Selenium‐sensitive miRNA‐181a‐5p targeting SBP2 regulates selenoproteins expression in cartilage

Abstract Selenium (Se) deficiency brings about defects in the biosynthesis of several selenoproteins and has been associated with aberrant chondrogenesis. Selenocysteine (Sec) Insertion Sequence (SECIS) and SECIS binding protein 2 (SBP2) interaction is a very critical node for the metabolic balance between Se and selenoproteins. The Gpx1, Gpx4 and SelS have different binding affinities with SBP2 in cells. According to our results, both miR‐181a‐5p and SBP2 appeared to be selenium‐sensitive and regulated the expression of selenoproteins in C28/I2 cells under Se sufficient environment. However, they showed significantly opposite expression trend in Se deficiency rats cartilage and SeD C28/I2 cells. The SBP2 is a direct target gene of miR‐181a‐5p in C28/I2 cells as determined by reporter gene and off‐target experiments. And the miR‐181a‐5p could regulate SBP2 and the selenoproteins in C28/I2 cells. Depending upon the Se supply levels, C28/I2 cells were divided into three groups, that is normal Se, SeD and SeS, which underwent through a 7‐day Se deprivation process, then SBP2 was knocked‐down and overexpressed in all the groups. Moreover, the selected selenoproteins were down‐regulated in second‐generation low Se diet rat cartilage. The selenoproteins expression was decreased by Se deficiency which depended on the Selenium‐sensitive miR‐181a‐5p to participate and regulate SBP2 at post‐transcriptional level. It involves a series of antioxidant and ECM (extracellular matrix) genes, to overcome the ROS‐related stress for the protection of essential physiological functions and to maintain the balance between anabolism and catabolism of the cartilage.

activity of selenoproteins (Sel), named after the Se. [1][2][3] Sel are involved in various biological processes, such as antioxidative stress, anti-tumour and especially the development. 3,4 Sec is encoded into the Sel by the genetic code "UGA" that is commonly a termination codon in cells. 5 Sel biosynthesis is regulated by several special cistrans elements and trans-acting factors, such as selenocysteine insertion sequence (SECIS) and SECIS binding protein 2 (SECISBP2 or SBP2). 6,7 SECIS is located at 3′-untranslated region (3′-UTR) of Sel mRNA and can bind with SBP2. SBP2 functions to carry Sec-tRNA Sec into ribosomal "A site" that recognizes the "UGA" as the codon for Sec during Sel synthesis. 6,7 Se deficiency leads to altered synthesis of several selenoproteins, affecting chondrogenesis. 8 This results in epiphyseal plate abnormalities and articular cartilage degradation. 9 Representatively, Kashin-Beck disease (KBD), an endemic osteoarthropathy in Se deficient regions of China, is characterized by pathological chondronecrosis in the deep zones of articular cartilage and growth plates from different peripheral joints of the endemic children. [10][11][12] Further, Se supplementation is able to protect the Se deficient rats from epiphysial growth plate abnormality and has been used in the prophylactics of KBD by unknown mechanisms. 9,13,14 Intriguingly, osteo-chondroprogenitor-specific deletion of the selenocysteinyl tRNA Sec gene leads to the phenotypes similar to KBD, particularly showing chondronecrosis and abnormal skeletal development in mice. 15 It is revealed that the termination codon "UGA" is subjected to in-sufficient Sec-tRNA Sec , because of which the inactive truncated selenoproteins are produced. 16 Parallelly, the short inactive fragment of TrxR1 with a two-amino acid-truncated C-terminal motif could make human lung carcinoma A549 cells die. 17 However, it is poorly understood how Se regulates the selenoprotein biosynthesis in cartilage. Particularly, to find the first-hand factor of Se-sensitivity is of great importance to understand the mechanism. In addition, there is little known about the regulatory pathway mediated by SBP2 from Se deficiency to selenoprotein biosynthesis in cartilage.
On the other hand, it has been observed that Dicer-deficient mice suffer from a severe deficiency in bone development with proliferating chondrocytes and expansion of the hypertrophic region in the limb bud, 18,19 suggesting that miRNAs regulate both proliferation and differentiation of growth plate chondrocytes. MiRNAs have been demonstrated to be associated with both cartilage homoeostasis and development, especially the cartilage-specific miR-140. 20,21 About 30 miRNAs are differently expressed during the development of rats femoral articular cartilage. 22 For instance, miR-337 could influence cartilage-specific gene expression in chondrocytes. 23 It would be very exhilarating if we could find a "selenium-sensitive miRNA" to regulate the expression of SBP2 in cartilage.
In this study, miR-181a-5p, one of the differently expressed miR-NAs during cartilage development, 22 was predicted as a target of hSBP2with TargetScanHuman7.1. Coincidentally, it could repress the expression of Cyclin A2(CCAN2) and Aggrecan(ACAN) in chicken chondrocytes that may act as a negative feedback for cartilage homoeostasis. 24 However, to understand the physiological roles of miR-181a-5p in cartilage, further investigation was required. GPx1, GPX4 and SELS were selected as selenium phenotype markers in cartilage or chondrocytes, as they not only are regulated in cartilage by Se deficiency and Se supply, 9,25 but also are the representatives of different subcellular localizations: cytoplasm, mitochondria and endoplasmic reticulum, respectively. Hence, the detailed regulatory relationship among "low-selenium, miRNA, SBP2 and selenoproteins in cartilage" was investigated during this study.

| Rats
The inbred Dark Agouti (DA) rats 9,22,23 were raised in a climate controlled environment, housed in polystyrene cages containing wood shavings and were fed standard rodent chow and water ad libitum, in the SPF animal house of the Department of Biochemistry and Molecular Biology, Xi'an Jiaotong University, Health Science Center.
The DA rats originated from the Section of Medical Inflammation Research, Lund University, Sweden. The two generation low-selenium rat model was established as described previously, including Se deficient diet group (SeD) and Se sufficient diet group (SeS). 9 The experimenters were blinded to the Se condition while processing data and making exclusion decisions. All procedures were in accordance with the Animals (Scientific Procedures) Act, 1986 (UK) (amended 2013). All sections of this report adhere to the ARRIVE Guidelines for reporting animal research. 26 A completed ARRIVE guidelines checklist is included in Checklist S.

| Histological staining
Knee joints fixed with 4% paraformaldehyde (PFA) were tenderly decalcified in 10% EDTA liquid for 4 weeks. Subsequently paraffinembedded, dissected into 5-μm-thick pieces and stained to observe the morphological changes in the epiphyseal plate. All the sections were then stained with conventional haematoxylin and eosin (H&E), safranin O and fast green dyes.

| Immunohistochemistry staining
After intrinsic peroxidase activity, the articular cartilage sections were blocked with 3% hydrogen peroxide (H 2 O 2 ) and then incubated MIN ET AL.
Rabbit IgG was used as a negative control.

| Total RNA extraction and quantitative PCR analysis
Total RNA was isolated from rats articular cartilage or C28/I2 cell samples using the TRIzol ® method (Invitrogen, USA). Two microgram of each total RNA from articular cartilage was timely reversed to cDNA according to the manufacturer's instructions (RevertAid ™ ; Fermentas, Canada) and stored at −20°C until used. In addition, miRNA-cDNA was obtained using One Step PrimeScript ® miRNA-cDNA Synthesis Kit (Takara, Japan). Because the cartilage were limited and contained very less RNA in one sample, it could not satisfy the test of every gene.
The mRNA or miRNA expression was tested by real-time quantitative PCR (RT-qPCR), which was performed on iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) with SYBR ® Premix Ex Taq ™ II (TaKaRa, Japan). The relative gene expression was normalized by GAPDH, while let-7a was used as the internal reference of miR-181a-5p. The procedure of miRNA-cDNA qPCR was two-step amplification:pre-degeneration 95°C 10 second; PCR amplification: 95°C 5 second, 60°C 20 second, 40 cycles. The information of primers is depicted in Tables 1-3. T A B L E 1 Information of miRNA-181a-5p for real-time PCR

| Statistical analysis
Data were presented as means ± SEM. The statistical significance of pathological data was calculated by Mann-Whitney U test. Means of two groups were compared using Student's t test, and statistical significance was considered as P < 0.05 in all tests (*P < 0.05, **P < 0.01and **P < 0.001). All analyses were performed using the software GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).

| Both miR-181a-5p and SBP2 are seleniumsensitive in cartilage and chondrocytes
Firstly, the expressions of miR-181a-5p, Sbp2 and SBP2 were detected in articular cartilage of the low Se diet rat model. 9 In SeD rat cartilage, miR-181a-5p showed remarkable up-regulation as compared to the SeS group (P = 0.0006, Figure 1A). Conversely, the expression of SBP2 was significantly reduced in SeD group compared with SeS group, both at mRNA and protein levels (P = 0.0066, Figure 1B  Further, a seven-day-term low medium cell culture was performed as described previously to induce the low Se condition in vitro. 25 MiR-181a-5p expressed at significantly elevated levels in SeD group compared with Se supplement cultured C28/I2 cells (P = 0.0035, Figure 1D), indicating that miR-181a-5p was Se susceptive in chondrocytes, which was in line with the results obtained from the rat model, presented above. Meanwhile, SBP2and SBP2 were significantly down-regulated, contrarily (P = 0.0335, Figure 1E and F). It is evident from the above results that both miR-181a-5p and SBP2 are selenium-sensitive and are regulated by Se supplement.

I2 cells
Subsequently dual-luciferase reporter gene analysis showed that, the relative activity of Rluc/Fluc of SBP2 WT group was reduced that hsa-mir-181a-5 p could not bind with SBP2-3′-UTR because of the mutation in the predicted binding sites (P < 0.0001, Figure 2B).
Then, mimic-miR-181a-5p and inhibitor-miR-181a-5p were used to alter the level of miR-181a-5p, and the relative activity of dual- is a direct target gene of miR-181a-5p.

| Both miR-181a-5p and SBP2 could regulate the biosynthesis of three selenoproteins in chondrocytes
The mimic or inhibitor of miR-181a-5p was used in normal C28/I2

| SBP2 regulates selenoproteins in different Se conditions
In order to further explore the role of SBP2 in cartilage, the knockdown or overexpression of SBP2 was performed in C28/I2 cells by si-SBP2 or ad-SBP2, respectively. Under the Se deficient or Se replenished situations, the chondrocytes were treated with si-SBP2 or Ad-SBP2, respectively, in order to observe the expression of the above three selected selenoproteins.
In SeD groups of si-SBP2, the expression of SELS was very low, even GPX1 and GPX4 were difficult to detect, no matter transfected with si-SBP2 or not. All concerned proteins could be visibly detected with Se supplement, showing a reduced tendency in the si-SBP2 chondrocytes ( Figure 4A).
In the SBP2 overexpression chondrocytes, both SeD and SeS groups showed a remarkable elevation in the expression of SBP2.
Interestingly, the GPX1, GPX4 and SELS were robustly up-regulated compared with control group despite selenium deficient conditions.
After the selenium was replenished in to the selenium deficient chondrocytes, the selected selenoproteins expression was rescued significantly. On the other hand, GPX4 showed a little response to the SBP2 overexpression in selenium deficient or supplement chondrcocytes ( Figure 4B).

| DISCUSSION
To investigate whether miR-181a-5p is involved in cartilage injury process induced by Se deficiency, we established the Se deficiency and Se sufficiency models using C28/I2 human chondrocytes and inbred DA rats. 9 The phenotypes of the two generation Se deficiency rats included extracellular matrix (ECM) metabolism, aberration of articular cartilage, epiphyseal dysplasia, delayed skeletal development and the secondary osteoarthritis. 9,27 These phenotypes match exactly with KBD for its Se-deficient nutritional status. [10][11][12] Moreover, the main features of epiphyseal dysplasia are the Colla-gen2α1(COL2α1) and ACAN metabolic imbalances and chondrocytes apoptosis interrelated to abnormal proliferation and differentiation in the Se deficient diet fed rats, 9,27 similar to the symptoms of KBD patients. [10][11][12] Conformably, miR-181a-5p down-regulated the ACAN in chondrocytes through the unknown mechanism. 24 Predictably, miR-181a-5p was up-regulated in Se deficiency both in vivo and Se deficiency, suggesting that miR-181a-5p is susceptive to Se supply in cartilage.
Next, we used mimic and inhibitor sequences of miR-181a-5p to up-or down-regulate its expression in C28/I2 cells. The Sbp2/SBP2 showed a significant negative correlation with the miR-181a-5p. Further, we proved that SBP2 is a new, direct target of miR-181a-5p in chondrocytes. These results establish that Selenium-sensitive miR-181a-5p can regulate the expression of three selected selenoproteins GPx1, GPX4 and SELS to a variable extent through its target gene SBP2 in cartilage.
Meanwhile, the three selenoproteins were out-of-step regulated by miR-181a-5p mediated by SBP2, because their SECIS had the different affinity and binding efficiency with SBP2, 6,7,28-31 and SBP2 affects the expression of selenoproteins at mRNA levels. 25,[28][29][30] Unexpectedly, GPX4 would not be regulated by miR-181a-5p and SBP2 after their knockdown and overexpression in C28/I2 cells. Several mechanisms, such as activated NF-Y and Sp1, were involved in transcriptional and post-transcriptional regulation of GPX4. 32,33 Further, its expression is augmented through post-transcriptional modification by Grsf1 (guanine-rich sequence-binding factor 1) which is the Gpx4 mRNA-binding protein. 34 On contrary, the Gpx4expression is decreased after Grsf1 knockdown in mice and leads to developmental retardation of the brain which is similar to the Gpx4 +/− mice. 34,35 It is implied that there are multiple modulators and a very complicated regulatory mechanism from Se supplementation to selenoproteins expression.
On the other hand, being the crucial antioxidant enzymes in vivo, both Gpx1 and Gpx4 were up-regulated in cartilage tissue of the second-generation Se deficient rats, after they were given the Se sufficient diet, and the two selenoproteins were up-regulated at post-transcriptional and translational levels. 25  GPx1, GPX4 and SELS, which further play complex roles in the cartilage ( Figure 6).
In conclusion, the three selenoproteins expression was decreased by Se deficiency which depended on the participation of miR-181a-5p to down-regulate SBP2 at post-transcriptional level. It involves a series of antioxidant and ECM genes, to overcome the ROS-related stress for the protection of essential physiological functions and to maintain the balance between anabolism and catabolism of the cartilage. Thus, Se deficiency, the risk factor condition, gives rise to epiphyseal dysplasia in DA rats, similar to KBD patients.
Overall, this study provides the first comprehensive evidence that "Selenium → miR-181a-5p → SBP2 → selenoproteins" phenomenon exists in cartilage. Therefore, our data suggest that miR-181a-5p, SBP2 and three pivotal selenoproteins can be used to develop novel diagnostic and therapeutic strategies for cartilage diseases.

ACKNOWLEDG EMENTS
This work was supported by grants from the National Natural Center.

CONF LICT OF I NTEREST
The authors have no conflicts of interest to declare.