- Email: [email protected]
Epigenetic silencing of miRNA-143 regulates apoptosis by targeting BCL2 in human intervertebral disc degeneration
Accepted Manuscript Epigenetic silencing of miRNA-143 regulates apoptosis by targeting BCL2 in human intervertebral disc degeneration
Kangcheng Zhao, Yukun Zhang, Kang Liang, Yu Song, Kun Wang, Shuai Li, Xinghuo Wu, Wenbin Hua, Zengwu Shao, Shuhua Yang, Cao Yang PII: DOI: Reference:
S0378-1119(17)30565-6 doi: 10.1016/j.gene.2017.07.043 GENE 42067
To appear in:
Gene
Received date: Revised date: Accepted date:
9 December 2016 7 July 2017 13 July 2017
Please cite this article as: Kangcheng Zhao, Yukun Zhang, Kang Liang, Yu Song, Kun Wang, Shuai Li, Xinghuo Wu, Wenbin Hua, Zengwu Shao, Shuhua Yang, Cao Yang , Epigenetic silencing of miRNA-143 regulates apoptosis by targeting BCL2 in human intervertebral disc degeneration, Gene (2017), doi: 10.1016/j.gene.2017.07.043
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Epigenetic silencing of miRNA-143 regulates apoptosis by targeting BCL2 in human intervertebral disc degeneration
1
Kangcheng Zhao , Yukun Zhang1, Kang Liang, Yu Song, Kun Wang, Shuai Li, Xinghuo
RI
PT
Wu, Wenbin Hua, Zengwu Shao, Shuhua Yang, Cao Yang*
SC
Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong
NU
University of Science and Technology, Wuhan, 430022, China
CE
PT E
D
These authors contributed equally to this work.
AC
1
MA
* Corresponding author: Cao Yang
ACCEPTED MANUSCRIPT
Abstract Accumulating evidence indicates that microRNAs can regulate the apoptosis of various cells. Apoptosis of nucleus pulposus cells plays an important role in the progression of intervertebral disc degeneration. The aim of this study is to investigate whether
PT
microRNA-143 (miRNA-143) is involved in the progression of intervertebral disc
RI
degeneration. In this study, the expression of miRNA-143 and its biological modulatory
SC
effects were examined. Messenger RNA and protein expression of miRNA-143 and B-cell lymphoma-2 (BCL2) in both normal and degenerative disc tissues was determined by using
NU
RT-PCR and western-blot assays. After miRNA-143 transfection, BCL2 expression and NP
MA
cell apoptosis were assessed by using RT-PCR, western-blot, and flow cytometry. The relationship between miRNA-143 and BCL2 was assessed by bioinformatics and dual assays.
Epigenetic
regulation
of
miRNA-143
was
determined
by
D
luciferase
PT E
methylation-specific PCR and the effect of hypomethylation using 5-AZA. In this study, miRNA-143 expression significantly increased, while that of BCL2 decreased in degenerative
CE
disc specimens. In addition, CpG islands in the promoter region of miRNA-143 were
AC
hypomethylated in degenerative disc tissues. Furthermore, bioinformatics analysis and luciferase reporter assay indicated that BCL2 was a target gene of miRNA-143, and miRNA-143 suppressed BCL2 messenger RNA (mRNA) and protein expression. MiRNAmiRNA-143 overexpression enhances apoptosis of nucleus pulposus cells, while miRNA-143 inhibitor had the opposite effect. BCL2 knockdown reversed the effects of the miRNA-143 inhibitor on nucleus pulposus apoptosis. Our results suggest that miRNA-143 promotes the progression of nucleus pulposus apoptosis by directly targeting BCL2,
ACCEPTED MANUSCRIPT providing a potential therapeutic target for the treatment of intervertebral disc degeneration disease.
Keywords: Intervertebral disc degeneration; BCL2; miRNA-143; apoptosis; methylation;
AC
CE
PT E
D
MA
NU
SC
RI
PT
human nucleus pulposus
ACCEPTED MANUSCRIPT
1. Introduction Lower back pain (LBP) is one of the most common diseases, affecting the quality of life of patients and becoming a socioeconomic burden worldwide. The causes of LBP include aging, abnormal mechanical loading, trauma, and genetic predisposition. However, for many
PT
patients, the major cause of LBP is intervertebral disc degeneration (IVDD) (Chen et al., 2015;
RI
Le Maitre et al., 2015). IVDD is characterized by extracellular matrix degradation and
SC
decreased number of viable cells (Jiang et al., 2013; Vergroesen et al., 2015), which is mainly attributed to an imbalance of cell apoptosis. The intervertebral disc presents a complex
NU
structure with three sections: the nucleus pulposus (NP), end plate, and annulus fibrosus (AF).
MA
NP cells play a crucial role in maintaining the normal physiological function via the synthesis of extracellular matrix (ECM) components to resist mechanical loads (Wang et al., 2015a).
D
The decrease of NP cells associated with apoptosis is considered as an initial trigger for
PT E
IVDD (Ding et al., 2013; Ma et al., 2015). Apoptosis plays a crucial role in the pathological process of cell death (Wang et al., 2015a). Studies demonstrated that aberrant cell apoptosis is
CE
responsible for many degenerative diseases such as Alzheimer’s disease, osteoarthritis, IVDD,
AC
and cancer (Li et al., 2015a; Gupta et al., 2016; Yan et al., 2016). MicroRNAs (miRNAs) are small noncoding RNAs (17–25 nucleotides), which regulate gene expression, resulting in the inhibition of translation or degradation. They play important regulatory roles in multitudinous biological cellular processes such as differentiation, proliferation, and apoptosis (Bartel, 2004; Sayed and Abdellatif, 2011; Wang et al., 2015a; Liu et al., 2016). Recent studies demonstrated that miRNAs such as miRNA-494, miRNA-27a, miRNA-155, and miRNA-15b play important regulatory roles in the progression of IVDD
ACCEPTED MANUSCRIPT (Wang et al., 2011; Wang et al., 2015a; Wang et al., 2015b; Cai et al., 2017). MiRNA-143 is also broadly involved in cellular processes such as proliferation, invasion, apoptosis, and senescence (Zhang et al., 2013; Sekar et al., 2016). Aberrant miRNA-143 expression is observed in many tumors such as gastric, prostate, and colorectal cancer (Ji, 2010; Qian et al.,
PT
2013), indicating a potentially regulatory role in tumor progression. However, the expression
RI
and the role of miRNA-143 in degenerative disc NP cells are still unclear.
SC
BCL2 gene is located on chromosome 18, which was discovered as part of the t(14;18) chromosomal translocation in patients with follicular lymphoma and diffuse large B cell
NU
lymphoma (DLBCL). The protein encoded by BCL2 supports cell survival by blocking
MA
apoptosis rather than by increasing the cell division rate (Hardwick and Soane, 2013; de Araujo Junior et al., 2015). Data support that apoptosis is involved in pathological IVDD (Ma
D
et al., 2015). Therefore, BCL2 overexpression in human NP may slow down the degeneration
PT E
of IVD by preventing apoptosis of NP cells. DNA methylation is an important epigenetic process regulating the expression of target
CE
genes and usually results in gene silencing by inhibiting the binding of transcription factors or
AC
recruitment of methyl-binding proteins and the associated chromatin remodeling factors. The CpG islands distributed in the human genome play crucial roles in the methylation process. The gene silencing function of miRNA promoter CpG islands is a common mechanism during tumorigenesis (Pronina et al., 2016). Considering that the imbalance of cell apoptosis caused by dysfunction of miRNA expression is the common mechanism of tumorigenesis and disc degeneration, methylation may be an important regulatory mechanism. Considering the important roles of miRNAs and BCL2 in the pathogenesis of IVDD and
ACCEPTED MANUSCRIPT the putative BCL2 binding site for miRNA-143, we hypothesized that miRNA-143 might affect IVDD via BCL2. In addition, we investigated whether methylation, which is involved in various diseases and impacts on many miRNA promoters (Long et al., 2014; Li et al., 2015b), was involved in the association between miRNA-143 and NP cell apoptosis.
PT
Thus, this study aimed to identify whether miRNA-143 expression increased in
RI
degenerative disc tissues, and whether it targets BCL2 to induce apoptosis. Our study might
SC
provide insights useful for the development of novel therapies for clinical LBP treatment.
NU
2. Materials and Methods
MA
2.1 Ethics statement
The study was approved by the ethics committee of Tongji Medical College, Huazhong
D
University of Science and Technology (HUST). All IVD specimens were obtained in
PT E
accordance with the World Medical Association Declaration of Helsinki Ethical Principles for
CE
Medical Research Involving Human Subjects. All patients signed a written informed consent.
AC
2.1 Sample collection
All subjects were vertebral column spine surgery patients admitted to The Union Hospital Orthopaedic Hospital of HUST (Wuhan, China). The normal human IVD tissues were obtained from patients with idiopathic scoliosis classified as Pfirrmann grade I or II according to Magnetic Resonance Imaging (MRI) (n = 10; mean age, 22.3 ± 4.25; range, 17–40). The IVDD tissues were obtained from the LBP patients with disc degeneration classified as Pfirrmann grade IV according to MRI (n = 10; mean age, 25.8 ± 5.32, 25–58).
ACCEPTED MANUSCRIPT
2.3 Isolation and culture of human degenerative NP cells The human NP tissues were microscopically isolated from IVD tissues using a scalpel, under sterile conditions. These NP tissues were then washed with PBS twice and cut into 1
PT
mm3 fragments. These fragments were digested by 0.25% trypsin solution containing 1%
RI
ethylenediaminetetraacetic acid (EDTA) for 30 min and type II collagenase (Invitrogen,
SC
Carlsbad, CA, USA) for 3–4 hours at 37C. Tissue debris was removed by passing through a 200 m filter and then the NP cells were resuspended in DMEM/F12 containing 15% fetal
NU
bovine serum (FBS), 100 g/mL streptomycin, 100 U/mL penicillin, and 1%
MA
L-glutamine and incubated at 37C in an atmosphere containing 5% CO2. When at 80–90% confluence, cells were digested by 0.25% trypsin solution and subcultured in culture
2.4 Cell transfection
PT E
D
flasks. The third passage of NP cells was used for all experiments.
CE
MiRNA-143 mimic, miRNA-143 mimic-scr, miRNA-143 inhibitor, miRNA-143
AC
inhibitor-scr, BCL2 siRNA, and BCL2 siRNA-scr were purchased from RiboBio (Guangzhou, China). MiRNA-143 mimic and miRNA-143 inhibitor were used to promote and inhibit miRNA-143 expression, respectively. BCL2 siRNA was used to inhibit BCL2 expression. They were transfected into NP cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 48 hours, NP cells were harvested and used for further analyses.
ACCEPTED MANUSCRIPT
2.5 RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from human NP tissues using TRIZOL reagent (Aidlab, Beijing China) according to the manufacturer’s instruction. The purity and concentration of RNA were measured by using an ultraviolet spectrophotometer (Shunyu, shanghai, China).
PT
qRT-PCR was conducted to quantify miRNA-143, BCL2, and caspase-3/9 expression using
RI
the 7500 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA) in a final
SC
volume of 20 L. The primers used for qRT-PCR are listed in Table 1. All experiments were performed in triplicate. The quantitative miRNA data were normalized to U6 and mRNA
NU
expression of BCL2, caspase-3, and caspase-9 was normalized to that of -actin. The
MA
fold-change in gene expression relative to the control was calculated by the 2-△△Ct method.
D
2.6 Western blot analysis
PT E
NP specimens were randomly selected and protein concentrations were determined by using the BCA assay (Beyotime, Shanghai, China). Human NP cells were washed with PBS at
CE
4C (pH = 7.2–7.3) and lysed by using RIPA lysis Buffer (Beyotime) on ice. Protein
AC
concentration was measured as described above. Protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Finally, membranes were treated with ECL reagent (ThermoFisher Scientific, Waltham, MA, USA) and visualized. GAPDH was used as an internal control. The experiment was repeated three times.
ACCEPTED MANUSCRIPT
2.7 miRNA-143 target prediction and 3-UTR luciferase reporter assay TargetScan (www.targetscan.org) was used to predict the target genes of miRNA-143. BCL2 contained a putative miRNA-143 binding site. BCL2 3-Untranslated Region (3-UTR) luciferase reporter was inserted into the 3-UTR region of pmir-Report construct (Promega,
PT
Madison, WI, USA). The BCL2 3-UTR luciferase reporter deleted for the miRNA-143 target
RI
site was created by PCR amplification of the BCL2 3-UTR luciferase reporter construct
SC
lacking the miRNA-143 bind target site. Normal NP cells (5 103) were co-transfected with luciferase reporter plasmid, internal control prl-tk-renilla-luciferase plasmid, and the indicated
NU
RNAs. After 48 hours, luciferase activity was measured by using the Dual-Luciferase
MA
Reporter Assay System (Promega) according to the manufacturer’s instructions. Firefly
D
luciferase activity was normalized to Renilla luciferase activity.
PT E
2.8 Methylation assay
We detected the methylation status of miRNA-143 promoter in degenerative and normal
CE
disc tissue extracts by using methylation-specific PCR (MSP) analysis. Genomic DNA was
AC
isolated from degenerative and normal disc tissues by using DNA extraction kit (Promega) according to the manufacturer’s instructions. The primer sequences used for MSP were as follows:
methylated,
forward:
ATTAGTTAGGTATGGTGGTGTACGT
and
reverse:
TTTAAAACAAAATCTCCCTCTATCG;
unmethylated,
forward:
ATTAGTTAGGTATGGTGGTGTATGT
and
reverse:
TTAAAACAAAATCTCCCTCTATCAC. The PCR products were electrophoresed on a 3% agarose gel.
ACCEPTED MANUSCRIPT
2.9 Flow cytometry Apoptosis was detected by using the Annexin V-APC apoptosis detection kit (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s instructions. Cells
PT
positively stained for annexin V-APC only and for both Annexin V-APC and PI represented
SC
RI
apoptotic cells.
2.10 Statistical analysis
NU
Data are expressed as means ± standard deviation (SD). Statistical analysis was
MA
performed by using the SPSS 18.0 (SPSS, Chicago, IL, USA). Comparisons of differences between groups parameters were performed by using Student’s t-test or ANOVA. P values
PT E
D
less than 0.05 were considered statistically significant.
3. Results
CE
3.1 Differential expression of miRNA-143 and BCL2 in degenerative disc tissues
AC
compared to that in normal disc tissues To determine the biological roles of miRNA-143 and BCL2 in human IVDD, we compared the expression of miRNA-143 and BCL2 mRNA in human normal and degenerative NPs by RT-PCR (Fig. 1A and B). MiRNA-143 expression was up-regulated, while that of BCL2 mRNA was down-regulated in degenerative disc tissues compared to those in normal disc tissues. MiRNA-143 and BCL2 expression negatively correlated (Fig. 1C). Furthermore, BCL2 protein expression was significantly decreased in degenerative disc
ACCEPTED MANUSCRIPT tissues compared to that in normal disc tissues (Fig. 1D and E). These results suggest that miRNA-143 and BCL2 are, at least in part, involved in human disc degeneration development.
PT
3.2 miRNA-143 modulates BCL2 expression and promotes NP cell apoptosis
RI
NP cells were transfected with scrambled control oligo or with miRNA-143 mimic,
SC
which presented high transfection efficiency (Fig. 2A). BCL2 expression was decreased in cells transfected with miRNA-143 mimics compared to control cells and cells treated with the
NU
inhibitor (Fig. 2B). Moreover, BCL2 protein expression was significantly decreased in the
MA
miRNA-143 mimic-transfected group compared to that in control and inhibitor groups (Fig. 2C and D). Furthermore, caspase-3 and caspase-9 expression increased in the miRNA-143
D
mimic-transfected group. It has been reported that extracellular-signal-regulated kinase 5
PT E
(ERK5) is a target of miRNA-143 (Akao et al., 2009). To investigate the effect of miRNA-143 in degenerative NP cells, we detected ERK5 expression. ERK5 protein
CE
expression was decreased in miRNA-143 mimic-transfected cells and increased in
AC
miRNA-143 inhibitor-transfected cells (Fig. 2E and F). To intuitively verify the regulatory relationship between miRNA-143 and NP cell apoptosis, we used flow cytometry (Fig. 2G and H). Apoptosis was significantly increased in the miRNA-143 mimic-transfected group compared to that in the control and inhibitor-transfected groups. These results demonstrated that miRNA-143 promoted NP cell apoptosis.
ACCEPTED MANUSCRIPT
3.3 miRNA-143 regulates NP cell apoptosis by directly targeting BCL2 Using TargetScan prediction, we determined that BCL2, an important molecule protecting many cell types from apoptosis, contains two miRNA-143 target sites (Fig. 3A). To verify the relationship between BCL2 and miRNA-143, a dual luciferase reporter system was
PT
employed. Luciferase activity was decreased in the miRNA-143 co-transfected group and this
RI
miRNA-143-induced decrease in luciferase activity was partially weakened when either of the
SC
binding sites was deleted (Fig. 3B). BCL2 expression was directly suppressed by miRNA-143 in NP cells (Fig. 3C, D, and E). These results demonstrate that miRNA-143 directly regulates
NU
BCL2 expression and suggest that miRNA-143 may implement its pro-apoptosis function by
MA
inhibiting BCL2 expression.
To verify this hypothesis, we used a BCL2 siRNA. The results showed that BCL2
D
mRNA and protein expression levels decreased in the BCL2 siRNA group (Fig. 3C, 3D and
PT E
E), while the mRNA and protein expression of caspase-3 and caspase-9 increased in the BCL2 siRNA group (Fig. 3C, 3D and E), indicating that caspase-3 and caspase-9 expression
CE
is regulated by BCL2. Flow cytometry indicated that apoptosis of NP cells increased in the
AC
BCL2 siRNA group (Fig. 3F and G). These results indicated that NP cell apoptosis was at least partially regulated by BCL2. In contrast, when using the miRNA-143 inhibitor, BCL2 expression increased, while that of both caspase-3 and caspase-9 decreased (Fig. 3C, D, and E). Apoptosis of NP cells also decreased in the miRNA-143 inhibitor group (Fig. 3F and G). Addition of the BCL2 siRNA into the miRNA-143 inhibitor group abrogated these effects (Fig. 3F and G). These results confirmed that miRNA-143 promotes apoptosis of NP cells by directly inhibiting BCL2 expression.
ACCEPTED MANUSCRIPT
3.4 Demethylation enhances miRNA-143 expression level and induces NP cell apoptosis MSP assay showed that the methylation level of degenerative NP tissues was decreased compared to that in normal NP tissues (Fig. 4A). The expression of miRNA-143 was increased in the 5-AZA treatment groups compared to the control groups (Fig. 4B). BCL2
PT
mRNA and protein expression decreased, while the mRNA and protein expression of
RI
caspase-3 and caspase-9 increased in the 5-AZA treatment groups compared to that in the
SC
5-AZA untreated groups (Fig. 4C and 4D). In addition, miRNA-143 expression was not affected by 5-AZA in the miRNA-143 inhibitor-treated groups. To analyze whether BCL2 is
NU
directly regulated by the demethylation drug, 5-AZA, we used miRNA-143 inhibitor to
MA
dislodge miRNA-143 expression in the cytoplasm. BCL2 expression did not significantly change compared to the 5-AZA untreated groups in the miRNA-143 inhibitor groups. Similar
D
results were obtained for caspase-3/9 (fig. 4C and 4D). The results confirmed that
PT E
methylation regulated miRNA-143 expression and NP cell apoptosis.
CE
4．Discussion
AC
Previous studies showed that NP cell apoptosis plays an important role in the pathological process of IVDD (Guo et al., 2016; Wang et al., 2016). Increasing evidence showed that miRNAs regulate diverse biological and pathological processes, including cell growth, differentiation, apoptosis, and carcinogenesis (Ma et al., 2007; Chen et al., 2012; Zhou et al., 2015). The role of miRNA-143 in regulating cell apoptosis in cancer has been studied (Ji, 2010; Gregersen et al., 2012; Shimbo et al., 2014), but the expression and role of miRNA-143 in human NP cell apoptosis are unclear.
ACCEPTED MANUSCRIPT In this study, we determined that miRNA-143 expression is significantly higher in degenerative NP tissues than in normal NP tissues. In addition, we demonstrated that miRNA-143 enhances NP cell apoptosis. In the process of NP cell apoptosis, a number of changes are observed such as senescence, apoptosis, and autophagy. Apoptosis is one of the
PT
most important factors in the process of NP cell death. BCL2 is known as a critical
RI
anti-apoptosis gene in mammalian cells. BCL2 protein displays genetic alterations in various
SC
tissues (Araujo Junior et al., 2013; Afroze et al., 2016; Borkan, 2016), helping the cells evade apoptosis by inhibiting pro-apoptotic genes and promoting anti-apoptotic genes. Aberrant
NU
expression of apoptosis-related genes is known as a critical mechanism for disc degeneration
MA
(Jiang et al., 2014; Zhang et al., 2016). Recent studies showed that BCL2 is expressed in NP tissues and plays an important role in the progression of NP cell apoptosis (Ding et al., 2012;
D
Miyazaki et al., 2015; Shi et al., 2015). Additionally, recent studies suggest that miRNA-143
PT E
modulates BCL2 expression (Ji, 2010; Shi et al., 2015; Li et al., 2016). We speculated that miRNA-143 contributed to apoptosis of NP cells by targeting BCL2.
CE
In this study, we found that miRNA-143 significantly regulated BCL2 expression. Thus,
AC
we focused on the function of miRNA-143 in the process of NP cell apoptosis. Our results demonstrated that miRNA-143 mimics significantly decreased BCL2 expression and increased NP cell apoptosis, while miRNA-143 inhibitors had the opposite effect. Our results indicate that miRNA-143 regulates BCL2 expression, thereby inducing NP cell apoptosis. A previous study showed that EKR5 is a target of miRNA-143 (Akao et al., 2009). Our results confirmed that miRNA-143 regulates ERK5 expression in degenerative human NP cells. The results demonstrate successful targeted effects of miRNA-143. Furthermore, the present
ACCEPTED MANUSCRIPT results indicated that deregulated miRNA-143 may enhance apoptosis in human IVDD by inhibiting BCL2, implying that miRNA-143 may have an etiological and therapeutic role in IVDD. We still do not know why miRNA-143 is up-regulated in degenerative discs. Epigenetics
PT
influence gene expression and phenotype manifestation without any changes in the DNA
RI
sequence. The aberrant CpG hyper-methylation constitutes a mechanism for miRNA silencing
SC
(Suzuki et al., 2012). However, the distribution of CpGs is not uniform. Although previous studies demonstrated that the expression of miRNA-143 is regulated by methylation (Dou et
NU
al., 2012), whether miRNA-143 expression and the process of disc degeneration are affected
MA
by methylation remains unclear.
In this study, we found that miRNA-143 methylation in degenerative NP cells was lower
D
than that in normal NP cells. Additionally, miRNA-143 expression increased when the gene
PT E
was de-methylated by 5-AZA treatment, and caspase-3/9 mRNA and protein expression increased, while that of BCL2 decreased. In addition, BCL2 and caspase-3/9 expression was
CE
not affected by 5-AZA after miRNA-143 depletion in the degenerative NP cells. Thus, the
AC
hypo-methylation status of NP cells may modulate miRNA-143 expression and induce NP cell apoptosis in IVDD. In conclusion, we demonstrated that miRNA-143 overexpression induced a decrease in the number of NP cells by reducing BCL2 expression in human degenerative NP cells and that miRNA-143 expression and function were regulated by NP cell methylation. We provide unequivocal evidence that miRNA-143 expression is up-regulated in the degenerative NP tissues in vivo and it plays a critical role in the process of human NP cell apoptosis by directly
ACCEPTED MANUSCRIPT targeting BCL2 in vitro for the first time. In addition, hyper-methylation status was observed in degenerative human NP cells in vivo, and miRNA-143 expression and function were regulated by DNA methylation status in human NP cells in vitro. Although further studies are needed especially in vivo studies, our results suggest that miRNA-143 plays a pivotal role in
PT
the pathogenesis of IVDD and may become a candidate for the development of RNA-based
SC
RI
treatments against disc degeneration.
NU
Conflicts of interest
MA
The authors declared no conflict of interest with respect to the research, authorship, and
PT E
Acknowledgments
D
publication of this article.
This study was supported by the National Natural Science Foundation of China
AC
CE
(No.81072187, No.81541056 and U1603121).
ACCEPTED MANUSCRIPT References Afroze, S.H., Kalagiri, R.R., Reyes, M., Zimmerman, J.D., Beeram, M.R., Drever, N., Zawieja, D.C., Kuehl, T.J. and Uddin, M.N., 2016. Apoptotic and stress signaling markers are augmented in preeclamptic placenta and umbilical cord. BBA Clin 6, 25-30.
PT
Akao, Y., Nakagawa, Y., Iio, A. and Naoe, T., 2009. Role of microRNA-143 in Fas-mediated apoptosis in
RI
human T-cell leukemia Jurkat cells. Leuk Res 33, 1530-8.
SC
Araujo Junior, R.F., Oliveira, A.L., Pessoa, J.B., Garcia, V.B., Guerra, G.C., Soares, L.A., Souza, T.P., Petrovick, P.R. and Araujo, A.A., 2013. Maytenus ilicifolia dry extract protects normal cells,
NU
induces apoptosis and regulates Bcl-2 in human cancer cells. Exp Biol Med (Maywood) 238,
MA
1251-8.
Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-97.
D
Borkan, S.C., 2016. The Role of BCL-2 Family Members in Acute Kidney Injury. Semin Nephrol 36,
PT E
237-50.
Cai, P., Yang, T., Jiang, X., Zheng, M., Xu, G. and Xia, J., 2017. Role of miR-15a in intervertebral disc
CE
degeneration through targeting MAP3K9. Biomed Pharmacother 87, 568-574.
AC
Chen, H., Wang, J., Hu, B., Wu, X., Chen, Y., Li, R. and Yuan, W., 2015. MiR-34a promotes Fas-mediated cartilage endplate chondrocyte apoptosis by targeting Bcl-2. Mol Cell Biochem 406, 21-30. Chen, L., Zhang, J., Feng, Y., Li, R., Sun, X., Du, W., Piao, X., Wang, H., Yang, D., Sun, Y., Li, X., Jiang, T., Kang, C., Li, Y. and Jiang, C., 2012. MiR-410 regulates MET to influence the proliferation and invasion of glioma. Int J Biochem Cell Biol 44, 1711-7. de Araujo Junior, R.F., Leitao Oliveira, A.L., de Melo Silveira, R.F., de Oliveira Rocha, H.A., de Franca Cavalcanti, P. and de Araujo, A.A., 2015. Telmisartan induces apoptosis and regulates Bcl-2 in
ACCEPTED MANUSCRIPT human renal cancer cells. Exp Biol Med (Maywood) 240, 34-44. Ding, F., Shao, Z.W. and Xiong, L.M., 2013. Cell death in intervertebral disc degeneration. Apoptosis 18, 777-85. Ding, F., Shao, Z.W., Yang, S.H., Wu, Q., Gao, F. and Xiong, L.M., 2012. Role of mitochondrial pathway in
PT
compression-induced apoptosis of nucleus pulposus cells. Apoptosis 17, 579-90.
RI
Dou, L., Zheng, D., Li, J., Li, Y., Gao, L., Wang, L. and Yu, L., 2012. Methylation-mediated repression of
SC
microRNA-143 enhances MLL-AF4 oncogene expression. Oncogene 31, 507-17. Gregersen, L.H., Jacobsen, A., Frankel, L.B., Wen, J., Krogh, A. and Lund, A.H., 2012. MicroRNA-143
NU
down-regulates Hexokinase 2 in colon cancer cells. BMC Cancer 12, 232.
MA
Guo, J., Shao, M., Lu, F., Jiang, J. and Xia, X., 2016. Role of Sirt1 Plays in Nucleus Pulposus Cells and Intervertebral Disc Degeneration. Spine (Phila Pa 1976).
D
Gupta, V.K., Chitranshi, N., Gupta, V.B., Golzan, M., Dheer, Y., Wall, R.V., Georgevsky, D., King, A.E.,
PT E
Vickers, J.C., Chung, R. and Graham, S., 2016. Amyloid beta accumulation and inner retinal degenerative changes in Alzheimer's disease transgenic mouse. Neurosci Lett 623, 52-6.
CE
Hardwick, J.M. and Soane, L., 2013. Multiple functions of BCL-2 family proteins. Cold Spring Harb
AC
Perspect Biol 5.
Ji, 2010. microRNA-143, down-regulated in osteosarcoma, promotes apoptosis and suppresses tumorigenicity by targeting Bcl-2. Oncology Reports 24. Jiang, L., Zhang, X., Zheng, X., Ru, A., Ni, X., Wu, Y., Tian, N., Huang, Y., Xue, E., Wang, X. and Xu, H., 2013. Apoptosis, senescence, and autophagy in rat nucleus pulposus cells: Implications for diabetic intervertebral disc degeneration. J Orthop Res 31, 692-702. Jiang, W., Zhang, X., Hao, J., Shen, J., Fang, J., Dong, W., Wang, D., Zhang, X., Shui, W., Luo, Y., Lin, L.,
ACCEPTED MANUSCRIPT Qiu, Q., Liu, B. and Hu, Z., 2014. SIRT1 protects against apoptosis by promoting autophagy in degenerative human disc nucleus pulposus cells. Sci Rep 4, 7456. Le Maitre, C.L., Binch, A.L., Thorpe, A.A. and Hughes, S.P., 2015. Degeneration of the intervertebral disc with new approaches for treating low back pain. J Neurosurg Sci 59, 47-61.
PT
Li, W.H., Wu, H.J., Li, Y.X., Pan, H.G., Meng, T. and Wang, X., 2016. MicroRNA-143 promotes apoptosis
RI
of osteosarcoma cells by caspase-3 activation via targeting Bcl-2. Biomed Pharmacother 80,
SC
8-15.
Li, Z., Yu, X., Shen, J., Chan, M.T. and Wu, W.K., 2015a. MicroRNA in intervertebral disc degeneration.
NU
Cell Prolif 48, 278-83.
MA
Li, Z., Zhang, G., Li, D., Jie, Z., Chen, H., Xiong, J., Liu, Y., Cao, Y., Jiang, M., Le, Z. and Tan, S., 2015b. Methylation-associated silencing of miR-495 inhibit the migration and invasion of human
D
gastric cancer cells by directly targeting PRL-3. Biochem Biophys Res Commun 456, 344-50.
PT E
Liu, J., Zhang, J., Li, Y., Wang, L., Sui, B. and Dai, D., 2016. MiR-455-5p acts as a novel tumor suppressor in gastric cancer by down-regulating RAB18. Gene 592, 308-15.
CE
Long, X.R., He, Y., Huang, C. and Li, J., 2014. MicroRNA-148a is silenced by hypermethylation and
AC
interacts with DNA methyltransferase 1 in hepatocellular carcinogenesis. Int J Oncol 44, 1915-22.
Ma, L., Teruya-Feldstein, J. and Weinberg, R.A., 2007. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449, 682-8. Ma, X., Lin, Y., Yang, K., Yue, B., Xiang, H. and Chen, B., 2015. Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro. Int J Mol Med 36, 186-94.
ACCEPTED MANUSCRIPT Miyazaki, S., Kakutani, K., Yurube, T., Maeno, K., Takada, T., Zhang, Z., Kurakawa, T., Terashima, Y., Ito, M., Ueha, T., Matsushita, T., Kuroda, R., Kurosaka, M. and Nishida, K., 2015. Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells. Arthritis Res Ther
PT
17, 253.
RI
Pronina, I.V., Loginov, V.I., Burdennyy, A.M., Fridman, M.V., Kazubskaya, T.P., Dmitriev, A.A. and Braga,
SC
E.A., 2016. Expression and DNA methylation alterations of seven cancer-associated 3p genes and their predicted regulator miRNAs (miR-129-2, miR-9-1) in breast and ovarian cancers.
NU
Gene 576, 483-91.
MA
Qian, X., Yu, J., Yin, Y., He, J., Wang, L., Li, Q., Zhang, L.Q., Li, C.Y., Shi, Z.M., Xu, Q., Li, W., Lai, L.H., Liu, L.Z. and Jiang, B.H., 2013. MicroRNA-143 inhibits tumor growth and angiogenesis and
D
sensitizes chemosensitivity to oxaliplatin in colorectal cancers. Cell Cycle 12, 1385-94.
PT E
Sayed, D. and Abdellatif, M., 2011. MicroRNAs in development and disease. Physiol Rev 91, 827-87. Sekar, D., Venugopal, B., Sekar, P. and Ramalingam, K., 2016. Role of microRNA 21 in diabetes and
CE
associated/related diseases. Gene 582, 14-8.
AC
Shi, L., Teng, H., Zhu, M., Li, C., Huang, K., Chen, B.I., Dai, Y. and Wang, J., 2015. Paeoniflorin inhibits nucleus pulposus cell apoptosis by regulating the expression of Bcl-2 family proteins and caspase-9 in a rabbit model of intervertebral disc degeneration. Exp Ther Med 10, 257-262. Shimbo, K., Miyaki, S., Ishitobi, H., Kato, Y., Kubo, T., Shimose, S. and Ochi, M., 2014. Exosome-formed synthetic microRNA-143 is transferred to osteosarcoma cells and inhibits their migration. Biochem Biophys Res Commun 445, 381-7. Suzuki, H., Maruyama, R., Yamamoto, E. and Kai, M., 2012. DNA methylation and microRNA
ACCEPTED MANUSCRIPT dysregulation in cancer. Mol Oncol 6, 567-78. Vergroesen, P.P., Kingma, I., Emanuel, K.S., Hoogendoorn, R.J., Welting, T.J., van Royen, B.J., van Dieen, J.H. and Smit, T.H., 2015. Mechanics and biology in intervertebral disc degeneration: a vicious circle. Osteoarthritis Cartilage 23, 1057-70.
PT
Wang, C., Wang, W.J., Yan, Y.G., Xiang, Y.X., Zhang, J., Tang, Z.H. and Jiang, Z.S., 2015a. MicroRNAs:
RI
New players in intervertebral disc degeneration. Clin Chim Acta 450, 333-41.
SC
Wang, H.Q., Yu, X.D., Liu, Z.H., Cheng, X., Samartzis, D., Jia, L.T., Wu, S.X., Huang, J., Chen, J. and Luo, Z.J., 2011. Deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral
NU
disc degeneration by targeting FADD and caspase-3. J Pathol 225, 232-42.
MA
Wang, J., Chen, H., Cao, P., Wu, X., Zang, F., Shi, L., Liang, L. and Yuan, W., 2016. Inflammatory cytokines induce caveolin-1/beta-catenin signalling in rat nucleus pulposus cell apoptosis
D
through the p38 MAPK pathway. Cell Prolif 49, 362-72.
PT E
Wang, T., Li, P., Ma, X., Tian, P., Han, C., Zang, J., Kong, J. and Yan, H., 2015b. MicroRNA-494 inhibition protects nucleus pulposus cells from TNF-alpha-induced apoptosis by targeting JunD.
CE
Biochimie 115, 1-7.
AC
Yan, S., Wang, M., Zhao, J., Zhang, H., Zhou, C., Jin, L., Zhang, Y., Qiu, X., Ma, B. and Fan, Q., 2016. MicroRNA-34a affects chondrocyte apoptosis and proliferation by targeting the SIRT1/p53 signaling pathway during the pathogenesis of osteoarthritis. Int J Mol Med 38, 201-9. Zhang, F., Zhao, X., Shen, H. and Zhang, C., 2016. Molecular mechanisms of cell death in intervertebral disc degeneration (Review). Int J Mol Med 37, 1439-48. Zhang, J., Sun, Q., Zhang, Z., Ge, S., Han, Z.G. and Chen, W.T., 2013. Loss of microRNA-143/145 disturbs cellular growth and apoptosis of human epithelial cancers by impairing the MDM2-p53
ACCEPTED MANUSCRIPT feedback loop. Oncogene 32, 61-9. Zhou, J., Wu, S., Chen, Y., Zhao, J., Zhang, K., Wang, J. and Chen, S., 2015. microRNA-143 is associated with the survival of ALDH1+CD133+ osteosarcoma cells and the chemoresistance of
AC
CE
PT E
D
MA
NU
SC
RI
PT
osteosarcoma. Exp Biol Med (Maywood) 240, 867-75.
ACCEPTED MANUSCRIPT Fig. 1 Differential expression of miRNA-143 and BCL2 in degenerative NP tissues compared with that in normal NP tissues. (A) miRNA-143 expression in normal and degenerative NP tissues was examined by PCR. (B) BCL2 mRNA expression in normal and degenerative NP tissues. (C) Relationship between miRNA-143 and BCL2 expression. (D) and (E) BCL2
PT
protein expression in normal and degenerative NP tissues. Results are shown as mean ± SD.
SC
RI
Data are representative of three independent experiments. *P < 0.05 vs. normal tissues
Fig. 2 MiRNA-143 modulates BCL2 expression and promotes NP cell apoptosis. (A)
NU
MiRNA-143 expression was examined by RT-PCR. (B) The mRNA expression of BCL2 and
MA
caspase-3/9 was examined by RT-PCR. (C) (D) Protein expression of BCL2 and caspase-3/9. (E) Protein levels of ERK5. (F) Quantitative analysis of the protein expression of ERK5. (G)
D
Flow cytometry analysis of NP cell apoptosis. (H) Percentage of apoptotic NP cells. Results
PT E
are shown as mean ± SD. Data are representative of three independent experiments. *P < 0.05
CE
vs. miR-143 mimic NC, #P < 0.05 vs. miR-143 inhibitor NC
AC
Fig. 3 MiRNA-143 promotes the apoptosis of NP cells by directly targeting BCL2. (A) Human BCL2 may be a molecular target of miRNA-143. (B) Firefly luciferase activity measured and normalized by Renilla luciferase activity. Normal NP cells were co-transfected with empty vector, human BCL2 3-UTR firefly luciferase reporter plasmid, or either one or both deletions of the putative target sites as indicated, together with pTK-Renilla-luciferase plasmids, control, or miRNA-143 mimic as indicated. (C) mRNA expression of BCL2 and caspase-3/9 in NP cells. (D) Protein levels of BCL2 and caspase-3/9. (E) Quantitative analysis
ACCEPTED MANUSCRIPT of the protein expression of BCL2 and caspase-3/9 in NP cells. (F) Flow cytometry. (G) Percentage of apoptotic NP cells. Results are shown as mean ± SD. Data are representative of three independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. siRNA scr and P < 0.05 vs.
PT
miR-143 inhibitor + BCL2 siRNA
RI
Fig. 4 Demethylation enhances miRNA-143 expression and induces NP cell apoptosis. (A)
SC
Hypo-methylation status was detected in degenerative NP tissues compared to normal NP tissues by MSP experiment. (B) MiRNA-143 expression was up-regulated after 5-AZA
NU
treatment compared to the control group. (C) mRNA expression of BCL2 and caspase-3/9
MA
was detected by RT-PCR. (D) Protein expression of BCL2 and caspase-3/9. (E) Quantitative analysis of BCL2 and caspase-3/9 protein expression. Results are shown as mean ± SD. Data
PT E
D
are representative of three independent experiments. *P < 0.05 vs. 5-AZA untreated groups.
Fig. 5 Schematic model illustrating how miRNA-143 regulates NP cell apoptosis via BCL2
CE
and the effect of methylation. During the transcriptional progress from DNA to miRNA-143,
AC
the increase in DNA promoter methylation status results in decreased miRNA-143 expression. miRNA-143 inhibits the translation from BCL2 mRNA to BCL2 protein by combining with BCL2 3-UTR. BCL2 decreases NP cell apoptosis by inhibiting caspase-3/9 activation.
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
Fig. 1
AC
CE
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
Fig. 2
AC
CE
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
Fig. 3
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
Fig. 4
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
Fig. 5
ACCEPTED MANUSCRIPT Table 1 Sequences of primers used for RT-PCR Gene
Oligonucleotide sequence Forward (5-3)
Product
Reverse (5-3)
size
GCCTTCTTTGAGTTCGGTGG
GAAATCAAACAGAGGCCGCA
192
Caspase-3
ACTGGACTGTGGCATTGAGA GCACAAAGCGACTGGATGAA
162
Caspase-9
CCTGTGTCGGTCGAGAAGAT
TGGGTGTGGGCAAACTAGAT
247
U6
CGCTTCGGCAGCACATATAC
AAATATGGAACGCTTCACGA
100
β-actin
AGCGAGCATCCCCCAAAGTT GGGCACGAAGGCTCATCATT
285
AC
CE
PT E
D
MA
NU
SC
PT
BCL-2
RI
(bp)
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
Graphical abstract
ACCEPTED MANUSCRIPT
Abbreviation list
Lower back pain
IVDD
Intervertebral disc degeneration
NP
Nucleus pulposus
AF
Annulus fibrosus
ECM
Extracellular matrix
MiRNA
MicroRNA
BCL2
B-cell lymphoma-2
HUST
Huazhong University of Science and Technology
MA
NU
SC
RI
PT
LBP
Intervertebral disc
MRI
Magnetic Resonance Imaging
D
IVD
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
PVDF
Polyvinylidene fluoride
3-UTR
3’- Untranslated Regions
mRNA ERK5
CE
Quantitative real-time polymerase chain reaction
AC
qRT-PCR
PT E
SDS-PAGE
Messenger RNA Extracellular-signal-regulated kinase 5
ACCEPTED MANUSCRIPT Highlights： 1. A high expression of miRNA-143 was observed in human IVDD tissues. 2. MiR-143 promotes the nucleus pulposus apoptosis in human IVDD by directly targeting BCL2. 3. BCL2 decreases nucleus pulposus cell apoptosis via restraint caspase-3/9.
PT
4. A high frequency of miRNA-143 methylation was observed in human IVDD tissues.
AC
CE
PT E
D
MA
NU
SC
RI
5. DNA methylation of miR-143 regulates the expression of miR-143 in IVDD
Kangcheng Zhao, Yukun Zhang, Kang Liang, Yu Song, Kun Wang, Shuai Li, Xinghuo Wu, Wenbin Hua, Zengwu Shao, Shuhua Yang, Cao Yang PII: DOI: Reference:
S0378-1119(17)30565-6 doi: 10.1016/j.gene.2017.07.043 GENE 42067
To appear in:
Gene
Received date: Revised date: Accepted date:
9 December 2016 7 July 2017 13 July 2017
Please cite this article as: Kangcheng Zhao, Yukun Zhang, Kang Liang, Yu Song, Kun Wang, Shuai Li, Xinghuo Wu, Wenbin Hua, Zengwu Shao, Shuhua Yang, Cao Yang , Epigenetic silencing of miRNA-143 regulates apoptosis by targeting BCL2 in human intervertebral disc degeneration, Gene (2017), doi: 10.1016/j.gene.2017.07.043
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Epigenetic silencing of miRNA-143 regulates apoptosis by targeting BCL2 in human intervertebral disc degeneration
1
Kangcheng Zhao , Yukun Zhang1, Kang Liang, Yu Song, Kun Wang, Shuai Li, Xinghuo
RI
PT
Wu, Wenbin Hua, Zengwu Shao, Shuhua Yang, Cao Yang*
SC
Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong
NU
University of Science and Technology, Wuhan, 430022, China
CE
PT E
D
These authors contributed equally to this work.
AC
1
MA
* Corresponding author: Cao Yang
ACCEPTED MANUSCRIPT
Abstract Accumulating evidence indicates that microRNAs can regulate the apoptosis of various cells. Apoptosis of nucleus pulposus cells plays an important role in the progression of intervertebral disc degeneration. The aim of this study is to investigate whether
PT
microRNA-143 (miRNA-143) is involved in the progression of intervertebral disc
RI
degeneration. In this study, the expression of miRNA-143 and its biological modulatory
SC
effects were examined. Messenger RNA and protein expression of miRNA-143 and B-cell lymphoma-2 (BCL2) in both normal and degenerative disc tissues was determined by using
NU
RT-PCR and western-blot assays. After miRNA-143 transfection, BCL2 expression and NP
MA
cell apoptosis were assessed by using RT-PCR, western-blot, and flow cytometry. The relationship between miRNA-143 and BCL2 was assessed by bioinformatics and dual assays.
Epigenetic
regulation
of
miRNA-143
was
determined
by
D
luciferase
PT E
methylation-specific PCR and the effect of hypomethylation using 5-AZA. In this study, miRNA-143 expression significantly increased, while that of BCL2 decreased in degenerative
CE
disc specimens. In addition, CpG islands in the promoter region of miRNA-143 were
AC
hypomethylated in degenerative disc tissues. Furthermore, bioinformatics analysis and luciferase reporter assay indicated that BCL2 was a target gene of miRNA-143, and miRNA-143 suppressed BCL2 messenger RNA (mRNA) and protein expression. MiRNAmiRNA-143 overexpression enhances apoptosis of nucleus pulposus cells, while miRNA-143 inhibitor had the opposite effect. BCL2 knockdown reversed the effects of the miRNA-143 inhibitor on nucleus pulposus apoptosis. Our results suggest that miRNA-143 promotes the progression of nucleus pulposus apoptosis by directly targeting BCL2,
ACCEPTED MANUSCRIPT providing a potential therapeutic target for the treatment of intervertebral disc degeneration disease.
Keywords: Intervertebral disc degeneration; BCL2; miRNA-143; apoptosis; methylation;
AC
CE
PT E
D
MA
NU
SC
RI
PT
human nucleus pulposus
ACCEPTED MANUSCRIPT
1. Introduction Lower back pain (LBP) is one of the most common diseases, affecting the quality of life of patients and becoming a socioeconomic burden worldwide. The causes of LBP include aging, abnormal mechanical loading, trauma, and genetic predisposition. However, for many
PT
patients, the major cause of LBP is intervertebral disc degeneration (IVDD) (Chen et al., 2015;
RI
Le Maitre et al., 2015). IVDD is characterized by extracellular matrix degradation and
SC
decreased number of viable cells (Jiang et al., 2013; Vergroesen et al., 2015), which is mainly attributed to an imbalance of cell apoptosis. The intervertebral disc presents a complex
NU
structure with three sections: the nucleus pulposus (NP), end plate, and annulus fibrosus (AF).
MA
NP cells play a crucial role in maintaining the normal physiological function via the synthesis of extracellular matrix (ECM) components to resist mechanical loads (Wang et al., 2015a).
D
The decrease of NP cells associated with apoptosis is considered as an initial trigger for
PT E
IVDD (Ding et al., 2013; Ma et al., 2015). Apoptosis plays a crucial role in the pathological process of cell death (Wang et al., 2015a). Studies demonstrated that aberrant cell apoptosis is
CE
responsible for many degenerative diseases such as Alzheimer’s disease, osteoarthritis, IVDD,
AC
and cancer (Li et al., 2015a; Gupta et al., 2016; Yan et al., 2016). MicroRNAs (miRNAs) are small noncoding RNAs (17–25 nucleotides), which regulate gene expression, resulting in the inhibition of translation or degradation. They play important regulatory roles in multitudinous biological cellular processes such as differentiation, proliferation, and apoptosis (Bartel, 2004; Sayed and Abdellatif, 2011; Wang et al., 2015a; Liu et al., 2016). Recent studies demonstrated that miRNAs such as miRNA-494, miRNA-27a, miRNA-155, and miRNA-15b play important regulatory roles in the progression of IVDD
ACCEPTED MANUSCRIPT (Wang et al., 2011; Wang et al., 2015a; Wang et al., 2015b; Cai et al., 2017). MiRNA-143 is also broadly involved in cellular processes such as proliferation, invasion, apoptosis, and senescence (Zhang et al., 2013; Sekar et al., 2016). Aberrant miRNA-143 expression is observed in many tumors such as gastric, prostate, and colorectal cancer (Ji, 2010; Qian et al.,
PT
2013), indicating a potentially regulatory role in tumor progression. However, the expression
RI
and the role of miRNA-143 in degenerative disc NP cells are still unclear.
SC
BCL2 gene is located on chromosome 18, which was discovered as part of the t(14;18) chromosomal translocation in patients with follicular lymphoma and diffuse large B cell
NU
lymphoma (DLBCL). The protein encoded by BCL2 supports cell survival by blocking
MA
apoptosis rather than by increasing the cell division rate (Hardwick and Soane, 2013; de Araujo Junior et al., 2015). Data support that apoptosis is involved in pathological IVDD (Ma
D
et al., 2015). Therefore, BCL2 overexpression in human NP may slow down the degeneration
PT E
of IVD by preventing apoptosis of NP cells. DNA methylation is an important epigenetic process regulating the expression of target
CE
genes and usually results in gene silencing by inhibiting the binding of transcription factors or
AC
recruitment of methyl-binding proteins and the associated chromatin remodeling factors. The CpG islands distributed in the human genome play crucial roles in the methylation process. The gene silencing function of miRNA promoter CpG islands is a common mechanism during tumorigenesis (Pronina et al., 2016). Considering that the imbalance of cell apoptosis caused by dysfunction of miRNA expression is the common mechanism of tumorigenesis and disc degeneration, methylation may be an important regulatory mechanism. Considering the important roles of miRNAs and BCL2 in the pathogenesis of IVDD and
ACCEPTED MANUSCRIPT the putative BCL2 binding site for miRNA-143, we hypothesized that miRNA-143 might affect IVDD via BCL2. In addition, we investigated whether methylation, which is involved in various diseases and impacts on many miRNA promoters (Long et al., 2014; Li et al., 2015b), was involved in the association between miRNA-143 and NP cell apoptosis.
PT
Thus, this study aimed to identify whether miRNA-143 expression increased in
RI
degenerative disc tissues, and whether it targets BCL2 to induce apoptosis. Our study might
SC
provide insights useful for the development of novel therapies for clinical LBP treatment.
NU
2. Materials and Methods
MA
2.1 Ethics statement
The study was approved by the ethics committee of Tongji Medical College, Huazhong
D
University of Science and Technology (HUST). All IVD specimens were obtained in
PT E
accordance with the World Medical Association Declaration of Helsinki Ethical Principles for
CE
Medical Research Involving Human Subjects. All patients signed a written informed consent.
AC
2.1 Sample collection
All subjects were vertebral column spine surgery patients admitted to The Union Hospital Orthopaedic Hospital of HUST (Wuhan, China). The normal human IVD tissues were obtained from patients with idiopathic scoliosis classified as Pfirrmann grade I or II according to Magnetic Resonance Imaging (MRI) (n = 10; mean age, 22.3 ± 4.25; range, 17–40). The IVDD tissues were obtained from the LBP patients with disc degeneration classified as Pfirrmann grade IV according to MRI (n = 10; mean age, 25.8 ± 5.32, 25–58).
ACCEPTED MANUSCRIPT
2.3 Isolation and culture of human degenerative NP cells The human NP tissues were microscopically isolated from IVD tissues using a scalpel, under sterile conditions. These NP tissues were then washed with PBS twice and cut into 1
PT
mm3 fragments. These fragments were digested by 0.25% trypsin solution containing 1%
RI
ethylenediaminetetraacetic acid (EDTA) for 30 min and type II collagenase (Invitrogen,
SC
Carlsbad, CA, USA) for 3–4 hours at 37C. Tissue debris was removed by passing through a 200 m filter and then the NP cells were resuspended in DMEM/F12 containing 15% fetal
NU
bovine serum (FBS), 100 g/mL streptomycin, 100 U/mL penicillin, and 1%
MA
L-glutamine and incubated at 37C in an atmosphere containing 5% CO2. When at 80–90% confluence, cells were digested by 0.25% trypsin solution and subcultured in culture
2.4 Cell transfection
PT E
D
flasks. The third passage of NP cells was used for all experiments.
CE
MiRNA-143 mimic, miRNA-143 mimic-scr, miRNA-143 inhibitor, miRNA-143
AC
inhibitor-scr, BCL2 siRNA, and BCL2 siRNA-scr were purchased from RiboBio (Guangzhou, China). MiRNA-143 mimic and miRNA-143 inhibitor were used to promote and inhibit miRNA-143 expression, respectively. BCL2 siRNA was used to inhibit BCL2 expression. They were transfected into NP cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 48 hours, NP cells were harvested and used for further analyses.
ACCEPTED MANUSCRIPT
2.5 RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from human NP tissues using TRIZOL reagent (Aidlab, Beijing China) according to the manufacturer’s instruction. The purity and concentration of RNA were measured by using an ultraviolet spectrophotometer (Shunyu, shanghai, China).
PT
qRT-PCR was conducted to quantify miRNA-143, BCL2, and caspase-3/9 expression using
RI
the 7500 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA) in a final
SC
volume of 20 L. The primers used for qRT-PCR are listed in Table 1. All experiments were performed in triplicate. The quantitative miRNA data were normalized to U6 and mRNA
NU
expression of BCL2, caspase-3, and caspase-9 was normalized to that of -actin. The
MA
fold-change in gene expression relative to the control was calculated by the 2-△△Ct method.
D
2.6 Western blot analysis
PT E
NP specimens were randomly selected and protein concentrations were determined by using the BCA assay (Beyotime, Shanghai, China). Human NP cells were washed with PBS at
CE
4C (pH = 7.2–7.3) and lysed by using RIPA lysis Buffer (Beyotime) on ice. Protein
AC
concentration was measured as described above. Protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Finally, membranes were treated with ECL reagent (ThermoFisher Scientific, Waltham, MA, USA) and visualized. GAPDH was used as an internal control. The experiment was repeated three times.
ACCEPTED MANUSCRIPT
2.7 miRNA-143 target prediction and 3-UTR luciferase reporter assay TargetScan (www.targetscan.org) was used to predict the target genes of miRNA-143. BCL2 contained a putative miRNA-143 binding site. BCL2 3-Untranslated Region (3-UTR) luciferase reporter was inserted into the 3-UTR region of pmir-Report construct (Promega,
PT
Madison, WI, USA). The BCL2 3-UTR luciferase reporter deleted for the miRNA-143 target
RI
site was created by PCR amplification of the BCL2 3-UTR luciferase reporter construct
SC
lacking the miRNA-143 bind target site. Normal NP cells (5 103) were co-transfected with luciferase reporter plasmid, internal control prl-tk-renilla-luciferase plasmid, and the indicated
NU
RNAs. After 48 hours, luciferase activity was measured by using the Dual-Luciferase
MA
Reporter Assay System (Promega) according to the manufacturer’s instructions. Firefly
D
luciferase activity was normalized to Renilla luciferase activity.
PT E
2.8 Methylation assay
We detected the methylation status of miRNA-143 promoter in degenerative and normal
CE
disc tissue extracts by using methylation-specific PCR (MSP) analysis. Genomic DNA was
AC
isolated from degenerative and normal disc tissues by using DNA extraction kit (Promega) according to the manufacturer’s instructions. The primer sequences used for MSP were as follows:
methylated,
forward:
ATTAGTTAGGTATGGTGGTGTACGT
and
reverse:
TTTAAAACAAAATCTCCCTCTATCG;
unmethylated,
forward:
ATTAGTTAGGTATGGTGGTGTATGT
and
reverse:
TTAAAACAAAATCTCCCTCTATCAC. The PCR products were electrophoresed on a 3% agarose gel.
ACCEPTED MANUSCRIPT
2.9 Flow cytometry Apoptosis was detected by using the Annexin V-APC apoptosis detection kit (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s instructions. Cells
PT
positively stained for annexin V-APC only and for both Annexin V-APC and PI represented
SC
RI
apoptotic cells.
2.10 Statistical analysis
NU
Data are expressed as means ± standard deviation (SD). Statistical analysis was
MA
performed by using the SPSS 18.0 (SPSS, Chicago, IL, USA). Comparisons of differences between groups parameters were performed by using Student’s t-test or ANOVA. P values
PT E
D
less than 0.05 were considered statistically significant.
3. Results
CE
3.1 Differential expression of miRNA-143 and BCL2 in degenerative disc tissues
AC
compared to that in normal disc tissues To determine the biological roles of miRNA-143 and BCL2 in human IVDD, we compared the expression of miRNA-143 and BCL2 mRNA in human normal and degenerative NPs by RT-PCR (Fig. 1A and B). MiRNA-143 expression was up-regulated, while that of BCL2 mRNA was down-regulated in degenerative disc tissues compared to those in normal disc tissues. MiRNA-143 and BCL2 expression negatively correlated (Fig. 1C). Furthermore, BCL2 protein expression was significantly decreased in degenerative disc
ACCEPTED MANUSCRIPT tissues compared to that in normal disc tissues (Fig. 1D and E). These results suggest that miRNA-143 and BCL2 are, at least in part, involved in human disc degeneration development.
PT
3.2 miRNA-143 modulates BCL2 expression and promotes NP cell apoptosis
RI
NP cells were transfected with scrambled control oligo or with miRNA-143 mimic,
SC
which presented high transfection efficiency (Fig. 2A). BCL2 expression was decreased in cells transfected with miRNA-143 mimics compared to control cells and cells treated with the
NU
inhibitor (Fig. 2B). Moreover, BCL2 protein expression was significantly decreased in the
MA
miRNA-143 mimic-transfected group compared to that in control and inhibitor groups (Fig. 2C and D). Furthermore, caspase-3 and caspase-9 expression increased in the miRNA-143
D
mimic-transfected group. It has been reported that extracellular-signal-regulated kinase 5
PT E
(ERK5) is a target of miRNA-143 (Akao et al., 2009). To investigate the effect of miRNA-143 in degenerative NP cells, we detected ERK5 expression. ERK5 protein
CE
expression was decreased in miRNA-143 mimic-transfected cells and increased in
AC
miRNA-143 inhibitor-transfected cells (Fig. 2E and F). To intuitively verify the regulatory relationship between miRNA-143 and NP cell apoptosis, we used flow cytometry (Fig. 2G and H). Apoptosis was significantly increased in the miRNA-143 mimic-transfected group compared to that in the control and inhibitor-transfected groups. These results demonstrated that miRNA-143 promoted NP cell apoptosis.
ACCEPTED MANUSCRIPT
3.3 miRNA-143 regulates NP cell apoptosis by directly targeting BCL2 Using TargetScan prediction, we determined that BCL2, an important molecule protecting many cell types from apoptosis, contains two miRNA-143 target sites (Fig. 3A). To verify the relationship between BCL2 and miRNA-143, a dual luciferase reporter system was
PT
employed. Luciferase activity was decreased in the miRNA-143 co-transfected group and this
RI
miRNA-143-induced decrease in luciferase activity was partially weakened when either of the
SC
binding sites was deleted (Fig. 3B). BCL2 expression was directly suppressed by miRNA-143 in NP cells (Fig. 3C, D, and E). These results demonstrate that miRNA-143 directly regulates
NU
BCL2 expression and suggest that miRNA-143 may implement its pro-apoptosis function by
MA
inhibiting BCL2 expression.
To verify this hypothesis, we used a BCL2 siRNA. The results showed that BCL2
D
mRNA and protein expression levels decreased in the BCL2 siRNA group (Fig. 3C, 3D and
PT E
E), while the mRNA and protein expression of caspase-3 and caspase-9 increased in the BCL2 siRNA group (Fig. 3C, 3D and E), indicating that caspase-3 and caspase-9 expression
CE
is regulated by BCL2. Flow cytometry indicated that apoptosis of NP cells increased in the
AC
BCL2 siRNA group (Fig. 3F and G). These results indicated that NP cell apoptosis was at least partially regulated by BCL2. In contrast, when using the miRNA-143 inhibitor, BCL2 expression increased, while that of both caspase-3 and caspase-9 decreased (Fig. 3C, D, and E). Apoptosis of NP cells also decreased in the miRNA-143 inhibitor group (Fig. 3F and G). Addition of the BCL2 siRNA into the miRNA-143 inhibitor group abrogated these effects (Fig. 3F and G). These results confirmed that miRNA-143 promotes apoptosis of NP cells by directly inhibiting BCL2 expression.
ACCEPTED MANUSCRIPT
3.4 Demethylation enhances miRNA-143 expression level and induces NP cell apoptosis MSP assay showed that the methylation level of degenerative NP tissues was decreased compared to that in normal NP tissues (Fig. 4A). The expression of miRNA-143 was increased in the 5-AZA treatment groups compared to the control groups (Fig. 4B). BCL2
PT
mRNA and protein expression decreased, while the mRNA and protein expression of
RI
caspase-3 and caspase-9 increased in the 5-AZA treatment groups compared to that in the
SC
5-AZA untreated groups (Fig. 4C and 4D). In addition, miRNA-143 expression was not affected by 5-AZA in the miRNA-143 inhibitor-treated groups. To analyze whether BCL2 is
NU
directly regulated by the demethylation drug, 5-AZA, we used miRNA-143 inhibitor to
MA
dislodge miRNA-143 expression in the cytoplasm. BCL2 expression did not significantly change compared to the 5-AZA untreated groups in the miRNA-143 inhibitor groups. Similar
D
results were obtained for caspase-3/9 (fig. 4C and 4D). The results confirmed that
PT E
methylation regulated miRNA-143 expression and NP cell apoptosis.
CE
4．Discussion
AC
Previous studies showed that NP cell apoptosis plays an important role in the pathological process of IVDD (Guo et al., 2016; Wang et al., 2016). Increasing evidence showed that miRNAs regulate diverse biological and pathological processes, including cell growth, differentiation, apoptosis, and carcinogenesis (Ma et al., 2007; Chen et al., 2012; Zhou et al., 2015). The role of miRNA-143 in regulating cell apoptosis in cancer has been studied (Ji, 2010; Gregersen et al., 2012; Shimbo et al., 2014), but the expression and role of miRNA-143 in human NP cell apoptosis are unclear.
ACCEPTED MANUSCRIPT In this study, we determined that miRNA-143 expression is significantly higher in degenerative NP tissues than in normal NP tissues. In addition, we demonstrated that miRNA-143 enhances NP cell apoptosis. In the process of NP cell apoptosis, a number of changes are observed such as senescence, apoptosis, and autophagy. Apoptosis is one of the
PT
most important factors in the process of NP cell death. BCL2 is known as a critical
RI
anti-apoptosis gene in mammalian cells. BCL2 protein displays genetic alterations in various
SC
tissues (Araujo Junior et al., 2013; Afroze et al., 2016; Borkan, 2016), helping the cells evade apoptosis by inhibiting pro-apoptotic genes and promoting anti-apoptotic genes. Aberrant
NU
expression of apoptosis-related genes is known as a critical mechanism for disc degeneration
MA
(Jiang et al., 2014; Zhang et al., 2016). Recent studies showed that BCL2 is expressed in NP tissues and plays an important role in the progression of NP cell apoptosis (Ding et al., 2012;
D
Miyazaki et al., 2015; Shi et al., 2015). Additionally, recent studies suggest that miRNA-143
PT E
modulates BCL2 expression (Ji, 2010; Shi et al., 2015; Li et al., 2016). We speculated that miRNA-143 contributed to apoptosis of NP cells by targeting BCL2.
CE
In this study, we found that miRNA-143 significantly regulated BCL2 expression. Thus,
AC
we focused on the function of miRNA-143 in the process of NP cell apoptosis. Our results demonstrated that miRNA-143 mimics significantly decreased BCL2 expression and increased NP cell apoptosis, while miRNA-143 inhibitors had the opposite effect. Our results indicate that miRNA-143 regulates BCL2 expression, thereby inducing NP cell apoptosis. A previous study showed that EKR5 is a target of miRNA-143 (Akao et al., 2009). Our results confirmed that miRNA-143 regulates ERK5 expression in degenerative human NP cells. The results demonstrate successful targeted effects of miRNA-143. Furthermore, the present
ACCEPTED MANUSCRIPT results indicated that deregulated miRNA-143 may enhance apoptosis in human IVDD by inhibiting BCL2, implying that miRNA-143 may have an etiological and therapeutic role in IVDD. We still do not know why miRNA-143 is up-regulated in degenerative discs. Epigenetics
PT
influence gene expression and phenotype manifestation without any changes in the DNA
RI
sequence. The aberrant CpG hyper-methylation constitutes a mechanism for miRNA silencing
SC
(Suzuki et al., 2012). However, the distribution of CpGs is not uniform. Although previous studies demonstrated that the expression of miRNA-143 is regulated by methylation (Dou et
NU
al., 2012), whether miRNA-143 expression and the process of disc degeneration are affected
MA
by methylation remains unclear.
In this study, we found that miRNA-143 methylation in degenerative NP cells was lower
D
than that in normal NP cells. Additionally, miRNA-143 expression increased when the gene
PT E
was de-methylated by 5-AZA treatment, and caspase-3/9 mRNA and protein expression increased, while that of BCL2 decreased. In addition, BCL2 and caspase-3/9 expression was
CE
not affected by 5-AZA after miRNA-143 depletion in the degenerative NP cells. Thus, the
AC
hypo-methylation status of NP cells may modulate miRNA-143 expression and induce NP cell apoptosis in IVDD. In conclusion, we demonstrated that miRNA-143 overexpression induced a decrease in the number of NP cells by reducing BCL2 expression in human degenerative NP cells and that miRNA-143 expression and function were regulated by NP cell methylation. We provide unequivocal evidence that miRNA-143 expression is up-regulated in the degenerative NP tissues in vivo and it plays a critical role in the process of human NP cell apoptosis by directly
ACCEPTED MANUSCRIPT targeting BCL2 in vitro for the first time. In addition, hyper-methylation status was observed in degenerative human NP cells in vivo, and miRNA-143 expression and function were regulated by DNA methylation status in human NP cells in vitro. Although further studies are needed especially in vivo studies, our results suggest that miRNA-143 plays a pivotal role in
PT
the pathogenesis of IVDD and may become a candidate for the development of RNA-based
SC
RI
treatments against disc degeneration.
NU
Conflicts of interest
MA
The authors declared no conflict of interest with respect to the research, authorship, and
PT E
Acknowledgments
D
publication of this article.
This study was supported by the National Natural Science Foundation of China
AC
CE
(No.81072187, No.81541056 and U1603121).
ACCEPTED MANUSCRIPT References Afroze, S.H., Kalagiri, R.R., Reyes, M., Zimmerman, J.D., Beeram, M.R., Drever, N., Zawieja, D.C., Kuehl, T.J. and Uddin, M.N., 2016. Apoptotic and stress signaling markers are augmented in preeclamptic placenta and umbilical cord. BBA Clin 6, 25-30.
PT
Akao, Y., Nakagawa, Y., Iio, A. and Naoe, T., 2009. Role of microRNA-143 in Fas-mediated apoptosis in
RI
human T-cell leukemia Jurkat cells. Leuk Res 33, 1530-8.
SC
Araujo Junior, R.F., Oliveira, A.L., Pessoa, J.B., Garcia, V.B., Guerra, G.C., Soares, L.A., Souza, T.P., Petrovick, P.R. and Araujo, A.A., 2013. Maytenus ilicifolia dry extract protects normal cells,
NU
induces apoptosis and regulates Bcl-2 in human cancer cells. Exp Biol Med (Maywood) 238,
MA
1251-8.
Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-97.
D
Borkan, S.C., 2016. The Role of BCL-2 Family Members in Acute Kidney Injury. Semin Nephrol 36,
PT E
237-50.
Cai, P., Yang, T., Jiang, X., Zheng, M., Xu, G. and Xia, J., 2017. Role of miR-15a in intervertebral disc
CE
degeneration through targeting MAP3K9. Biomed Pharmacother 87, 568-574.
AC
Chen, H., Wang, J., Hu, B., Wu, X., Chen, Y., Li, R. and Yuan, W., 2015. MiR-34a promotes Fas-mediated cartilage endplate chondrocyte apoptosis by targeting Bcl-2. Mol Cell Biochem 406, 21-30. Chen, L., Zhang, J., Feng, Y., Li, R., Sun, X., Du, W., Piao, X., Wang, H., Yang, D., Sun, Y., Li, X., Jiang, T., Kang, C., Li, Y. and Jiang, C., 2012. MiR-410 regulates MET to influence the proliferation and invasion of glioma. Int J Biochem Cell Biol 44, 1711-7. de Araujo Junior, R.F., Leitao Oliveira, A.L., de Melo Silveira, R.F., de Oliveira Rocha, H.A., de Franca Cavalcanti, P. and de Araujo, A.A., 2015. Telmisartan induces apoptosis and regulates Bcl-2 in
ACCEPTED MANUSCRIPT human renal cancer cells. Exp Biol Med (Maywood) 240, 34-44. Ding, F., Shao, Z.W. and Xiong, L.M., 2013. Cell death in intervertebral disc degeneration. Apoptosis 18, 777-85. Ding, F., Shao, Z.W., Yang, S.H., Wu, Q., Gao, F. and Xiong, L.M., 2012. Role of mitochondrial pathway in
PT
compression-induced apoptosis of nucleus pulposus cells. Apoptosis 17, 579-90.
RI
Dou, L., Zheng, D., Li, J., Li, Y., Gao, L., Wang, L. and Yu, L., 2012. Methylation-mediated repression of
SC
microRNA-143 enhances MLL-AF4 oncogene expression. Oncogene 31, 507-17. Gregersen, L.H., Jacobsen, A., Frankel, L.B., Wen, J., Krogh, A. and Lund, A.H., 2012. MicroRNA-143
NU
down-regulates Hexokinase 2 in colon cancer cells. BMC Cancer 12, 232.
MA
Guo, J., Shao, M., Lu, F., Jiang, J. and Xia, X., 2016. Role of Sirt1 Plays in Nucleus Pulposus Cells and Intervertebral Disc Degeneration. Spine (Phila Pa 1976).
D
Gupta, V.K., Chitranshi, N., Gupta, V.B., Golzan, M., Dheer, Y., Wall, R.V., Georgevsky, D., King, A.E.,
PT E
Vickers, J.C., Chung, R. and Graham, S., 2016. Amyloid beta accumulation and inner retinal degenerative changes in Alzheimer's disease transgenic mouse. Neurosci Lett 623, 52-6.
CE
Hardwick, J.M. and Soane, L., 2013. Multiple functions of BCL-2 family proteins. Cold Spring Harb
AC
Perspect Biol 5.
Ji, 2010. microRNA-143, down-regulated in osteosarcoma, promotes apoptosis and suppresses tumorigenicity by targeting Bcl-2. Oncology Reports 24. Jiang, L., Zhang, X., Zheng, X., Ru, A., Ni, X., Wu, Y., Tian, N., Huang, Y., Xue, E., Wang, X. and Xu, H., 2013. Apoptosis, senescence, and autophagy in rat nucleus pulposus cells: Implications for diabetic intervertebral disc degeneration. J Orthop Res 31, 692-702. Jiang, W., Zhang, X., Hao, J., Shen, J., Fang, J., Dong, W., Wang, D., Zhang, X., Shui, W., Luo, Y., Lin, L.,
ACCEPTED MANUSCRIPT Qiu, Q., Liu, B. and Hu, Z., 2014. SIRT1 protects against apoptosis by promoting autophagy in degenerative human disc nucleus pulposus cells. Sci Rep 4, 7456. Le Maitre, C.L., Binch, A.L., Thorpe, A.A. and Hughes, S.P., 2015. Degeneration of the intervertebral disc with new approaches for treating low back pain. J Neurosurg Sci 59, 47-61.
PT
Li, W.H., Wu, H.J., Li, Y.X., Pan, H.G., Meng, T. and Wang, X., 2016. MicroRNA-143 promotes apoptosis
RI
of osteosarcoma cells by caspase-3 activation via targeting Bcl-2. Biomed Pharmacother 80,
SC
8-15.
Li, Z., Yu, X., Shen, J., Chan, M.T. and Wu, W.K., 2015a. MicroRNA in intervertebral disc degeneration.
NU
Cell Prolif 48, 278-83.
MA
Li, Z., Zhang, G., Li, D., Jie, Z., Chen, H., Xiong, J., Liu, Y., Cao, Y., Jiang, M., Le, Z. and Tan, S., 2015b. Methylation-associated silencing of miR-495 inhibit the migration and invasion of human
D
gastric cancer cells by directly targeting PRL-3. Biochem Biophys Res Commun 456, 344-50.
PT E
Liu, J., Zhang, J., Li, Y., Wang, L., Sui, B. and Dai, D., 2016. MiR-455-5p acts as a novel tumor suppressor in gastric cancer by down-regulating RAB18. Gene 592, 308-15.
CE
Long, X.R., He, Y., Huang, C. and Li, J., 2014. MicroRNA-148a is silenced by hypermethylation and
AC
interacts with DNA methyltransferase 1 in hepatocellular carcinogenesis. Int J Oncol 44, 1915-22.
Ma, L., Teruya-Feldstein, J. and Weinberg, R.A., 2007. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449, 682-8. Ma, X., Lin, Y., Yang, K., Yue, B., Xiang, H. and Chen, B., 2015. Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro. Int J Mol Med 36, 186-94.
ACCEPTED MANUSCRIPT Miyazaki, S., Kakutani, K., Yurube, T., Maeno, K., Takada, T., Zhang, Z., Kurakawa, T., Terashima, Y., Ito, M., Ueha, T., Matsushita, T., Kuroda, R., Kurosaka, M. and Nishida, K., 2015. Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells. Arthritis Res Ther
PT
17, 253.
RI
Pronina, I.V., Loginov, V.I., Burdennyy, A.M., Fridman, M.V., Kazubskaya, T.P., Dmitriev, A.A. and Braga,
SC
E.A., 2016. Expression and DNA methylation alterations of seven cancer-associated 3p genes and their predicted regulator miRNAs (miR-129-2, miR-9-1) in breast and ovarian cancers.
NU
Gene 576, 483-91.
MA
Qian, X., Yu, J., Yin, Y., He, J., Wang, L., Li, Q., Zhang, L.Q., Li, C.Y., Shi, Z.M., Xu, Q., Li, W., Lai, L.H., Liu, L.Z. and Jiang, B.H., 2013. MicroRNA-143 inhibits tumor growth and angiogenesis and
D
sensitizes chemosensitivity to oxaliplatin in colorectal cancers. Cell Cycle 12, 1385-94.
PT E
Sayed, D. and Abdellatif, M., 2011. MicroRNAs in development and disease. Physiol Rev 91, 827-87. Sekar, D., Venugopal, B., Sekar, P. and Ramalingam, K., 2016. Role of microRNA 21 in diabetes and
CE
associated/related diseases. Gene 582, 14-8.
AC
Shi, L., Teng, H., Zhu, M., Li, C., Huang, K., Chen, B.I., Dai, Y. and Wang, J., 2015. Paeoniflorin inhibits nucleus pulposus cell apoptosis by regulating the expression of Bcl-2 family proteins and caspase-9 in a rabbit model of intervertebral disc degeneration. Exp Ther Med 10, 257-262. Shimbo, K., Miyaki, S., Ishitobi, H., Kato, Y., Kubo, T., Shimose, S. and Ochi, M., 2014. Exosome-formed synthetic microRNA-143 is transferred to osteosarcoma cells and inhibits their migration. Biochem Biophys Res Commun 445, 381-7. Suzuki, H., Maruyama, R., Yamamoto, E. and Kai, M., 2012. DNA methylation and microRNA
ACCEPTED MANUSCRIPT dysregulation in cancer. Mol Oncol 6, 567-78. Vergroesen, P.P., Kingma, I., Emanuel, K.S., Hoogendoorn, R.J., Welting, T.J., van Royen, B.J., van Dieen, J.H. and Smit, T.H., 2015. Mechanics and biology in intervertebral disc degeneration: a vicious circle. Osteoarthritis Cartilage 23, 1057-70.
PT
Wang, C., Wang, W.J., Yan, Y.G., Xiang, Y.X., Zhang, J., Tang, Z.H. and Jiang, Z.S., 2015a. MicroRNAs:
RI
New players in intervertebral disc degeneration. Clin Chim Acta 450, 333-41.
SC
Wang, H.Q., Yu, X.D., Liu, Z.H., Cheng, X., Samartzis, D., Jia, L.T., Wu, S.X., Huang, J., Chen, J. and Luo, Z.J., 2011. Deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral
NU
disc degeneration by targeting FADD and caspase-3. J Pathol 225, 232-42.
MA
Wang, J., Chen, H., Cao, P., Wu, X., Zang, F., Shi, L., Liang, L. and Yuan, W., 2016. Inflammatory cytokines induce caveolin-1/beta-catenin signalling in rat nucleus pulposus cell apoptosis
D
through the p38 MAPK pathway. Cell Prolif 49, 362-72.
PT E
Wang, T., Li, P., Ma, X., Tian, P., Han, C., Zang, J., Kong, J. and Yan, H., 2015b. MicroRNA-494 inhibition protects nucleus pulposus cells from TNF-alpha-induced apoptosis by targeting JunD.
CE
Biochimie 115, 1-7.
AC
Yan, S., Wang, M., Zhao, J., Zhang, H., Zhou, C., Jin, L., Zhang, Y., Qiu, X., Ma, B. and Fan, Q., 2016. MicroRNA-34a affects chondrocyte apoptosis and proliferation by targeting the SIRT1/p53 signaling pathway during the pathogenesis of osteoarthritis. Int J Mol Med 38, 201-9. Zhang, F., Zhao, X., Shen, H. and Zhang, C., 2016. Molecular mechanisms of cell death in intervertebral disc degeneration (Review). Int J Mol Med 37, 1439-48. Zhang, J., Sun, Q., Zhang, Z., Ge, S., Han, Z.G. and Chen, W.T., 2013. Loss of microRNA-143/145 disturbs cellular growth and apoptosis of human epithelial cancers by impairing the MDM2-p53
ACCEPTED MANUSCRIPT feedback loop. Oncogene 32, 61-9. Zhou, J., Wu, S., Chen, Y., Zhao, J., Zhang, K., Wang, J. and Chen, S., 2015. microRNA-143 is associated with the survival of ALDH1+CD133+ osteosarcoma cells and the chemoresistance of
AC
CE
PT E
D
MA
NU
SC
RI
PT
osteosarcoma. Exp Biol Med (Maywood) 240, 867-75.
ACCEPTED MANUSCRIPT Fig. 1 Differential expression of miRNA-143 and BCL2 in degenerative NP tissues compared with that in normal NP tissues. (A) miRNA-143 expression in normal and degenerative NP tissues was examined by PCR. (B) BCL2 mRNA expression in normal and degenerative NP tissues. (C) Relationship between miRNA-143 and BCL2 expression. (D) and (E) BCL2
PT
protein expression in normal and degenerative NP tissues. Results are shown as mean ± SD.
SC
RI
Data are representative of three independent experiments. *P < 0.05 vs. normal tissues
Fig. 2 MiRNA-143 modulates BCL2 expression and promotes NP cell apoptosis. (A)
NU
MiRNA-143 expression was examined by RT-PCR. (B) The mRNA expression of BCL2 and
MA
caspase-3/9 was examined by RT-PCR. (C) (D) Protein expression of BCL2 and caspase-3/9. (E) Protein levels of ERK5. (F) Quantitative analysis of the protein expression of ERK5. (G)
D
Flow cytometry analysis of NP cell apoptosis. (H) Percentage of apoptotic NP cells. Results
PT E
are shown as mean ± SD. Data are representative of three independent experiments. *P < 0.05
CE
vs. miR-143 mimic NC, #P < 0.05 vs. miR-143 inhibitor NC
AC
Fig. 3 MiRNA-143 promotes the apoptosis of NP cells by directly targeting BCL2. (A) Human BCL2 may be a molecular target of miRNA-143. (B) Firefly luciferase activity measured and normalized by Renilla luciferase activity. Normal NP cells were co-transfected with empty vector, human BCL2 3-UTR firefly luciferase reporter plasmid, or either one or both deletions of the putative target sites as indicated, together with pTK-Renilla-luciferase plasmids, control, or miRNA-143 mimic as indicated. (C) mRNA expression of BCL2 and caspase-3/9 in NP cells. (D) Protein levels of BCL2 and caspase-3/9. (E) Quantitative analysis
ACCEPTED MANUSCRIPT of the protein expression of BCL2 and caspase-3/9 in NP cells. (F) Flow cytometry. (G) Percentage of apoptotic NP cells. Results are shown as mean ± SD. Data are representative of three independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. siRNA scr and P < 0.05 vs.
PT
miR-143 inhibitor + BCL2 siRNA
RI
Fig. 4 Demethylation enhances miRNA-143 expression and induces NP cell apoptosis. (A)
SC
Hypo-methylation status was detected in degenerative NP tissues compared to normal NP tissues by MSP experiment. (B) MiRNA-143 expression was up-regulated after 5-AZA
NU
treatment compared to the control group. (C) mRNA expression of BCL2 and caspase-3/9
MA
was detected by RT-PCR. (D) Protein expression of BCL2 and caspase-3/9. (E) Quantitative analysis of BCL2 and caspase-3/9 protein expression. Results are shown as mean ± SD. Data
PT E
D
are representative of three independent experiments. *P < 0.05 vs. 5-AZA untreated groups.
Fig. 5 Schematic model illustrating how miRNA-143 regulates NP cell apoptosis via BCL2
CE
and the effect of methylation. During the transcriptional progress from DNA to miRNA-143,
AC
the increase in DNA promoter methylation status results in decreased miRNA-143 expression. miRNA-143 inhibits the translation from BCL2 mRNA to BCL2 protein by combining with BCL2 3-UTR. BCL2 decreases NP cell apoptosis by inhibiting caspase-3/9 activation.
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
Fig. 1
AC
CE
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
Fig. 2
AC
CE
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
Fig. 3
PT E
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
Fig. 4
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
Fig. 5
ACCEPTED MANUSCRIPT Table 1 Sequences of primers used for RT-PCR Gene
Oligonucleotide sequence Forward (5-3)
Product
Reverse (5-3)
size
GCCTTCTTTGAGTTCGGTGG
GAAATCAAACAGAGGCCGCA
192
Caspase-3
ACTGGACTGTGGCATTGAGA GCACAAAGCGACTGGATGAA
162
Caspase-9
CCTGTGTCGGTCGAGAAGAT
TGGGTGTGGGCAAACTAGAT
247
U6
CGCTTCGGCAGCACATATAC
AAATATGGAACGCTTCACGA
100
β-actin
AGCGAGCATCCCCCAAAGTT GGGCACGAAGGCTCATCATT
285
AC
CE
PT E
D
MA
NU
SC
PT
BCL-2
RI
(bp)
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
Graphical abstract
ACCEPTED MANUSCRIPT
Abbreviation list
Lower back pain
IVDD
Intervertebral disc degeneration
NP
Nucleus pulposus
AF
Annulus fibrosus
ECM
Extracellular matrix
MiRNA
MicroRNA
BCL2
B-cell lymphoma-2
HUST
Huazhong University of Science and Technology
MA
NU
SC
RI
PT
LBP
Intervertebral disc
MRI
Magnetic Resonance Imaging
D
IVD
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
PVDF
Polyvinylidene fluoride
3-UTR
3’- Untranslated Regions
mRNA ERK5
CE
Quantitative real-time polymerase chain reaction
AC
qRT-PCR
PT E
SDS-PAGE
Messenger RNA Extracellular-signal-regulated kinase 5
ACCEPTED MANUSCRIPT Highlights： 1. A high expression of miRNA-143 was observed in human IVDD tissues. 2. MiR-143 promotes the nucleus pulposus apoptosis in human IVDD by directly targeting BCL2. 3. BCL2 decreases nucleus pulposus cell apoptosis via restraint caspase-3/9.
PT
4. A high frequency of miRNA-143 methylation was observed in human IVDD tissues.
AC
CE
PT E
D
MA
NU
SC
RI
5. DNA methylation of miR-143 regulates the expression of miR-143 in IVDD