Targeting of CDKN1B by miR‐222‐3p may contribute to the development of intervertebral disc degeneration

MicroRNAs (miRNAs) are small endogenous non‐coding RNAs that can negatively regulate the expression of their complementary mRNA targets, and have been implicated in various pathophysiological processes. In this study, we examined the effect of miR‐222‐3p on intervertebral disc degeneration (IDD). We found that expression of miR‐222‐3p was significantly higher in IDD tissues than in normal intervertebral disc tissue, and report that overexpression of miR‐222‐3p remarkably increased apoptosis and reduced proliferation of nucleus pulposus (NP) cells. In addition, miR‐222‐3p promoted secretion of matrix metalloproteinase‐3, and decreased collagen type II and aggrecan production. Cyclin‐dependent kinase inhibitor 1B (CDKN1B) was identified as a direct target of negative regulation by miR‐222‐3p in NP cells, and expression of miR‐222‐3p was found to be negatively correlated with that of CDKN1B in IDD tissue. Finally, we observed that transfection with miR‐222‐3p dramatically reduced CDKN1B expression in NP cells. In conclusion, miR‐222‐3p may be involved in IDD development, possibly through targeting CDKN1B.

MicroRNAs (miRNAs) are small endogenous non-coding RNAs that can negatively regulate the expression of their complementary mRNA targets, and have been implicated in various pathophysiological processes. In this study, we examined the effect of miR-222-3p on intervertebral disc degeneration (IDD). We found that expression of miR-222-3p was significantly higher in IDD tissues than in normal intervertebral disc tissue, and report that overexpression of miR-222-3p remarkably increased apoptosis and reduced proliferation of nucleus pulposus (NP) cells. In addition, miR-222-3p promoted secretion of matrix metalloproteinase-3, and decreased collagen type II and aggrecan production. Cyclin-dependent kinase inhibitor 1B (CDKN1B) was identified as a direct target of negative regulation by miR-222-3p in NP cells, and expression of miR-222-3p was found to be negatively correlated with that of CDKN1B in IDD tissue. Finally, we observed that transfection with miR-222-3p dramatically reduced CDKN1B expression in NP cells. In conclusion, miR-222-3p may be involved in IDD development, possibly through targeting CDKN1B.
Because of chronic low back pain and considerable economic expense, intervertebral disc degeneration (IDD) is a heavy load on society [1]. Growing evidence has confirmed that gradual degeneration of the nucleus pulposus (NP) is responsible for the development of IDD, and the imbalance of extracellular matrix (ECM) breakdown and abnormal synthesis is a major cause of IDD [2,3]. NP cells are the most important cells of the intervertebral disc, and their aberrant activity is one of the leading cause of IDD [4]. However, the pathological process of IDD and its potential mechanism remain unknown. Thus, finding specific targets on NP cells would help to better understand the development of IDD.
MicroRNAs (miRNAs) are small endogenous noncoding RNAs. By binding to the 3 0 -untranslated region (3 0 -UTR) of the target mRNAs, miRNAs negatively regulate of the expression of complementary mRNA targets [5]. miRNAs have been implicated in various cell pathophysiological processes, such as cell proliferation, apoptosis, and ECM metabolism. Accumulating evidence indicates that miRNAs are frequently dysregulated in the development of IDD. Although in recent years several miRNAs have come to be regarded as key players in the pathogenesis of IDD, the roles of many other miRNAs that are associated with pathogenesis of IDD remain to be elucidated miR-222-3p has been implicated in several diseases, such as cancers [6][7][8], hypertrophic scar [9], and cardiovascular diseases [10]; however, its role in IDD remains unclear. Cyclin-dependent kinase inhibitor 1B (CDKN1B), also named p27kip1, has been reported to inhibit cell cycle progression G1-S transitions, and phosphorylation of CDKN1B at different sites altered its distribution in the nucleus and cytoplasm in different cancers [11,12]. CDKN1B was also a target gene of miR-222-3p in several cancers [13,14], but the regulation by miR-222-3p of CDKN1B in NP cells remains unknown. Therefore, the aim of this study was to examine the effect and mechanism of miR-222-3p in IDD in targeting CDKN1B, and our results will provide a new therapeutic target for the treatment of IDD.

Microarray data
The miRNA expression dataset of GSE19943 [15] was downloaded from the Gene Expression Omnibus (GEO) database. This dataset has six samples, including three IDD NP tissues and three normal NP tissues. The microarray data were generated based on the GPL19446 platform (Exiqon human miRCURY LNA TM microRNA Array V11.0, Duesseldorf, Germany). The NP tissues in the normal group were grade I and in the IDD group grades IV and V by Pfirrmann grading [16].

Collection of IDD tissue
The intervertebral disc tissues were collected from 30 IDD patients who underwent lumbar spine surgery from October 2017 to June 2018 in the Third Affiliated Hospital of Guangxi Medical University. IDD assessment was based on the criteria of Pfirrmann grading using MRI examination [16]. Another 10 normal intervertebral disc tissues were obtained from patients who had traumatic lumbar fracture. The study protocols were approved by the ethics committee of Third Affiliated Hospital of Guangxi Medical University. All the procedures were in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects, with signed written informed consent.

NP cell isolation and culture
Human NP cells were obtained and cultured as previously described [17]. The third passage of NP cells was used for further tests.

miR-222-3p transfection
miR-222-3p mimic and inhibitors were chemically synthesized and purchased from GenePharma (Shanghai, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for transaction as per the manufacturer's instructions. The NP cells were seeded at 1 9 10 5 per well on 24-well plates and then transfected with 80 ng plasmid, 5 ng Renilla luciferase vector pRL-SV40, 50 nM miR-222-3p mimics and inhibitors by using Lipofectamine 2000. The final working concentration of miRNA was 100 nM. Experiments except the luciferase test were all conducted after 12 h of transfection.

RNA extraction and quantitative real-time PCR
RNA extraction and quantitative real-time PCR (qRT-PCR) were carried out using a general protocol of our laboratory [17]. U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal control for miR-222-3p and CDKN1B, respectively. The primer sequences of miR-222-3p, CDKN1B, U6 and GAPDH are listed in Table 1. The relative expression levels of miR-222-3p and CDKN1B were calculated using the 2 ÀDDCq method.

Western blotting
Protein extraction and western blotting were performed according to the general protocol in our laboratory [18]. Antibodies to CDKN1B (1 : 1000; Abcam, Cambridge, UK) and GAPDH (1: 2500; Abcam) were used as primary antibodies.

Cell proliferation and apoptosis assay
Cell proliferation assay was conducted using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) kit as instructed by the manufacturer. Cell apoptosis assays were quantified using an FITC Annexin V Apoptosis Detection Kit (Solarbio, Beijing, China) as previously described [17].

Immunofluorescence microscopy
Cell immunofluorescence staining of collagen type II was observed and acquired using an epifluorescence microscope (Olympus BX53, Tokyo, Japan) equipped with a camera.

Luciferase reporter assay
The binding site in the 3 0 -UTR of CDKN1B, including CDKN1B wild-type and CDKN1B mutant (MT) were cloned from human genomic DNA and then inserted into the KpnI and SacI sites of the pGL3 promoter vector (Realgene, Nanjing, China) in a dual-luciferase reporter assay. After transfection for 48 h, the cells were collected and measured using a Dual-Luciferase Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.

Statistical analysis
Data are shown as mean AE SD. Student's t test and oneway ANOVA followed by Tukey's post hoc test were used to assess the statistical significance for numerical data (including the miR-222-3p expression in Table 2) using SPSS STATISTICS v. 19.0 (IBM Corp., Armonk, NY, USA). Statistical significance was set at P < 0.05.

miR-222-3p increased in IDD tissues
By analyzing the GSE19943 dataset, which included three IDD tissues and three normal intervertebral disc tissues, it was revealed that miR-222-3p was up-regulated significantly in IDD tissues in contrast to normal intervertebral disc tissues (Fig. 1A, P < 0.05). In the clinical tissues, using the qRT-PCR method, we found that the expression of miR-222-3p was greatly higher in IDD tissues than in normal intervertebral disc tissues as well (Fig. 1B, P < 0.01).

miR-222-3p correlated with IDD stage
Among the clinical intervertebral disc tissues, 10 intervertebral disc tissues in the normal group were grade I, and in the IDD group, six tissues were grade IV and 24 were grade V. No significant difference was detected between normal and IDD tissues with regard to age and gender (P > 0.05). We then analyzed the miR-222-3p with the clinical parameters of IDD and found that miR-222-3p expression was up-regulated in the advanced grade of IDD (P < 0.05). There was no significant difference for age and gender in the IDD patients (P > 0.05; Table 2.

miR-222-3p suppresses NP cell proliferation and induces apoptosis
To examine the effect of miR-222-3p on the phenotype of NP cells, miR-222-3p mimics and inhibitors were transfected into NP cells. The expression of miR-222-3p was elevated significantly or decreased after transfecting using miR-222-3p mimics or inhibitors, respectively ( Fig. 2A). The CCK-8 assay showed that Table 1. Sequence of primers used in qRT-PCR.

Primer
Sequence (  proliferation of NP cells was significant increased after transfecting miR-222-3p inhibitors (Fig. 2B), and flow cytometry showed that the apoptosis rate of NP cells was significantly reduced after transfecting with miR-222-3p inhibitors (Fig. 2C). Collectedly, reduction of miR-222-3p was able to facilitate proliferation and suppress apoptosis of NP cells.

miR-222-3p inhibited ECM production of NP cells
Collagen type II (COL2A1) and aggrecan (ACAN) are common cytokines used to characterize ECM production in NP cells, whereas MMP-3 suppresses ECM synthesis [19,20]. In this study, when the miR-222-3p mimics and inhibitors were transfected into NP cells, we observed that the mRNA and protein level of COL2A1 and ACAN were decreased with miR-222-3p overexpression, while the mRNA and protein expression of MMP-3 was increased with overexpression of miR-222-3p ( Fig. 3A-C). In addition, the immunostaining also indicated that miR-222-3p could reduce COL2A1 expression (Fig. 3D). All these results demonstrated that miR-222-3p uptake can reduce the activity and production of ECM in NP cells.
Luciferase reporter assay confirmed that miR-222-3p directly targeted CDKN1B Three miRNA target gene databases (Targetscan, http://www.targetscan.org/mamm_31/) revealed that the miR-222-3p sequence has four binding sites for the 3 0 -UTR of CDKN1B, suggesting that CDKN1B may be a potential target gene of miR-222-3p (Fig. 4A). Then, through using the dual-luciferase reporter assay, we found that miR-222-3p overexpression significantly reduced the relative luciferase activity of the reporter gene for wild-type, but not mutant CDKN1B in NP cells (Fig. 4B), indicating that miR-222-3p directly targeted the 3 0 -UTR of CDKN1B in NP cells.

miR-222-3p negatively correlated with CDKN1B and reduced its expression
Using qRT-PCR method to measure the CDKN1B expression in IDD tissue, we observed a negatively correlated expression of miR-222-3p to CDKN1B (Fig. 5A). We then transfected NP cells with miR-222-3p mimics and inhibitor for 24 h and measured the CDKN1B mRNA and protein in NP cells. The results  indicated that CDKN1B mRNA and protein were significantly decreased after miR-222-3p mimic transfection ( Fig. 5B-D), indicating that miR-222-3p targeted and negatively regulates CDKN1B at the post-transcriptional level in NP cells.

Discussion
In this study, we measured the miRNAs in the GSE19943 dataset and clinical specimens using the qRT-PCR method. The results indicated that miR-222-3p was dramatically increased in IDD tissue compared with normal intervertebral disc. In addition, our results also indicated that expression of miR-222-3p was much higher in the advance grade of IDD compared with that in early grade, suggesting miR-222-3p is involved in the pathogenesis of IDD. We next examined the effect on the change of NP cell phenotype and observed that down-regulation of miR-222-3p could suppress NP cells apoptosis and induce proliferation, that it subsequently reduced the production of COL2A1 and ACAN, and that it increased the MMP-3 expression in NP cells. Our results demonstrated that CDKN1B is a direct targeted gene of miR-222-3p in NP cells, and CDKN1B was negatively correlated with miR-222-3p in IDD tissue. miR-222-3p expression was reported to be enhanced in breast cancer [21], gastric cancer [22], and lung cancer [23]; however, lower miR-222-3p levels were observed in severe myocardial fibrosis as compared to non-severe fibrosis [24]. Thus, the expression of miR-222-3p is varied in different diseases. With regard to the effect of miR-222-3p on cells, several studies have observed that miR-222-3p was associated with the proliferation, apoptosis, invasion and migration of some cancer cells [6,25]. Studies also showed that miR-222-3p is involved in regulation of mitochondrial dysfunction in response to transmissible gastroenteritis virus infection [26] and erythroid differentiation [27]. Our results showed that down-regulation of miR-222-3p could greatly inhibit apoptosis and enhance proliferation of NP cells, indicating that miR-222-3p expression was closely correlated with the NP cell phenotype.
In the current study, we also detected the expression of COL2A1, ACAN and MMP-3 after NP cells were transfected by miR-222-3p mimics and inhibitors. It is well known that the progressive loss of ECM is a hallmark of IDD, while MMP-3 is the main enzymes that degrades collagen II and expression of MMP-3 was increased in IDD tissue compared with healthy controls [19,20]. Moreover, other research reported that miR-222-3p was associated Fig. 4. Cyclin-dependent kinase inhibitor 1B was a direct target of miR-222-3p. (A) Targetscan database showed that miR-222-3p sequence has four binding sites for the 3 0 -UTR of CDKN1B. (B) Luciferase reporter assay showed that miR-222-3p significantly reduced the luciferase activity of wild-type, but not mutant CDKN1B in NP cells. Mean AE SD, n = 3; one-way ANOVA followed by Tukey's post hoc test was used to assess statistical significance: *P < 0.05, **P < 0.01. with ECM production in axial spondyloarthritis [28]. In agreement with the results for the phenotypic changes of NP cells, we observed that miR-222-3p overexpression significantly down-regulated the expression of COL2A1 and ACAN mRNA and protein levels, and up-regulated the MMP-3 mRNA and protein levels in NP cells. All in all, miR-222-3p will modulate the development of IDD through inhibiting the activity of NP cells.
Evidence has shown that miR-222-3p could target several genes, and a relationship of miR-222-3p-targeted CDKN1B has been implicated in breast cancer cells [29] and vascular smooth muscle cells [30], but this relationship has not been reported in NP cells. CDKN1B has high expression in nearly all the tissue of the body, and is involved in several forms of cell growth regulation including cell cycle, apoptosis, and phenotype expression [31]. The role of CDKN1B is influenced mainly by two mechanisms: the level of transcription and protein stability, and its subcellular localization [32]. Our work confirmed that miR-222-3p directly targeted CDKN1B in NP cells, and negatively regulated the CDKN1B level, which was also correlated to IDD. Therefore, this indicates that miR-222-3p targets CDKN1B then regulates IDD progression.

Conclusion
In this paper, our results revealed that miR-222-3p was significantly increased in IDD tissues and associated with IDD grade. Furthermore, CDKN1B was demonstrated to be the potential target of miR-222-3p, which facilitates IDD development. Our results provide a potential theory of IDD development, and a potential target for the treatment of IDD.