MicroRNA‐638 inhibits human aortic valve interstitial cell calcification by targeting Sp7

Abstract Calcific aortic valve disease (CAVD) is a complex heart valve disease involving a wide range of pathological changes. Emerging evidence indicates that osteogenic differentiation of human aortic valve interstitial cells (hAVICs) plays a key role in valve calcification. In this study, we aimed to investigate the function of miR‐638 in hAVICs osteogenesis. Both miRNA microarray assay and qRT‐PCR results demonstrating miR‐638 was obviously up‐regulated in calcific aortic valves compared with non‐calcific valves. We also proved that miR‐638 was significantly up‐regulated during hAVICs osteogenic differentiation. Overexpression of miR‐638 suppressed osteogenic differentiation of hAVICs in vitro, whereas down‐regulation of miR‐638 enhance the process. Target prediction analysis and dual‐luciferase reporter assay confirmed that Sp7 transcription factor (Sp7) was a direct target of miR‐638. Furthermore, knockdown of Sp7 inhibited osteogenic differentiation of hAVICs, which is similar to the results observed in up‐regulation miR‐638. Our data indicated that miR‐638 plays an inhibitory role in hAVICs osteogenic differentiation, which may act by targeting Sp7. MiR‐638 may be a potential therapeutic target for CAVD.

with accumulating evidence for heterotopic ossification. Mohler et al confirmed that heterotopic ossification consisting of mature lamellar bone formation and active bone remodelling is relatively common in end-stage calcific aortic valves. 12 Studies had demonstrated the presence of osteoblast-like cells, osteoblast-specific transcripts and osteogenic markers in calcified aortic valves. 13,14 In addition, extracellular bone matrix proteins, such as osteopontin, osteonectin and bone morphogenetic proteins (BMPs) that are believed to play a part in the calcific process, are also found in calcific aortic valves. 12,[15][16][17] These above results suggested that cellular osteogenic transdifferentiation may be correlated with the pathogenesis of CAVD.
The human aortic valves are composed of three small collagenous leaflets attached to the fibrous ring of the left ventricular outflow tract. The leaflets are composed of a dense extracellular matrix, usually divided into three layers: lamina fibrosa, lamina spongiosa and lamina ventricularis. All three layers are populated with aortic valve interstitial cells (AVICs), with the whole structure covered by a confluent monolayer of valve endothelial cells. 10,18 AVICs are the most predominant cells in aortic valves and play a key role in maintaining normal aortic valve structure and function. 19 AVICs are capable of undergoing a phenotypic transition to become osteoblast-like cells and elaborate bone matrix in response to osteogenic inductors during CAVD. 1,20,21 Multiple studies have also showed that osteogenic differentiation of AVICs is involved in the pathogenesis of CAVD. [22][23][24] MicroRNAs (MiRNAs) are small, single-stranded, non-coding RNAs that act as fine-tuners in the negative regulation of gene expression by binding to complementary sequences in the 3-untranslated region of targeted mRNA, thereby leading to either mRNA degradation or translational repression. [25][26][27] MiRNAs are involved in diverse physiological and pathological processes, including cell development, proliferation, apoptosis and differentiation. [28][29][30] Multiple miRNAs that regulate the process of osteogenic differentiation have been identified. For instance miRNA-214 promotes periodontal ligament stem cell osteoblastic differentiation by modulating Wnt/β-catenin signalling, 31 whereas miRNA-98 targets BMP-2 to inhibit osteogenic differentiation of human bone mesenchymal stromal cells. 32 Moreover, miRNA-22 is confirmed to enhance osteogenic differentiation and inhibit adipogenic differentiation of human adipose tissue-derived mesenchymal stromal cells by repressing histone deacetylase (HDAC6). 33 In our preliminary study, we found that miRNA-638 was significantly up-regulated in calcific aortic valves compared with non-calcific valves. 1 In the present research, we further investigated the function of miRNA-638 in osteogenic differentiation of hAVICs and identified target genes of miRNA-638. Our findings indicate that miRNA-638, which is up-regulated in osteogenic differentiation of hAVICs, inhibits the differentiation process by repressing Sp7.

| Ethics statements
The study protocol was approved by the Ethical Committee of the Affiliated Hospital of Qingdao University, and informed consents were obtained from the human donors. All experiments were performed in accordance with the relevant guidelines and regulations.

| Calcific aortic valve collection
Samples were obtained from 10 CAVD patients (Table 1), who had undergone cardiac surgery with implantation of a valve prosthesis. Exclusion criteria included non-stenotic, congenital aortic valve disease, rheumatic aortic valve disease, genetic disease and autoimmune disease. During the operation, two tissue Samples were taken from each patient for the follow-up study: one was calcific aortic valves that contained calcific nodules, and the other that serves as a control was adjacent non-calcific aortic valve tissues surrounding calcific valves of the same patient. All samples were resected during the operation and immediately placed in pairs in liquid nitrogen for the following research. At the same time, pathological examinations of tissue samples from 10 patients were performed to make sure the accuracy of tissues sampling and trimming.

| Microarray analysis
Calcific aortic valves and non-calcific valves from three CAVD patients were sent to carry on the miRNA microarray assay. Total RNA was extracted from tissues using the miRNAeasy Mini Kit

| MiRNA real-time quantitative PCR
MiRNA-638 was extracted using the miRVana extraction kit (Ambion). For miRNA-638 quantification, 10 ng total RNA was transcribed reversely and amplified using the miRNA reverse transcription and detection kit (Applied Biosystems, Inc). All results were normalized to U6 levels, which were determined by the ABI miRNA U6 assay kit (Applied Biosystems, Inc).

| hAVICs isolation and cell culture
Normal aortic valves (n = 5) were derived from patients who had undergone acute Stanford A aortic dissection. Primary hAVICs were prepared as described previously. 1,9,35 In brief, non-leaflet tissues were carefully eliminated after effective removal of the endothelial layer of aortic and ventricular aspects, then valves were immersed in 0.25% trypsin at 37°C for 5 minutes. The tissues were then cut into pieces and digested for an additional 2 hours at 37°C.
Primary hAVICs were obtained and seeded in growth medium (Dulbecco's Modified Eagle Medium supplemented with penicillin and streptomycin, mem non-essential amino acid, sodium pyruvate and 10% FBS) at 37°C under a 5% carbon dioxide atmosphere.
The purity of hAVICs was confirmed by microscopic examination and evaluation of expression of marker proteins. according to the manufacturer's instructions. Transfection efficiency was measure at day 3 in a preliminary test. Osteogenic differentiation was subsequently induced after transfection by culturing cells in osteogenic differentiation medium (growth medium supplemented with 500-ng/mL BMP-2, 100-nmol/L dexamethasone, 50-µg/mL ascorbic acid and 10-mmol/L β-glycerophosphate).

| mRNA quantitative real-time PCR
The mRNA expression of alkaline phosphatase (ALP), integrin binding sialoprotein (IBSP) and Sp7 were detected using qRT-PCR after os-

| Western blotting
The protein expression of ALP, IBSP and Sp7 were measured by using western blotting after osteogenic differentiation of hAVICs.
The transfected hAVICs samples were fixed in 4% paraformaldehyde for 30 minutes, and then blocked with 0.2% Triton X-100 and

| Dual luciferase reporter assay
The 3UTR of human gene Sp7 was amplified from human cDNA.
The wide-type fragment containing the predicted miRNA-638 binding site and its mutant fragment, designed to carry sites for SacI (5 end) and XbaI (3 end

| Alkaline phosphatase activity assay
The osteogenic phenotype was determined based on the ALP activity, which is an early osteoblastic differentiation marker. The ALP activity assay was conducted on day 3 or 7 after osteogenic differentiation of hAVICs. Cells were washed twice with phosphate-buffered saline solution (PBS) and lysed with 150 µL NP-40 lysis buffer (Beyotime, China). The cell lysates were quantified by an alkaline phosphatase assay kit (Beyotime) using p-nitrophenyl phosphate (pNPP) as the substrate. In the presence of magnesium ions, pNPP was hydrolysed by phosphatases to phosphate and p-nitrophenol.
The rate of p-nitrophenol liberation is proportional to the ALP activity and can be measured photometrically. The ALP activity was measured by spectrophotometer at 405 nm.

| Statistical analysis
Each experiment was repeated in triplicate at least three times.
Statistical analysis which was performed by using SPSS 16.0.
Data were presented as mean ± SD. Comparisons of parameters between two groups were evaluated by Students' t test.
Comparisons of parameters among more than two groups were analysed by one-way ANOVA, and comparisons of different parameters between each group were made by a post hoc analysis using a Bonferroni's test. Non-parametric Mann-Whitney U and Kruskal-Wallis tests were performed when the sample size was smaller. Differences at P < 0.05 were considered to be statistically significant.

| Expression level of miRNA-638 is up-regulated in human calcific aortic valves
In order to identify the dysregulated miRNAs in CAVD pathogenesis, miRNA microarray assay was conducted to analyse the expres-

| Primary culture and phenotype identification of hAVICs
The primary hAVICs began to grow with adherence at about 2 days of primary culture. After 6 days, the adherent cells were flat and spindle-shaped. The cells grew slowly and reached to about 70% confluences after 14 days of culture. During subsequent cell passages, the cell density was high and the cells were swirling and radial arranged (Figure 2A).
To further verify the isolated cells were hAVICs, two marker proteins associated with hAVICs were detected by immunohistochemical staining. The results showed hAVICs from three passages were positive for vimentin and α-SMA (91% and 71% respectively) ( Figure 2B-D).

| Expression level of miRNA-638 increases during osteogenic differentiation of hAVICs
To explore the role of miRNA-638 in osteogenic differentiation of hAVICs, these cells were harvested at different time points in the process of osteogenic differentiation (0, 3, 6 and 9 days), and miRNA-638 expression level was analysed by qRT-PCR. Expression of miRNA-638 increased on day 3 compared with that of untreated control hAVICs (day 0) and remained high until day 9 ( Figure 3A).
This result indicates that miRNA-638 might negatively regulate osteogenic differentiation of hAVICs.

| MiRNA-638 directly targets Sp7
To reveal the molecular mechanism by which miRNA-638 regulates osteogenic differentiation of hAVICs, TargetScan was used to predict potential target genes of miRNA-638. Among these candidates, Sp7 was found to contain a specific miRNA-638 binding sites in its 3UTR ( Figure 4C). During induction of osteogenic differentiation, Sp7 mRNA and protein levels decreased gradually until reaching a peak at day 6, then subsequently increased gradually ( Figure 4A,B) in an inverse trend from that of miRNA-638 levels ( Figure 3A). Next, we studied whether miRNA-638 regulates Sp7 expression during osteogenic differentiation of hAVICs. Our results showed that Sp7 mRNA and protein levels were significantly decreased by overexpression of miRNA-638. On the contrary, Sp7 mRNA and protein levels were markedly increased by reduction in miRNA-638 ( Figure 5A,B). These data suggested that miRNA-638 attenuated Sp7 expression during osteogenic differentiation of hAVICs.
To further confirm whether miRNA-638 directly targets Sp7, we

| Sp7 down-regulation inhibits osteogenic differentiation of hAVICs
To investigate the functional effect of Sp7 on osteogenic differentiation of hAVICs, we suppressed Sp7 expression by transfecting hAVICs with SiRNA against Sp7 (Si-Sp7). As shown in Figure 5A and ALP activity ( Figure 3E,F).

| Sp7 knockdown could block the effect of miRNA-638 during osteogenic differentiation of hAVICs
To confirm that the function of miRNA-638 during osteogenic differentiation of hAVICs is mediated by repressing Sp7, we transfected miRNA-638 inhibitors into hAVICs after Sp7 knockdown, and then proceeded with osteogenic differentiation. As is shown by western blotting (Figure 5C), miRNA-638 inhibitors could accelerate osteogenic differentiation in Si-Sp7 negative control (Si-NC) group, but differentiation in the presence of inhibitors is abolished after Sp7 knockdown.
These results demonstrated that deletion of Sp7 could block the effect of miRNA-638 inhibitors, further indicating that miRNA-638 regulates osteogenic differentiation of hAVICs through targeting Sp7.
F I G U R E 3 Overexpression of miRNA-638 inhibits osteogenic differentiation of hAVICs, whereas downexpression of miRNA-638 promotes the process. A, The endogenous expression level of miRNA-638 was measured by qRT-PCR at different time points during osteogenic differentiation (0, 3, 6 and 9 days) of hAVICs. The data, normalized to U6, are averages of three independent experiments (mean ± SD). *P < 0.05 versus day 0. B, The expression of miRNA-638 in hAVICs transfected with miRNA mimic or inhibitor at day 3 was determined by qRT-PCR compared with miR-NC or miR-NCI group. The data, normalized to U6, are averages of three independent experiments (mean ± SD). *P < 0.05 versus miR-NC or miR-NCI group. C and D, The mRNA expression of osteogenic specific genes ALP and IBSP after miRNA-638 overexpression or downexpression at day 7 was analysed by qRT-PCR. The data, normalized to GAPDH, are averages of three independent experiments (mean ± SD). *P < 0.05. E, ALP activity at day 3 after osteogenic differentiation of hAVICs was measure in each group. The data are averages of three independent experiments (mean ± SD). *P < 0.05. F, ALP activity at day 7 after osteogenic differentiation of hAVICs was measure in each group. The data are averages of three independent experiments (mean ± SD). *P < 0.05. G and H, Western blotting results showed the protein expression of ALP and IBSP at day 14 after osteogenic differentiation of hAVICs transfected with miRNA-638 mimic or inhibitor. The data, normalized to GAPDH, are averages of three independent experiments (mean ± SD). *P < 0.05 versus miR-NC or miR-NCI group. I, Schematic diagram depicting the mechanism underlying aortic valve calcification in this study. '┥': inhibit, '→': promote F I G U R E 4 miRNA-638 directly targets Sp7. A and B, mRNA and protein expression levles of Sp7 were determined by qRT-PCR and western blotting at different time points (0, 3, 6 and 9 days) during osteogenic differentiation of hAVICs. The data, normalized to GAPDH, are averages of three independent experiments (mean ± SD). *P < 0.05. C, The single binding sites of miRNA-638 (unshaded) in the 3UTR of Sp7 was predicted by TargetScan software. D, Schematic of putative miRNA-638 target site in human Sp7 3UTR and the corresponding mutant nucleotides were coloured red. E, Dual-luciferase assay after transfection of M-miR-638, NC-miR-638 with wide-type Sp7 3UTR or mutant Sp7 3UTR. The data represent the mean ± SD of three independent experiments. *P < 0.05s

| D ISCUSS I ON
Calcific aortic valve disease is one of the cardiovascular diseases which causing significant morbidity and mortality, especially in the elderly, and calcification plays an important role in the pathogenesis of this disease. 1,38 However, there are still no effective pharmacological treatments to prevent or treat this disease. Osteogenic differentiation of hAVICs has been confirmed to be closely associated with the pathological process of CAVD. 1,9,[38][39][40] Therefore, investigating osteogenic differentiation of hAVICs may lead to a better understanding of the pathogenesis of CAVD and improve treatment options. Recently, mounting evidence has shown that miRNAs could play a vital role in osteogenic differentiation of hAVICs.
In this study, we first screened out the differentially expressed miRNAs in CAVD pathogenesis using miRNA microarray assay. Then MiRNA-141 was identified to inhibit the osteogenic differentiation of porcine VICs through a BMP-dependent pathway. 40 In contrast to previous studies, our study firstly used miRNA microarray assay to explore the real miRNAs involved in CAVD pathogenesis. This may make our results more representative of the actual pathological process of CAVD. Finally, miRNA-638 was found and confirmed to negatively regulate the osteogenic differentiation of hAVICs.
miRNA-638 was expressed in human and non-human primates. In previous studies, dysregulation of miRNA-638 had been described as a cohort of human tumours, including non-small cell lung cancer, 45 54 In Sp7-null embryos, cartilage was formed normally, but the embryos totally lacked bone formation. 55 In recent years, the co-function of miRNAs and Sp7 during bone formation and osteogenic differentiation has also been investigated. miRNA-637 was identified to maintain the balance between adipocytes and osteoblasts by directly targeting Sp7. 37 miRNA-143 and miRNA-145 could inhibit osteogenic differentiation by targeting Sp7 and form a feedback loop with KLF4 and Sp7 in odontoblasts. 57,58 In our study, we demonstrated that miRNA-638 inhibits osteogenesis of hAVICs by targeting Sp7.
We also found that down-regulation of Sp7 suppressed osteogenic differentiation, similar to the effect of miRNA-638 overexpression. Moreover, effects of miRNA-638 inhibitor on osteogenic differentiation of hAVICs could be reversed by Sp7 SiRNA. These results provided evidence that miRNA-638 inhibited osteogenic differentiation of hAVICs by negatively regulating Sp7.
In conclusion, we demonstrated that miRNA-638 negatively regulated osteogenic differentiation of hAVICs by directly targeting Sp7. As far as we know, this is the first report to study the regulatory role of miRNA-638 for human aortic valve calcification. Our study indicated that miRNA-638 and Sp7 might be potential therapeutic targets for the management of CAVD.

ACK N OWLED G EM ENTS
We would like to extend a special thanks to all the patients who provided specimen and thank Guangzhou RiboBio Technology Co. Ltd.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.