, Baoding, Hebei, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2018 Accepted 30 September 2018 Available online xxx
MicroRNA-199 has been reported to play a potential role in the apoptosis of Human nucleus pulposus cells. However, the effect of miR-199 in regulating Human nucleus pulposus cell injury induced by TNF-a has not been previously illustrated. This study searched to probe the effect and the molecular mechanism of miR-199 on Human nucleus pulposus cell injury induced by TNF-a. Using the TNF-a model of Human nucleus pulposus cell in vitro, we found that miR-199 was extremely decreased in Human nucleus pulposus cells after TNF-a treatment. Knockdown the expression of miR-199 by recombinant adenoassociated viral vector infection markedly promoted the apoptosis of Human nucleus pulposus cells induced by TNF-a treatment, whereas miR-199 overexpression significantly decreased Human nucleus pulposus cell apoptosis. Both Dual-luciferase reporter and western blot assay proved that MAP3K5 was a direct target gene of miR-199, and miR-199 inhibited the expression of MAP3K5 via binding to its 30 -UTR. Furthermore, we proved that overexpression of miR-199 could inhibit the expression of MAP3K5 at the transcription and translation levels, whereas the inhibition of miR-199 could upregulate the expression of MAP3K5. Moreover, MAP3K5 was highly expressed in TNF-a treated Human nucleus pulposus cells and the apoptosis rate induced by TNF-a was associated with the increase in MAP3K5 expression. Importantly, knockdown the expression of MAP3K5 apparently abrogated the inhibitory effect of miR199 mimics on TNF-a induced Human nucleus pulposus cell apoptosis. In conclusion, these results indicate that upregulation of miR-199 could inhibit Human nucleus pulposus cells injury through downregulation of MAP3K5 expression, providing an important molecular target mechanism for Human nucleus pulposus cells injury. © 2018 Elsevier Inc. All rights reserved.
Keywords: miR-199 TNF-a Human nucleus pulposus cell Apoptosis MAP3K5
1. Introduction Intervertebral disc degeneration (IVDD) is one of most serious spinal degenerative diseases, which leads to disability, low health related quality of life and economic burden [1,2]. However, the mechanisms underlying IVDD have not yet been fully elucidated. IVDD is characterized by decreased nucleus pulposus (NP) cell quantity and function, which lead to NP injury [3,4]. One of the most important causes of IVDD is thought to be the excessive apoptosis of NP cells, and recent evidence has demonstrated that TNF-a may induce NP cells apoptosis [3], demonstrating that
* Corresponding author. Orthopedics Department, The Third Hospital of Hebei Medical University, No.139 Ziqiang road, Shijiazhuang, 050051, Hebei Province, China. E-mail address: [email protected] (W. Ding).
apoptosis of NP cells plays an important role in the development of IVDD. Therefore, the control of apoptosis is of great significance for the treatment of NP cell TNF-a injury. MicroRNAs (miRNAs), a kind of endogenous noncoding RNAs composed of about 20e24 nucleotides, regulate intracellular gene posttranscriptional expression by interacting with the 30 -untranslated region (30 -UTR) of the target mRNA resulting in translational repression or degradation [4]. A large number of studies have proved that miRNAs play a key role in the development and progression of various diseases, including TNF-a-induced injury [5]. MiRNAs have brought innovative investigations into the regulation of TNF-a-induced injuries. It has been reported that miRNA-103/ 107 regulated TNF-a-induced injury by targeting cell death ligands just like tumor necrosis factor (TNF) or Fas-associating protein with a novel death domain (FADD) [6]. MiR-199 regulates GATA-binding protein 4 (GATA-4) expression on the myocardial cells to decrease TNF-a-induced injuries [7e9]. However, the role of
https://doi.org/10.1016/j.bbrc.2018.09.194 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
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W. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
miRNAs in regulating TNF-a-induced injury is still mainly unknown and requires intensive investigation [10e12]. MiR-199 was considered to play a potential role in apoptosis of NP cells. However, the specific role of miR-199 in apoptosis during TNF-a-induced injury in NP cells has not been previously published. MAP3K5, mitogen-activated protein kinase kinase kinase 5, a serine/threonine protein kinase, is a member of the MAPKKK family (also known as mitogen-activated protein kinase kinase kinase 5, MAPKKK5) and is proved to be activated by a number of stresses, such as endoplasmic reticulum stress (ERS), nitrosative stress or reactive oxygen species (ROS) and cell death ligands like Fasassociating protein with a novel death domain (FADD) and TNF [13]. MAP3K5 has been reported to be associated with apoptosis progress in numerous tissues, such as brain, muscle and heart tissues. MAP3K5 was previously proved to play an important role as an upstream regulatory elements of p38 and JNK in myocardial apoptosis [14]. Knockdown the expression of MAP3K5 has an antiapoptotic effect on myocardium and cancer cells [15]. Although researches in the last decades proved that MAP3K5 is an important regulatory element of apoptosis, the association between MAP3K5 and miRNAs has rarely been investigated.
2.5. LDH activity measurement The lactate dehydrogenase (LDH) activity in the culture medium was measured using an LDH assay kit (Dojindo Molecular Technologies, Shanghai, China) for the detection of cell injury according to the manufacturer's instructions. 2.6. TUNEL determination of Human nucleus pulposus cell apoptosis
2. Materials and methods
Cells were seeded in 6-well plates with placed slides. After different treatment, cells were washed with phosphate-buffered saline (PBS), the slides were immersed in 4% formaldehyde in PBS at 4 C and then in 3% H2O2 and in 0.5% Triton X-100 for 5e10 min at room temperature. Equilibration buffer (100 ml) was added, and the slides were incubated at room temperature for 10 min. TdT reaction mix (50 ml) was then added, and the cells were incubated for 60 min at 37 C. The slides were then immersed in 2 SSC for 15 min. The localized green fluorescence of apoptotic cells was detected by fluorescence microscopy. For the quantification of TUNEL-positive (apoptotic) cells, a minimum of 200 cells was counted per group, and the percentage of the positively labeled cells was calculated.
2.1. Cell culture and treatments
2.7. Quantitative real-time PCR (qPCR)
The Human nucleus pulposus cells were preserved in our laboratory and were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) at 37 C and 5% CO2.
Total RNA was extracted from Human nucleus pulposus cells with TRIzol reagent (Ambion, Waltham, MA, USA), and then reverse transcribed to cDNA using AMV reverse transcriptase is provided in a RT system kit (Promega, Fitchburg, WI, USA), then the cDNA was used as a template for amplification using a reverse transcription kit (Promega, Fitchburg, WI, USA). Quantitative realtime PCR was performed with specific primers of MAP3K5 (sense:50 -CGTAGCCTCTTGGTCCTTTATC-30 ,antisense:50 -GGAAGTC TTTCTGCTCTCCTTC-30 ); GAPDH(sense:50 -CATGGGTGTGAACCATGAGAA-30 ,antisense:50 -GGTCATGAGTCCTTCCACGAT-30 ) using the SYBR Green I real-time PCR kit (Promega, Fitchburg, WI, USA) and the relative gene expression was calculated via the 2-△△Ct method. GAPDH and U6 were used for the normalizing the expression of mRNA and miRNA. Each sample was detected in triplicate.
2.2. Cell transfection AAV (recombinant adenovirus-associated virus) vectors were constructed by GenePharma Technologies (GenePharma, Shanghai, China). Human nucleus pulposus cells were infected with AAV-miR Control (miR Ctrl), AAV-anti-miR Control (anti-miR Ctrl), AAV-miR199 (miR-199), AAV-anti-miR-199 (anti-miR-199), MAP3K5 expression plasmid (MAP3K5 plasmid), or empty plasmid vector (vector plasmid) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and incubated for 8 h. Cells were then subjected to TNF-a treatment and finally harvested for further analysis. 2.3. Human nucleus pulposus cells treated in TNF-a condition Human nucleus pulposus cells were cultured in TNF-a condition in vitro as an injury model. As previously described [5], the cells were cultured in TNF-a condition at 37 C in a humidified atmosphere with 5% O2 for different time points, and then harvested for further analysis. 2.4. MTT assay MTT assay kit (Dojindo Molecular Technologies, Shanghai, China) was carried for the measurement of cell viability. Generaly, Human nucleus pulposus cells were digested and then seeded in 96-well plates (5 103/well) for 12 h, after that, cells were infected with AAV related vectors and then subjected to TNF-a treatment for indicated times. Then the cells in each well were incubated with fresh culture medium containing MTT solution (5 mg/mL) for 4 h at 37 C. Finally, 100 mL of DMSO(dimethyl sulfoxide) were added to each well, and the absorbance was checked with an automatic microplate reader at 490 nm (Bio-Tek, Winooski, VA, USA).
2.8. Western blot detection Proteins isolated from Human nucleus pulposus cells were detected by SDS-PAGE and then transferred to a NC membrane (Millipore, Bedford, MA, USA). After blocking with BSA(5% in TBST) for 1.5 h at 37 C, the membranes were probed with the primary antibodies against MAP3K5, Bcl-2, Bax, Caspase-3, and cleaved Caspase-3 antibodies at 4 C for 12 h. The membranes were then washed with TBST(with 0.1% Tween-20) three times (each for 10 min) and incubated with corresponding secondary antibodies for 2 h at 37 C. The expressions of corresponding protein were detected using the enhanced chemiluminescent (ECL) system, and quantified using an image Quantity One System (Bio-Rad, Richmond, CA, USA). b-actin was used as control. 2.9. Luciferase reporter assay HEK293T cells were seeded into 24-well plates (1 104/well). The pGL3 luciferase promoter vectors (Promega, Madison, WI, USA) containing MAP3K5 mRNA 30 -UTR or 30 -UTR-mut (MAP3K5 mRNA mutated forms) were cotransfected with AAV-miR-199 or corresponding controls into HEK293 cells using Lipofectamine 3000 (Invitrogen, USA) for 24 h. Cells were then harvested and lysed on
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
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ice, and the luciferase activity was detected with the Luciferase reporter assay kit (Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. All detections were executed at least three times. The relative luciferase activities were normalized to the corresponding controls. 2.10. Statistical analysis All experiments were executed independently repeated at least three times. All results were demonstrated as means ± S.E.M. Student's t-test and one-way ANOVA analysis were used to determine the significance differences between two experimental groups with statistic software SPSS 20.0. Values of P < 0.05 were considered statistically significant.
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(P < 0.05), as shown in Fig. 1C, indicating that miR-199 may play a significant role in TNF-a injury in Human nucleus pulposus cells. There were no obvious differences in cell viability, LDH viability and miR-199 expression between TNF-a treatment for 24 h and TNF-a treatment for 48 h group, thus Human nucleus pulposus cells treated with TNF-a for 24 h were regard as TNF-a injury model. To investigate whether miR-199 plays an important role in protection against TNF-a injury, Human nucleus pulposus cells were stably transfected with miR-199 precursor or inhibitor by adenovirusmediated transfection. As shown in Fig. 1D, Human nucleus pulposus cells transfected with miR-199 obviously increased miR-199 expression, whereas the expression of miR-199 in Human nucleus pulposus cells with miR-199 inhibitor (anti-miR-199) was significantly decreased (P < 0.05). Non-transfected or negativetransfected cells were set up as controls.
3. Results 3.1. TNF-a decreases miR-199 expression in Human nucleus pulposus cells Results shown that the cell viability was decreased (Fig. 1A) and the LDH viability was highly increased (Fig. 1B) in Human nucleus pulposus cells subjected to TNF-a at 0, 6, 12, 24, 48 h differrnt time points (P < 0.05), demonstrating that Human nucleus pulposus cells suffered TNF-a injury. MiR-199 is significantly downregulated at 0, 6, 12, 24, 48 h differrnt time points under TNF-a treatment
3.2. The downregulation of miR-199 promotes Human nucleus pulposus cells apoptosis under TNF-a treatment The cell viability decreased (Fig. 2A) and the LDH viability increased induced by TNF-a (Fig. 2B) were obviously reversed by miR-199 transfection but reverted by anti-miR-199 transfection. The descending expression of miR-199 obviously increased TNF-ainduced cell apoptosis, whereas miR-199 overexpression significantly reduced Human nucleus pulposus cell apoptosis, as shown by TUNEL staining (Fig. 2C). Furthermore, the expression of pro-
Fig. 1. MiR-199 is downregulated in TNF-a-treated Human nucleus pulposus cells. Cell viability was highly decreased (A) and LDH viability was significantly increased (B) during 0e48 h TNF-a treatment in Human nucleus pulposus cells. (C) The relative expression of miR-199 was obviously downregulated during 0e48 h TNF-a treatment in Human nucleus pulposus cells. (D) Analysis of miR-199 expression in Human nucleus pulposus cells transfected with miR-199 or miR-199 inhibitor by qPCR. MiR-199 Ctrl, miR-199, anti-miR Ctrl, anti-miR-199, correspondingly, transfected with AAV control miRNA, AVV-miR-199, AAV-control anti-miRNA and AAV-anti-miR-199. *P < 0.05 vs. control, n ¼ 3.
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
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Fig. 2. The downregulation of miR-199 under TNF-a treatment promotes the apoptosis of Human nucleus pulposus cells in vitro. (A) MiR-199 increased cell viability in Human nucleus pulposus cell treated with TNF-a detected by MTT assay. (B) LDH viability was increased in Human nucleus pulposus cells under TNF-a by anti-miR-199 transfection. (C) MiR-199 inhibited apoptosis in Human nucleus pulposus cells after 24 h of TNF-a. (D) MiR-199 influenced pro- and anti-apoptotic relative protein expression in TNF-a treated Human nucleus pulposus cells detected by western blot. △P < 0.05 vs. control; *P < 0.05 vs. TNF-a for 24 h, n ¼ 3.
and anti-apoptotic relative proteins of the Bcl-2 and casepase family, were detected by western blot (Fig. 2D). TNF-a treatment promoted the expression of both Bax and cleaved-caspase-3, and the ratio of Bax/Bcl-2 increased as compared with the corresponding control groups (P < 0.05). This phenomenon was further enhanced in the case of miR-199 downregulation, while this phenomenon was partially reverted in the presence of miR-199 (P < 0.05). These results suggest that miR-199 plays a protective role in TNF-a-induced Human nucleus pulposus cell apoptosis.
3.3. MiR-199 targets MAP3K5 and reduces its expression in TNF-a treated Human nucleus pulposus cells To explore miR-199's biological function and its molecular mechanism, we investigated the possible target gene of miR-199 that contribute to TNF-a induced injury. Bio-informatics analysis indicated that MAP3K5, which is an important gene for TNF-ainduced Human nucleus pulposus cell injury, was also a target gene of miR-199 (Fig. 3A). To investigate the direct target association
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
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Fig. 3. MAP3K5 is a direct target gene of miR-199. (A) Presumed binding nucleotide sequences in the 30 -UTR of MAP3K5 mRNA with miR-199. (B) The interaction between the miR199 and MAP3K5 mRNA 30 -UTR detected by Dual-Luciferase Reporter Assay after transfection for 24 h (C and D) The mRNA and protein expression of MAP3K5 were detected by qPCR and western blot in Human nucleus pulposus cells transfected with miR-199 or anti-miR-199. Relative expression was measured using image analysis software.△P < 0.05 vs. control, n ¼ 3. (E and F) The expression of MAP3K5 in mRNA and protein levels in Human nucleus pulposus cells after TNF-a treatment. △P < 0.05 vs. control, n ¼ 3.
between miR-199 and MAP3K5, a dual-luciferase reporter assay system was conducted. The overexpression of miR-199 could significantly reduce the luciferase activity of the wild type MAP3K5 30 -UTR reporter gene, but not the mutant reporter gene (Fig. 3B), indicating that miR-199 targeted MAP3K5 by binding to its 30 -UTR area. Moreover, to prove whether miR-199 regulates MAP3K5 expression in TNF-a-treated Human nucleus pulposus cells, we
detected the expression of MAP3K5 by qPCR and western blot analysis in miR-199 or anti-miR-199-transfected Human nucleus pulposus cells. These results proved that overexpression of miR-199 could obviously reduce the expression of MAP3K5 both at the mRNA and protein levels, and inhibition the expression of miR-199 could promote MAP3K5 expression (Fig. 3C and D). Furthermore, we found that MAP3K5 was highly expressed in TNF-a treated
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
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Human nucleus pulposus cells (Fig. 3E and F). 3.4. MAP3K5 contributes to the effect of miR-199 induced cell apoptosis under TNF-a treatment To further investigate the molecular mechanism that miR-199
downregulation enhanced TNF-a-induced Human nucleus pulposus cell apoptosis, MAP3K5 expression plasmid and miR-199 were co-transfected into Human nucleus pulposus cells. As shown in Fig. 4A, the mRNA and protein expression of MAP3K5 decreased by miR-199 and was obviously reverted by MAP3K5 expression plasmid transfection in TNF-a treated Human nucleus pulposus
Fig. 4. MAP3K5 is involved in TNF-a-induced apoptosis in Human nucleus pulposus cells. (A and B) Detection of the expression of MAP3K5 in mRNA and protein levels in TNF-a treated Human nucleus pulposus cells co-transfected with miR-199 and MAP3K5. MAP3K5 overexpression reversed the protective effect of miR-199 on cell viability (C) and LDH activity (D) in TNF-a treated Human nucleus pulposus cells. (E and F) The inhibition effect of miR-199 on TNF-a-induce apoptosis was reverted when MAP3K5 expression plasmid was transfected. Control cells were transfected with control miRNA, miR-199 and empty vector cells were treated with miR-199 and empty plasmid vector, miR-199 and MAP3K5 cells were transfected with miR-199 and MAP3K5 expression plasmid. △P < 0.05 vs. miR-ctrl; *P < 0.05 vs. miR-199 or MAP3K5,n ¼ 3.
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
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cells (Fig. 4A and B). Additionally, Human nucleus pulposus cell viability was obviously decreased after MAP3K5 expression plasmid transfection (Fig. 4C). Also, the decrease in LDH viability caused by miR-199 was highly increased by MAP3K5 expression plasmid transfection (Fig. 4D). Importantly, the inhibitory effect of miR-199 on TNF-a induced-apoptosis was obviously inhibited by MAP3K5 expression plasmid transfection (Fig. 4E and F). These results demonstrated that the overexpression of MAP3K5 abolished the inhibitory effects of miR-199 on TNF-a-induced apoptosis of Human nucleus pulposus cells.
downregulated miRNA in TNF-a treated Human nucleus pulposus cells, plays a significant role in TNF-a-induced apoptosis in Human nucleus pulposus cells by regulating MAP3K5. Based on these results, miR-199 could serve as a potential target for the prevention of Human nucleus pulposus cells apoptosis under TNF-a, and the further research in vivo should be conducted.
4. Discussion
Transparency document
IVDD is characterized by an imbalance between survival and injury in the NP [3,4]. Inflammatory cytokines, such as TNF-a and IL1-b, facilitate NP injury either by directly inhibiting miRNAs expression or by increasing apoptosis related protein expression [3e5]. In the current study, we showed for the first time that MAP3K5 expression levels are elevated in degenerated human IVDs and plays critical role in the pathophysiology of inflammationdriven IVDD. Therefore, it is of great significant to demonstrate the potential molecular mechanisms of NP cell injury under TNF-a in IVDD. In the present research, it was proved that the expression of miR-199 was obviously decreased in Human nucleus pulposus cells after TNF-a treatment. Thus, we hypothesized that miR-199 might play a significant role in TNF-a treated Human nucleus pulposus cells and its upregulation or overexpression might attenuate TNFa-induced injury in Human nucleus pulposus cells. Our results demonstrated that the downregulation of miR-199 was correlated with an promotion of apoptosis in TNF-a treated Human nucleus pulposus cells. Furthermore, we identified that MAP3K5, which was expressed at high levels in TNF-a treated Human nucleus pulposus cells, was a direct target gene of miR-199. Additionally, MAP3K5 was confirmed to be involved in the effect of anti-miR-199 on promotion from TNF-a-induced apoptosis in Human nucleus pulposus cells. Recent researches have demonstrated that numerous miRNAs play important roles in the apoptosis induced by TNF-a. For example, the overexpression of miR-101 attenuated TNF-a-induced apoptosis through induction of autophagy [16e18]. It has also been reported that miR-25 family could inhibit TNF-a and ischemiainduced apoptosis in cerebral through inhibiting Fas/FasL expression [19]. MiR-199 was proved to mediate hepatocellular carcinoma cell apoptosis [20]. MiR-199 was also proved to be involved in regulating apoptosis in large B-cell lymphoma [21]. In this research, we further investigated the importance of miR-199 in TNF-ainduced Human nucleus pulposus cell apoptosis. MAP3K5, one of the most important apoptotic signal-regulating kinases, has been proved to participate in cell proliferation [22] and survival [23]. MAP3K5 was reported to be able to promote TNF-a-induced cardiomyocyte or myocardial stem cell injury [24]. Numerous researches have proved the damage effect of MAP3K5 in proapoptosis by regulating the expression of p38/JNK/NF-kB signaling [25,26]. We investigated and characterized the direct target sequence of the 30 -UTR of MAP3K5 by miR-199. MAP3K5 expression was promoted by miR-199 downregulation and downregulated by miR-199 overexpression. In addition, MAP3K5 overexpression plasmid exacerbate TNF-a induced apoptosis inhibited by miR-199, demonstrating that miR-199 may conduct functions in TNF-a induced apoptosis of Human nucleus pulposus cells by regulating MAP3K5. These findings imply that MAP3K5 plays a significant role in TNF-a-induced injury, and miR-199 might be a novel target for preventing NP cells injury under TNF-a. In conclusion, our study demonstrates that miR-199, an important
Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.09.194.
Conflicts of interest The authors declare that they have no conflict of interest.
References [1] S.M. Richardson, G. Kalamegam, P.N. Pushparaj, C. Matta, A. Memic, A. Khademhosseini, R. Mobasheri, F.L. Poletti, J.A. Hoyland, A. Mobasheri, Mesenchymal stem cells in regenerative medicine: focus on articular cartilage and intervertebral disc regeneration, Methods 99 (2016) 69e80. [2] N. Vo, L.J. Niedernhofer, L.A. Nasto, L. Jacobs, P.D. Robbins, J. Kang, C.H. Evans, An overview of underlying causes and animal models for the study of agerelated degenerative disorders of the spine and synovial joints, J. Orthop. Res.: Off. Pub. Orthop. Res. Soc. 31 (2013) 831e837. [3] G. Vadala, F. Russo, A. Di Martino, V. Denaro, Intervertebral disc regeneration: from the degenerative cascade to molecular therapy and tissue engineering, J. Tissue Eng. Regen. Med. 9 (2015) 679e690. [4] J. Liu, H. Tao, H. Wang, F. Dong, R. Zhang, J. Li, P. Ge, P. Song, H. Zhang, P. Xu, X. Liu, C. Shen, Biological behavior of human nucleus pulposus mesenchymal stem cells in response to changes in the acidic environment during intervertebral disc degeneration, Stem Cell. Dev. 26 (2017) 901e911. [5] E. Sonkoly, A. Pivarcsi, Advances in microRNAs: implications for immunity and inflammatory diseases, J. Cell Mol. Med. 13 (2009) 24e38. [6] C.J. Kearney, S.P. Cullen, G.A. Tynan, C.M. Henry, D. Clancy, E.C. Lavelle, S.J. Martin, Necroptosis suppresses inflammation via termination of TNF- or LPS-induced cytokine and chemokine production, Cell Death Differ. 22 (2015) 1313e1327. [7] V. Gupta, A.A. Khan, B.K. Sasi, N.R. Mahapatra, Molecular mechanism of monoamine oxidase A gene regulation under inflammation and ischemia-like conditions: key roles of the transcription factors GATA2, Sp1 and TBP, J. Neurochem. 134 (2015) 21e38. [8] S. Wang, M. Luo, Z. Zhang, J. Gu, J. Chen, K.M. Payne, Y. Tan, Y. Wang, X. Yin, X. Zhang, G.C. Liu, K. Wintergerst, Q. Liu, Y. Zheng, L. Cai, Zinc deficiency exacerbates while zinc supplement attenuates cardiac hypertrophy in high-fat diet-induced obese mice through modulating p38 MAPK-dependent signaling, Toxicol. Lett. 258 (2016) 134e146. [9] A. Briones-Herrera, D. Eugenio-Perez, J.G. Reyes-Ocampo, S. Rivera-Mancia, J. Pedraza-Chaverri, New highlights on the health-improving effects of sulforaphane, Food & function 9 (2018) 2589e2606. [10] P. Prajapati, L. Sripada, K. Singh, K. Bhatelia, R. Singh, R. Singh, TNF-alpha regulates miRNA targeting mitochondrial complex-I and induces cell death in dopaminergic cells, Biochim. Biophys. Acta 1852 (2015) 451e461. [11] T. Alexy, K. Rooney, M. Weber, W.D. Gray, C.D. Searles, TNF-alpha alters the release and transfer of microparticle-encapsulated miRNAs from endothelial cells, Physiol. Genom. 46 (2014) 833e840. [12] H. Li, X. Chen, L. Guan, Q. Qi, G. Shu, Q. Jiang, L. Yuan, Q. Xi, Y. Zhang, MiRNA181a regulates adipogenesis by targeting tumor necrosis factor-alpha (TNFalpha) in the porcine model, PloS One 8 (2013), e71568. [13] X. Xu, T. Liu, A. Zhang, X. Huo, Q. Luo, Z. Chen, L. Yu, Q. Li, L. Liu, Z.R. Lun, J. Shen, Reactive oxygen species-triggered trophoblast apoptosis is initiated by endoplasmic reticulum stress via activation of caspase-12, CHOP, and the JNK pathway in Toxoplasma gondii infection in mice, Infect. Immun. 80 (2012) 2121e2132. [14] L.A. Amos, F.Y. Ma, G.H. Tesch, J.T. Liles, D.G. Breckenridge, D.J. NikolicPaterson, Y. Han, ASK1 inhibitor treatment suppresses p38/JNK signalling with reduced kidney inflammation and fibrosis in rat crescentic glomerulonephritis, J. Cell Mol. Med. 22 (2018) 4522e4533. [15] J. Ma, D. Zhao, H. Lu, W. Huang, D. Yu, Apoptosis signal-regulating kinase 1 (ASK1) activation is involved in silver nanoparticles induced apoptosis of A549 lung cancer cell line, J. Biomed. Nanotechnol. 13 (2017) 349e354. [16] C.C. Wang, J.R. Yuan, C.F. Wang, N. Yang, J. Chen, D. Liu, J. Song, L. Feng, X.B. Tan, X.B. Jia, Anti-inflammatory effects of Phyllanthus emblica L on benzopyrene-induced precancerous lung lesion by regulating the IL-1beta/ mir-101/lin28B signaling pathway, Integr. Canc. Ther. 16 (2017) 505e515. [17] N. Zhang, J. Lei, H. Lei, X. Ruan, Q. Liu, Y. Chen, W. Huang, MicroRNA-101 overexpression by IL-6 and TNF-alpha inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp. Cell Res. 336
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
8
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(2015) 33e42. [18] H. Dong, Y. Sun, F. Shan, Q. Sun, B. Yang, Down-regulation of miR-101 contributes to rheumatic heart disease through up-regulating TLR2, Med. Sci. Mon. Int. Med. J. Exp. Clin. Res.: Int. Med. J. Exp. Clin. Res. 21 (2015) 1500e1506. [19] A.R. Kuhn, K. Schlauch, R. Lao, A.J. Halayko, W.T. Gerthoffer, C.A. Singer, MicroRNA expression in human airway smooth muscle cells: role of miR-25 in regulation of airway smooth muscle phenotype, Am. J. Respir. Cell Mol. Biol. 42 (2010) 506e513. [20] R.R. Kamel, K.S. Amr, M. Afify, Y.A. Elhosary, A.E. Hegazy, H.H. Fahim, W.M. Ezzat, Relation between microRNAs and apoptosis in hepatocellular carcinoma, Open access Macedonian J. Med. Sci. 4 (2016) 31e37. [21] K. Troppan, K. Wenzl, M. Pichler, B. Pursche, D. Schwarzenbacher, J. Feichtinger, G.G. Thallinger, C. Beham-Schmid, P. Neumeister, A. Deutsch, miR-199a and miR-497 are associated with better overall survival due to increased chemosensitivity in diffuse large B-cell lymphoma patients, Int. J. Mol. Sci. 16 (2015) 18077e18095. [22] Y. Guo, D. Lin, M. Zhang, X. Zhang, Y. Li, R. Yang, Y. Lu, X. Jin, M. Yang, M. Wang, S. Zhao, C. Quan, CLDN6-induced apoptosis via regulating ASK1p38/JNK signaling in breast cancer MCF-7 cells, Int. J. Oncol. 48 (2016)
2435e2444. [23] D.T. Cai, H. Jin, Q.X. Xiong, W.G. Liu, Z.G. Gao, G.X. Gu, Y.H. Qiu, ER stress and ASK1-JNK activation contribute to oridonin-induced apoptosis and growth inhibition in cultured human hepatoblastoma HuH-6 cells, Mol. Cell. Biochem. 379 (2013) 161e169. [24] H.F. Song, S. He, S.H. Li, W.J. Yin, J. Wu, J. Guo, Z.B. Shao, X.Y. Zhai, H. Gong, L. Lu, F. Wei, R.D. Weisel, J. Xie, R.K. Li, Aged human multipotent mesenchymal stromal cells can Be rejuvenated by neuron-derived neurotrophic factor and improve heart function after injury, JACC. Basic to translational science 2 (2017) 702e716. [25] Y. Hayakawa, Y. Hirata, H. Kinoshita, K. Sakitani, H. Nakagawa, W. Nakata, R. Takahashi, K. Sakamoto, S. Maeda, K. Koike, Differential roles of ASK1 and TAK1 in Helicobacter pylori-induced cellular responses, Infect. Immun. 81 (2013) 4551e4560. [26] J.H. Cho, J.H. Lee, E.J. Lee, D. Nam, B.S. Shim, M.Y. Song, S.S. Kim, S.H. Kim, S.H. Jung, W.S. Chung, K.S. Ahn, 8beta-hydroxy-3-oxopimar-15-ene exerts anti-inflammatory effects by inhibiting ROS-mediated activation of the TRAF6-ASK1-p38 signaling pathway, Immunopharmacol. Immunotoxicol. 35 (2013) 549e557.
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Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5 Wei Wang a, Zhao Guo b, Sidong Yang a, Hui Wang a, Wenyuan Ding a, * a b
Orthopedics Department, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China Orthopedics Department, Affiliated Hospital of Hebei University, Baoding, Hebei, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2018 Accepted 30 September 2018 Available online xxx
MicroRNA-199 has been reported to play a potential role in the apoptosis of Human nucleus pulposus cells. However, the effect of miR-199 in regulating Human nucleus pulposus cell injury induced by TNF-a has not been previously illustrated. This study searched to probe the effect and the molecular mechanism of miR-199 on Human nucleus pulposus cell injury induced by TNF-a. Using the TNF-a model of Human nucleus pulposus cell in vitro, we found that miR-199 was extremely decreased in Human nucleus pulposus cells after TNF-a treatment. Knockdown the expression of miR-199 by recombinant adenoassociated viral vector infection markedly promoted the apoptosis of Human nucleus pulposus cells induced by TNF-a treatment, whereas miR-199 overexpression significantly decreased Human nucleus pulposus cell apoptosis. Both Dual-luciferase reporter and western blot assay proved that MAP3K5 was a direct target gene of miR-199, and miR-199 inhibited the expression of MAP3K5 via binding to its 30 -UTR. Furthermore, we proved that overexpression of miR-199 could inhibit the expression of MAP3K5 at the transcription and translation levels, whereas the inhibition of miR-199 could upregulate the expression of MAP3K5. Moreover, MAP3K5 was highly expressed in TNF-a treated Human nucleus pulposus cells and the apoptosis rate induced by TNF-a was associated with the increase in MAP3K5 expression. Importantly, knockdown the expression of MAP3K5 apparently abrogated the inhibitory effect of miR199 mimics on TNF-a induced Human nucleus pulposus cell apoptosis. In conclusion, these results indicate that upregulation of miR-199 could inhibit Human nucleus pulposus cells injury through downregulation of MAP3K5 expression, providing an important molecular target mechanism for Human nucleus pulposus cells injury. © 2018 Elsevier Inc. All rights reserved.
Keywords: miR-199 TNF-a Human nucleus pulposus cell Apoptosis MAP3K5
1. Introduction Intervertebral disc degeneration (IVDD) is one of most serious spinal degenerative diseases, which leads to disability, low health related quality of life and economic burden [1,2]. However, the mechanisms underlying IVDD have not yet been fully elucidated. IVDD is characterized by decreased nucleus pulposus (NP) cell quantity and function, which lead to NP injury [3,4]. One of the most important causes of IVDD is thought to be the excessive apoptosis of NP cells, and recent evidence has demonstrated that TNF-a may induce NP cells apoptosis [3], demonstrating that
* Corresponding author. Orthopedics Department, The Third Hospital of Hebei Medical University, No.139 Ziqiang road, Shijiazhuang, 050051, Hebei Province, China. E-mail address: [email protected] (W. Ding).
apoptosis of NP cells plays an important role in the development of IVDD. Therefore, the control of apoptosis is of great significance for the treatment of NP cell TNF-a injury. MicroRNAs (miRNAs), a kind of endogenous noncoding RNAs composed of about 20e24 nucleotides, regulate intracellular gene posttranscriptional expression by interacting with the 30 -untranslated region (30 -UTR) of the target mRNA resulting in translational repression or degradation [4]. A large number of studies have proved that miRNAs play a key role in the development and progression of various diseases, including TNF-a-induced injury [5]. MiRNAs have brought innovative investigations into the regulation of TNF-a-induced injuries. It has been reported that miRNA-103/ 107 regulated TNF-a-induced injury by targeting cell death ligands just like tumor necrosis factor (TNF) or Fas-associating protein with a novel death domain (FADD) [6]. MiR-199 regulates GATA-binding protein 4 (GATA-4) expression on the myocardial cells to decrease TNF-a-induced injuries [7e9]. However, the role of
https://doi.org/10.1016/j.bbrc.2018.09.194 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
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miRNAs in regulating TNF-a-induced injury is still mainly unknown and requires intensive investigation [10e12]. MiR-199 was considered to play a potential role in apoptosis of NP cells. However, the specific role of miR-199 in apoptosis during TNF-a-induced injury in NP cells has not been previously published. MAP3K5, mitogen-activated protein kinase kinase kinase 5, a serine/threonine protein kinase, is a member of the MAPKKK family (also known as mitogen-activated protein kinase kinase kinase 5, MAPKKK5) and is proved to be activated by a number of stresses, such as endoplasmic reticulum stress (ERS), nitrosative stress or reactive oxygen species (ROS) and cell death ligands like Fasassociating protein with a novel death domain (FADD) and TNF [13]. MAP3K5 has been reported to be associated with apoptosis progress in numerous tissues, such as brain, muscle and heart tissues. MAP3K5 was previously proved to play an important role as an upstream regulatory elements of p38 and JNK in myocardial apoptosis [14]. Knockdown the expression of MAP3K5 has an antiapoptotic effect on myocardium and cancer cells [15]. Although researches in the last decades proved that MAP3K5 is an important regulatory element of apoptosis, the association between MAP3K5 and miRNAs has rarely been investigated.
2.5. LDH activity measurement The lactate dehydrogenase (LDH) activity in the culture medium was measured using an LDH assay kit (Dojindo Molecular Technologies, Shanghai, China) for the detection of cell injury according to the manufacturer's instructions. 2.6. TUNEL determination of Human nucleus pulposus cell apoptosis
2. Materials and methods
Cells were seeded in 6-well plates with placed slides. After different treatment, cells were washed with phosphate-buffered saline (PBS), the slides were immersed in 4% formaldehyde in PBS at 4 C and then in 3% H2O2 and in 0.5% Triton X-100 for 5e10 min at room temperature. Equilibration buffer (100 ml) was added, and the slides were incubated at room temperature for 10 min. TdT reaction mix (50 ml) was then added, and the cells were incubated for 60 min at 37 C. The slides were then immersed in 2 SSC for 15 min. The localized green fluorescence of apoptotic cells was detected by fluorescence microscopy. For the quantification of TUNEL-positive (apoptotic) cells, a minimum of 200 cells was counted per group, and the percentage of the positively labeled cells was calculated.
2.1. Cell culture and treatments
2.7. Quantitative real-time PCR (qPCR)
The Human nucleus pulposus cells were preserved in our laboratory and were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) at 37 C and 5% CO2.
Total RNA was extracted from Human nucleus pulposus cells with TRIzol reagent (Ambion, Waltham, MA, USA), and then reverse transcribed to cDNA using AMV reverse transcriptase is provided in a RT system kit (Promega, Fitchburg, WI, USA), then the cDNA was used as a template for amplification using a reverse transcription kit (Promega, Fitchburg, WI, USA). Quantitative realtime PCR was performed with specific primers of MAP3K5 (sense:50 -CGTAGCCTCTTGGTCCTTTATC-30 ,antisense:50 -GGAAGTC TTTCTGCTCTCCTTC-30 ); GAPDH(sense:50 -CATGGGTGTGAACCATGAGAA-30 ,antisense:50 -GGTCATGAGTCCTTCCACGAT-30 ) using the SYBR Green I real-time PCR kit (Promega, Fitchburg, WI, USA) and the relative gene expression was calculated via the 2-△△Ct method. GAPDH and U6 were used for the normalizing the expression of mRNA and miRNA. Each sample was detected in triplicate.
2.2. Cell transfection AAV (recombinant adenovirus-associated virus) vectors were constructed by GenePharma Technologies (GenePharma, Shanghai, China). Human nucleus pulposus cells were infected with AAV-miR Control (miR Ctrl), AAV-anti-miR Control (anti-miR Ctrl), AAV-miR199 (miR-199), AAV-anti-miR-199 (anti-miR-199), MAP3K5 expression plasmid (MAP3K5 plasmid), or empty plasmid vector (vector plasmid) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and incubated for 8 h. Cells were then subjected to TNF-a treatment and finally harvested for further analysis. 2.3. Human nucleus pulposus cells treated in TNF-a condition Human nucleus pulposus cells were cultured in TNF-a condition in vitro as an injury model. As previously described [5], the cells were cultured in TNF-a condition at 37 C in a humidified atmosphere with 5% O2 for different time points, and then harvested for further analysis. 2.4. MTT assay MTT assay kit (Dojindo Molecular Technologies, Shanghai, China) was carried for the measurement of cell viability. Generaly, Human nucleus pulposus cells were digested and then seeded in 96-well plates (5 103/well) for 12 h, after that, cells were infected with AAV related vectors and then subjected to TNF-a treatment for indicated times. Then the cells in each well were incubated with fresh culture medium containing MTT solution (5 mg/mL) for 4 h at 37 C. Finally, 100 mL of DMSO(dimethyl sulfoxide) were added to each well, and the absorbance was checked with an automatic microplate reader at 490 nm (Bio-Tek, Winooski, VA, USA).
2.8. Western blot detection Proteins isolated from Human nucleus pulposus cells were detected by SDS-PAGE and then transferred to a NC membrane (Millipore, Bedford, MA, USA). After blocking with BSA(5% in TBST) for 1.5 h at 37 C, the membranes were probed with the primary antibodies against MAP3K5, Bcl-2, Bax, Caspase-3, and cleaved Caspase-3 antibodies at 4 C for 12 h. The membranes were then washed with TBST(with 0.1% Tween-20) three times (each for 10 min) and incubated with corresponding secondary antibodies for 2 h at 37 C. The expressions of corresponding protein were detected using the enhanced chemiluminescent (ECL) system, and quantified using an image Quantity One System (Bio-Rad, Richmond, CA, USA). b-actin was used as control. 2.9. Luciferase reporter assay HEK293T cells were seeded into 24-well plates (1 104/well). The pGL3 luciferase promoter vectors (Promega, Madison, WI, USA) containing MAP3K5 mRNA 30 -UTR or 30 -UTR-mut (MAP3K5 mRNA mutated forms) were cotransfected with AAV-miR-199 or corresponding controls into HEK293 cells using Lipofectamine 3000 (Invitrogen, USA) for 24 h. Cells were then harvested and lysed on
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
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ice, and the luciferase activity was detected with the Luciferase reporter assay kit (Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. All detections were executed at least three times. The relative luciferase activities were normalized to the corresponding controls. 2.10. Statistical analysis All experiments were executed independently repeated at least three times. All results were demonstrated as means ± S.E.M. Student's t-test and one-way ANOVA analysis were used to determine the significance differences between two experimental groups with statistic software SPSS 20.0. Values of P < 0.05 were considered statistically significant.
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(P < 0.05), as shown in Fig. 1C, indicating that miR-199 may play a significant role in TNF-a injury in Human nucleus pulposus cells. There were no obvious differences in cell viability, LDH viability and miR-199 expression between TNF-a treatment for 24 h and TNF-a treatment for 48 h group, thus Human nucleus pulposus cells treated with TNF-a for 24 h were regard as TNF-a injury model. To investigate whether miR-199 plays an important role in protection against TNF-a injury, Human nucleus pulposus cells were stably transfected with miR-199 precursor or inhibitor by adenovirusmediated transfection. As shown in Fig. 1D, Human nucleus pulposus cells transfected with miR-199 obviously increased miR-199 expression, whereas the expression of miR-199 in Human nucleus pulposus cells with miR-199 inhibitor (anti-miR-199) was significantly decreased (P < 0.05). Non-transfected or negativetransfected cells were set up as controls.
3. Results 3.1. TNF-a decreases miR-199 expression in Human nucleus pulposus cells Results shown that the cell viability was decreased (Fig. 1A) and the LDH viability was highly increased (Fig. 1B) in Human nucleus pulposus cells subjected to TNF-a at 0, 6, 12, 24, 48 h differrnt time points (P < 0.05), demonstrating that Human nucleus pulposus cells suffered TNF-a injury. MiR-199 is significantly downregulated at 0, 6, 12, 24, 48 h differrnt time points under TNF-a treatment
3.2. The downregulation of miR-199 promotes Human nucleus pulposus cells apoptosis under TNF-a treatment The cell viability decreased (Fig. 2A) and the LDH viability increased induced by TNF-a (Fig. 2B) were obviously reversed by miR-199 transfection but reverted by anti-miR-199 transfection. The descending expression of miR-199 obviously increased TNF-ainduced cell apoptosis, whereas miR-199 overexpression significantly reduced Human nucleus pulposus cell apoptosis, as shown by TUNEL staining (Fig. 2C). Furthermore, the expression of pro-
Fig. 1. MiR-199 is downregulated in TNF-a-treated Human nucleus pulposus cells. Cell viability was highly decreased (A) and LDH viability was significantly increased (B) during 0e48 h TNF-a treatment in Human nucleus pulposus cells. (C) The relative expression of miR-199 was obviously downregulated during 0e48 h TNF-a treatment in Human nucleus pulposus cells. (D) Analysis of miR-199 expression in Human nucleus pulposus cells transfected with miR-199 or miR-199 inhibitor by qPCR. MiR-199 Ctrl, miR-199, anti-miR Ctrl, anti-miR-199, correspondingly, transfected with AAV control miRNA, AVV-miR-199, AAV-control anti-miRNA and AAV-anti-miR-199. *P < 0.05 vs. control, n ¼ 3.
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Fig. 2. The downregulation of miR-199 under TNF-a treatment promotes the apoptosis of Human nucleus pulposus cells in vitro. (A) MiR-199 increased cell viability in Human nucleus pulposus cell treated with TNF-a detected by MTT assay. (B) LDH viability was increased in Human nucleus pulposus cells under TNF-a by anti-miR-199 transfection. (C) MiR-199 inhibited apoptosis in Human nucleus pulposus cells after 24 h of TNF-a. (D) MiR-199 influenced pro- and anti-apoptotic relative protein expression in TNF-a treated Human nucleus pulposus cells detected by western blot. △P < 0.05 vs. control; *P < 0.05 vs. TNF-a for 24 h, n ¼ 3.
and anti-apoptotic relative proteins of the Bcl-2 and casepase family, were detected by western blot (Fig. 2D). TNF-a treatment promoted the expression of both Bax and cleaved-caspase-3, and the ratio of Bax/Bcl-2 increased as compared with the corresponding control groups (P < 0.05). This phenomenon was further enhanced in the case of miR-199 downregulation, while this phenomenon was partially reverted in the presence of miR-199 (P < 0.05). These results suggest that miR-199 plays a protective role in TNF-a-induced Human nucleus pulposus cell apoptosis.
3.3. MiR-199 targets MAP3K5 and reduces its expression in TNF-a treated Human nucleus pulposus cells To explore miR-199's biological function and its molecular mechanism, we investigated the possible target gene of miR-199 that contribute to TNF-a induced injury. Bio-informatics analysis indicated that MAP3K5, which is an important gene for TNF-ainduced Human nucleus pulposus cell injury, was also a target gene of miR-199 (Fig. 3A). To investigate the direct target association
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Fig. 3. MAP3K5 is a direct target gene of miR-199. (A) Presumed binding nucleotide sequences in the 30 -UTR of MAP3K5 mRNA with miR-199. (B) The interaction between the miR199 and MAP3K5 mRNA 30 -UTR detected by Dual-Luciferase Reporter Assay after transfection for 24 h (C and D) The mRNA and protein expression of MAP3K5 were detected by qPCR and western blot in Human nucleus pulposus cells transfected with miR-199 or anti-miR-199. Relative expression was measured using image analysis software.△P < 0.05 vs. control, n ¼ 3. (E and F) The expression of MAP3K5 in mRNA and protein levels in Human nucleus pulposus cells after TNF-a treatment. △P < 0.05 vs. control, n ¼ 3.
between miR-199 and MAP3K5, a dual-luciferase reporter assay system was conducted. The overexpression of miR-199 could significantly reduce the luciferase activity of the wild type MAP3K5 30 -UTR reporter gene, but not the mutant reporter gene (Fig. 3B), indicating that miR-199 targeted MAP3K5 by binding to its 30 -UTR area. Moreover, to prove whether miR-199 regulates MAP3K5 expression in TNF-a-treated Human nucleus pulposus cells, we
detected the expression of MAP3K5 by qPCR and western blot analysis in miR-199 or anti-miR-199-transfected Human nucleus pulposus cells. These results proved that overexpression of miR-199 could obviously reduce the expression of MAP3K5 both at the mRNA and protein levels, and inhibition the expression of miR-199 could promote MAP3K5 expression (Fig. 3C and D). Furthermore, we found that MAP3K5 was highly expressed in TNF-a treated
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Human nucleus pulposus cells (Fig. 3E and F). 3.4. MAP3K5 contributes to the effect of miR-199 induced cell apoptosis under TNF-a treatment To further investigate the molecular mechanism that miR-199
downregulation enhanced TNF-a-induced Human nucleus pulposus cell apoptosis, MAP3K5 expression plasmid and miR-199 were co-transfected into Human nucleus pulposus cells. As shown in Fig. 4A, the mRNA and protein expression of MAP3K5 decreased by miR-199 and was obviously reverted by MAP3K5 expression plasmid transfection in TNF-a treated Human nucleus pulposus
Fig. 4. MAP3K5 is involved in TNF-a-induced apoptosis in Human nucleus pulposus cells. (A and B) Detection of the expression of MAP3K5 in mRNA and protein levels in TNF-a treated Human nucleus pulposus cells co-transfected with miR-199 and MAP3K5. MAP3K5 overexpression reversed the protective effect of miR-199 on cell viability (C) and LDH activity (D) in TNF-a treated Human nucleus pulposus cells. (E and F) The inhibition effect of miR-199 on TNF-a-induce apoptosis was reverted when MAP3K5 expression plasmid was transfected. Control cells were transfected with control miRNA, miR-199 and empty vector cells were treated with miR-199 and empty plasmid vector, miR-199 and MAP3K5 cells were transfected with miR-199 and MAP3K5 expression plasmid. △P < 0.05 vs. miR-ctrl; *P < 0.05 vs. miR-199 or MAP3K5,n ¼ 3.
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cells (Fig. 4A and B). Additionally, Human nucleus pulposus cell viability was obviously decreased after MAP3K5 expression plasmid transfection (Fig. 4C). Also, the decrease in LDH viability caused by miR-199 was highly increased by MAP3K5 expression plasmid transfection (Fig. 4D). Importantly, the inhibitory effect of miR-199 on TNF-a induced-apoptosis was obviously inhibited by MAP3K5 expression plasmid transfection (Fig. 4E and F). These results demonstrated that the overexpression of MAP3K5 abolished the inhibitory effects of miR-199 on TNF-a-induced apoptosis of Human nucleus pulposus cells.
downregulated miRNA in TNF-a treated Human nucleus pulposus cells, plays a significant role in TNF-a-induced apoptosis in Human nucleus pulposus cells by regulating MAP3K5. Based on these results, miR-199 could serve as a potential target for the prevention of Human nucleus pulposus cells apoptosis under TNF-a, and the further research in vivo should be conducted.
4. Discussion
Transparency document
IVDD is characterized by an imbalance between survival and injury in the NP [3,4]. Inflammatory cytokines, such as TNF-a and IL1-b, facilitate NP injury either by directly inhibiting miRNAs expression or by increasing apoptosis related protein expression [3e5]. In the current study, we showed for the first time that MAP3K5 expression levels are elevated in degenerated human IVDs and plays critical role in the pathophysiology of inflammationdriven IVDD. Therefore, it is of great significant to demonstrate the potential molecular mechanisms of NP cell injury under TNF-a in IVDD. In the present research, it was proved that the expression of miR-199 was obviously decreased in Human nucleus pulposus cells after TNF-a treatment. Thus, we hypothesized that miR-199 might play a significant role in TNF-a treated Human nucleus pulposus cells and its upregulation or overexpression might attenuate TNFa-induced injury in Human nucleus pulposus cells. Our results demonstrated that the downregulation of miR-199 was correlated with an promotion of apoptosis in TNF-a treated Human nucleus pulposus cells. Furthermore, we identified that MAP3K5, which was expressed at high levels in TNF-a treated Human nucleus pulposus cells, was a direct target gene of miR-199. Additionally, MAP3K5 was confirmed to be involved in the effect of anti-miR-199 on promotion from TNF-a-induced apoptosis in Human nucleus pulposus cells. Recent researches have demonstrated that numerous miRNAs play important roles in the apoptosis induced by TNF-a. For example, the overexpression of miR-101 attenuated TNF-a-induced apoptosis through induction of autophagy [16e18]. It has also been reported that miR-25 family could inhibit TNF-a and ischemiainduced apoptosis in cerebral through inhibiting Fas/FasL expression [19]. MiR-199 was proved to mediate hepatocellular carcinoma cell apoptosis [20]. MiR-199 was also proved to be involved in regulating apoptosis in large B-cell lymphoma [21]. In this research, we further investigated the importance of miR-199 in TNF-ainduced Human nucleus pulposus cell apoptosis. MAP3K5, one of the most important apoptotic signal-regulating kinases, has been proved to participate in cell proliferation [22] and survival [23]. MAP3K5 was reported to be able to promote TNF-a-induced cardiomyocyte or myocardial stem cell injury [24]. Numerous researches have proved the damage effect of MAP3K5 in proapoptosis by regulating the expression of p38/JNK/NF-kB signaling [25,26]. We investigated and characterized the direct target sequence of the 30 -UTR of MAP3K5 by miR-199. MAP3K5 expression was promoted by miR-199 downregulation and downregulated by miR-199 overexpression. In addition, MAP3K5 overexpression plasmid exacerbate TNF-a induced apoptosis inhibited by miR-199, demonstrating that miR-199 may conduct functions in TNF-a induced apoptosis of Human nucleus pulposus cells by regulating MAP3K5. These findings imply that MAP3K5 plays a significant role in TNF-a-induced injury, and miR-199 might be a novel target for preventing NP cells injury under TNF-a. In conclusion, our study demonstrates that miR-199, an important
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Conflicts of interest The authors declare that they have no conflict of interest.
References [1] S.M. Richardson, G. Kalamegam, P.N. Pushparaj, C. Matta, A. Memic, A. Khademhosseini, R. Mobasheri, F.L. Poletti, J.A. Hoyland, A. Mobasheri, Mesenchymal stem cells in regenerative medicine: focus on articular cartilage and intervertebral disc regeneration, Methods 99 (2016) 69e80. [2] N. Vo, L.J. Niedernhofer, L.A. Nasto, L. Jacobs, P.D. Robbins, J. Kang, C.H. Evans, An overview of underlying causes and animal models for the study of agerelated degenerative disorders of the spine and synovial joints, J. Orthop. Res.: Off. Pub. Orthop. Res. Soc. 31 (2013) 831e837. [3] G. Vadala, F. Russo, A. Di Martino, V. Denaro, Intervertebral disc regeneration: from the degenerative cascade to molecular therapy and tissue engineering, J. Tissue Eng. Regen. Med. 9 (2015) 679e690. [4] J. Liu, H. Tao, H. Wang, F. Dong, R. Zhang, J. Li, P. Ge, P. Song, H. Zhang, P. Xu, X. Liu, C. Shen, Biological behavior of human nucleus pulposus mesenchymal stem cells in response to changes in the acidic environment during intervertebral disc degeneration, Stem Cell. Dev. 26 (2017) 901e911. [5] E. Sonkoly, A. Pivarcsi, Advances in microRNAs: implications for immunity and inflammatory diseases, J. Cell Mol. Med. 13 (2009) 24e38. [6] C.J. Kearney, S.P. Cullen, G.A. Tynan, C.M. Henry, D. Clancy, E.C. Lavelle, S.J. Martin, Necroptosis suppresses inflammation via termination of TNF- or LPS-induced cytokine and chemokine production, Cell Death Differ. 22 (2015) 1313e1327. [7] V. Gupta, A.A. Khan, B.K. Sasi, N.R. Mahapatra, Molecular mechanism of monoamine oxidase A gene regulation under inflammation and ischemia-like conditions: key roles of the transcription factors GATA2, Sp1 and TBP, J. Neurochem. 134 (2015) 21e38. [8] S. Wang, M. Luo, Z. Zhang, J. Gu, J. Chen, K.M. Payne, Y. Tan, Y. Wang, X. Yin, X. Zhang, G.C. Liu, K. Wintergerst, Q. Liu, Y. Zheng, L. Cai, Zinc deficiency exacerbates while zinc supplement attenuates cardiac hypertrophy in high-fat diet-induced obese mice through modulating p38 MAPK-dependent signaling, Toxicol. Lett. 258 (2016) 134e146. [9] A. Briones-Herrera, D. Eugenio-Perez, J.G. Reyes-Ocampo, S. Rivera-Mancia, J. Pedraza-Chaverri, New highlights on the health-improving effects of sulforaphane, Food & function 9 (2018) 2589e2606. [10] P. Prajapati, L. Sripada, K. Singh, K. Bhatelia, R. Singh, R. Singh, TNF-alpha regulates miRNA targeting mitochondrial complex-I and induces cell death in dopaminergic cells, Biochim. Biophys. Acta 1852 (2015) 451e461. [11] T. Alexy, K. Rooney, M. Weber, W.D. Gray, C.D. Searles, TNF-alpha alters the release and transfer of microparticle-encapsulated miRNAs from endothelial cells, Physiol. Genom. 46 (2014) 833e840. [12] H. Li, X. Chen, L. Guan, Q. Qi, G. Shu, Q. Jiang, L. Yuan, Q. Xi, Y. Zhang, MiRNA181a regulates adipogenesis by targeting tumor necrosis factor-alpha (TNFalpha) in the porcine model, PloS One 8 (2013), e71568. [13] X. Xu, T. Liu, A. Zhang, X. Huo, Q. Luo, Z. Chen, L. Yu, Q. Li, L. Liu, Z.R. Lun, J. Shen, Reactive oxygen species-triggered trophoblast apoptosis is initiated by endoplasmic reticulum stress via activation of caspase-12, CHOP, and the JNK pathway in Toxoplasma gondii infection in mice, Infect. Immun. 80 (2012) 2121e2132. [14] L.A. Amos, F.Y. Ma, G.H. Tesch, J.T. Liles, D.G. Breckenridge, D.J. NikolicPaterson, Y. Han, ASK1 inhibitor treatment suppresses p38/JNK signalling with reduced kidney inflammation and fibrosis in rat crescentic glomerulonephritis, J. Cell Mol. Med. 22 (2018) 4522e4533. [15] J. Ma, D. Zhao, H. Lu, W. Huang, D. Yu, Apoptosis signal-regulating kinase 1 (ASK1) activation is involved in silver nanoparticles induced apoptosis of A549 lung cancer cell line, J. Biomed. Nanotechnol. 13 (2017) 349e354. [16] C.C. Wang, J.R. Yuan, C.F. Wang, N. Yang, J. Chen, D. Liu, J. Song, L. Feng, X.B. Tan, X.B. Jia, Anti-inflammatory effects of Phyllanthus emblica L on benzopyrene-induced precancerous lung lesion by regulating the IL-1beta/ mir-101/lin28B signaling pathway, Integr. Canc. Ther. 16 (2017) 505e515. [17] N. Zhang, J. Lei, H. Lei, X. Ruan, Q. Liu, Y. Chen, W. Huang, MicroRNA-101 overexpression by IL-6 and TNF-alpha inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression, Exp. Cell Res. 336
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194
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W. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
(2015) 33e42. [18] H. Dong, Y. Sun, F. Shan, Q. Sun, B. Yang, Down-regulation of miR-101 contributes to rheumatic heart disease through up-regulating TLR2, Med. Sci. Mon. Int. Med. J. Exp. Clin. Res.: Int. Med. J. Exp. Clin. Res. 21 (2015) 1500e1506. [19] A.R. Kuhn, K. Schlauch, R. Lao, A.J. Halayko, W.T. Gerthoffer, C.A. Singer, MicroRNA expression in human airway smooth muscle cells: role of miR-25 in regulation of airway smooth muscle phenotype, Am. J. Respir. Cell Mol. Biol. 42 (2010) 506e513. [20] R.R. Kamel, K.S. Amr, M. Afify, Y.A. Elhosary, A.E. Hegazy, H.H. Fahim, W.M. Ezzat, Relation between microRNAs and apoptosis in hepatocellular carcinoma, Open access Macedonian J. Med. Sci. 4 (2016) 31e37. [21] K. Troppan, K. Wenzl, M. Pichler, B. Pursche, D. Schwarzenbacher, J. Feichtinger, G.G. Thallinger, C. Beham-Schmid, P. Neumeister, A. Deutsch, miR-199a and miR-497 are associated with better overall survival due to increased chemosensitivity in diffuse large B-cell lymphoma patients, Int. J. Mol. Sci. 16 (2015) 18077e18095. [22] Y. Guo, D. Lin, M. Zhang, X. Zhang, Y. Li, R. Yang, Y. Lu, X. Jin, M. Yang, M. Wang, S. Zhao, C. Quan, CLDN6-induced apoptosis via regulating ASK1p38/JNK signaling in breast cancer MCF-7 cells, Int. J. Oncol. 48 (2016)
2435e2444. [23] D.T. Cai, H. Jin, Q.X. Xiong, W.G. Liu, Z.G. Gao, G.X. Gu, Y.H. Qiu, ER stress and ASK1-JNK activation contribute to oridonin-induced apoptosis and growth inhibition in cultured human hepatoblastoma HuH-6 cells, Mol. Cell. Biochem. 379 (2013) 161e169. [24] H.F. Song, S. He, S.H. Li, W.J. Yin, J. Wu, J. Guo, Z.B. Shao, X.Y. Zhai, H. Gong, L. Lu, F. Wei, R.D. Weisel, J. Xie, R.K. Li, Aged human multipotent mesenchymal stromal cells can Be rejuvenated by neuron-derived neurotrophic factor and improve heart function after injury, JACC. Basic to translational science 2 (2017) 702e716. [25] Y. Hayakawa, Y. Hirata, H. Kinoshita, K. Sakitani, H. Nakagawa, W. Nakata, R. Takahashi, K. Sakamoto, S. Maeda, K. Koike, Differential roles of ASK1 and TAK1 in Helicobacter pylori-induced cellular responses, Infect. Immun. 81 (2013) 4551e4560. [26] J.H. Cho, J.H. Lee, E.J. Lee, D. Nam, B.S. Shim, M.Y. Song, S.S. Kim, S.H. Kim, S.H. Jung, W.S. Chung, K.S. Ahn, 8beta-hydroxy-3-oxopimar-15-ene exerts anti-inflammatory effects by inhibiting ROS-mediated activation of the TRAF6-ASK1-p38 signaling pathway, Immunopharmacol. Immunotoxicol. 35 (2013) 549e557.
Please cite this article in press as: W. Wang, et al., Upregulation of miR-199 attenuates TNF-a-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.194