Overexpression of miR-10a-5p facilitates the progression of osteoarthritis

Introduction

Osteoarthritis (OA), a leading cause of joint damage and disability, is associated with impaired quality of life, shortened working duration, and increased all-cause mortality [1–3]. The prevalence of OA continues to grow in recent years, which will further deteriorate with aging population and obesity epidemic [4–6]. A study based on the National Health Interview Survey indicates that approximately 14 million persons have symptomatic knee OA and more than half of them are younger than 65 years old, with high possibility for joint disability over the next 30 years [7]. Several pharmacological agents are helpful in improving the symptoms of early-stage OA, but they can hardly inhibit or block the pathological progression of OA [8]. Eventually, most of advanced-stage victims may undergo total joint replacement owing to serious afflicted joint pain and disability [9]. Mountains of studies suggest that aberrant expression of genes are associated with the initiation and progression of OA [10], but the exact pathogenesis remains to be further clarified.

Non-coding RNAs (ncRNAs) are a group of RNA molecules accounting for a large proportion of the RNA and there are several types of ncRNAs, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) [11, 12]. miRNA, one of the most widely-studied ncRNAs, suppresses the expression of target genes through binding to the 3’UTR of their corresponding mRNAs [13]. LncRNAs are a group of ncRNAs with lengths exceeding 200 nucleotides and can regulate gene expression at transcription and post-transcription level [14]. CircRNAs are a special type of ncRNAs with covalently closed ring structure originating from back-splicing of pre-mRNAs at the downstream 5' splice site and the 3' splice site [15]. The rapid development of RNA-sequencing and bioinformatics analysis reveal that numerous miRNAs, lncRNAs and circRNAs are identified in various human diseases, including OA [16–18]. Accumulating evidence indicated that complicated communication networks across mRNA, miRNAs, lncRNAs and circRNAs participated in the onset and progression of OA and acted as potential therapeutic targets for OA. Wang and coworkers found that miR-483-5p was upregulated in OA and inhibition of miR-483-5p attenuated the progression of OA via Matn3 and Timp2 in vivo [19]. LncRNA-TM1P3 was significantly over-expressed in OA and promoted extracellular matrix degradation by regulating miR-22/TGF-β signaling/MMP13 pathway [20]. CircSERPINE2 participated in regulating chondrocytes metabolism and apoptosis through miR-1271/ERG axis and overexpression of CircSERPINE2 repressed the progression of OA in the rabbit model [17]. Actually, the crosstalk across coding and noncoding RNAs has been attributed increasing importance since Salmena et al. firstly proposed the ceRNA (competing endogenous RNA) hypothesis in 2011 [21]. Of the ceRNA networks, miRNAs play a central role to connect coding and noncoding RNAs via microRNA response elements (MREs), which act as the critical communicate bridges [21]. Furthermore, increasing studies suggested that miRNAs participated in the development and progression of diseases through triggering alterations of the whole transcriptome. For instance, overexpression of circRNA-Filip1l induced by decrease of miRNA-1224 facilitated chronic inflammatory pain via upregulation of Ubr5 in an Ago2-dependent manner [22]. Ye and colleagues also revealed that overexpression of miR-145 suppressed breast cancer progression and induced alterations of mRNAs, miRNAs, lncRNAs and circRNAs [23]. Therefore, it is essential to further explore the network regulatory interactions across coding and noncoding RNAs induced by miRNAs, which may provide novel diagnostic biomarkers and potential drug targets for OA. miR-10a-5p is a member of miR-10 family and could regulate cell proliferation, apoptosis, and inflammatory factors in many inflammation-associated diseases, including atopic dermatitis and rheumatoid arthritis [24–26]. Previous studies also indicated that miR-10a-5p was over-expressed in OA [16, 27]. However, the potential roles and molecule mechanisms of miR-10a-5p in OA were not fully elucidated.

In the current study, we verified that miR-10a-5p was significantly upregulated in OA and acted as a potential promising biomarker. Furthermore, we found that miR-10a-5p inhibited chondrocyte proliferation and promoted chondrocyte apoptosis. To in-depth explore the mechanisms of miR-10a-5p in OA, we performed RNA sequence for the whole transcriptome. Subsequently, integrated bioinformatics analyses were employed to illuminate the alterations of the whole transcriptome induced by miR-10a-5p. The workflow of study design is shown in Figure 1. Our findings may open up a new sight into regulatory mechanism of miRNAs, which may provide novel therapeutic targets for OA.

Figure 1. The workflow of study design.

Results

miR-10a-5p is upregulated in OA and acts as a potential promising biomarker

To explore the potential roles of miR-10a-5p in OA, we firstly detected the relative expression level of miR-10a-5p in OA and normal articular cartilage using RT-qPCR (Figure 2A). The results indicated that miR-10a-5p was upregulated in OA articular cartilage. Previous studies indicated that miRNAs in PBMC acted as promising biomarkers [28, 29]. In the current study, we also explore whether miR-10a-5p can act as a potential biomarker in OA. The results showed that miR-10a-5p was upregulated in PBMC of OA patients and might act as a promising predictor for OA with an area under the curve of 0.84 (95% confidence interval 0.65–1.04, P=0.02; Figure 2B–2C). Consistently, we also verified that miR-10a-5p was significantly over-expressed in mouse OA model (Figure 2D–2E) and IL-1β induced chondrocytes (Figure 3A).

Figure 2. miR-10a-5p is upregulated in OA and acts as a potential promising biomarker. (A) The relative expression of miR-10a-5p in OA and control cartilage tissues analyzed by RT-qPCR (8 OA cartilage vs. 7 control cartilage, **P < 0.01); (B) The relative expression of miR-10a-5p in OA and control PBMC analyzed by RT-qPCR (8 OA cartilage vs. 8 control cartilage, *P < 0.05); (C) ROC analysis of miR-10a-5p in PBMC for the diagnosis of OA (AUC,0.84; P=0.02); Representative pictures of articular cartilage in sham group (D left) and DMM group (D right) stained by H&E. Scale bar, 200 um. (E) The relative expression of miR-10a-5p in sham group and DMM group analyzed by RT-qPCR (6 sham vs. 6 DMM cartilage, **P < 0.01).

Overexpression of miR-10a-5p inhibits chondrocyte proliferation and promotes chondrocyte apoptosis and cartilage matrix degradation

Subsequently, we evaluated the effect of miR-10a-5p on chondrocyte proliferation, apoptosis and metabolism after overexpressing miR-10a-5p in HC-a (Figure 3B). CCK-8 assay, EDU assay, and flow cytometry assay indicated that overexpression of miR-10a-5p inhibited chondrocyte proliferation (Figure 3C–3E) and promoted chondrocyte apoptosis (Figure 3F–3G). Also, we performed western blot to explore the effect of miR-10a-5p on extracellular matrix metabolism. Western blot results indicated that overexpression of miR-10a-5p inhibited the expression of COL2A1 and promoted the expression of MMP13 (Figure 3H). Taken together, these results demonstrated that miR-10a-5p may act as a significant contributing factor for OA.

Figure 3. miR-10a-5p acts as a significant contributing factor for OA. (A) The relative expression of COL2A1, MMP13, SOX9, and miR-10a-5p in control and IL-1β induced Hc-a analyzed by RT-qPCR (n=3; **P < 0.01, ****P < 0.0001). (B) The relative expression of miR-10a-5p after transfecting miR-10a-5p mimics, ***P < 0.001. (C) The effect of miR-10a-5p overexpression on cell proliferation detected by CCK8 assay (n=3; *P < 0.05, **P < 0.01, ***P < 0.001). (D, E) The effect of miR-10a-5p overexpression on cell proliferation detected by EDU assay (n=3; *P < 0.05). (F, G) The effect of miR-10a-5p overexpression on cell apoptosis detected by flow cytometry assay (n=3; ****P < 0.0001). (H) Effects of miR-10a-5p overexpression on Col2a1, MMP13, BAX, and GAPDH protein levels detected by western blot.

Overexpression of miR-10a-5p induces alteration of the whole transcriptome in human primary chondrocyte

To explore the potential mechanism of miR-10a-5p in OA, we performed the whole transcriptome sequencing to identify differentially-expressed genes on HC-a via miR-10a-5p overexpressing. These results indicated that overexpression of miR-10a-5p induced the alteration of the whole transcriptome, which involved 395 up-regulated and 278 down-regulated mRNAs (Figure 4A), 202 up-regulated and 223 down-regulated lncRNAs (Figure 4B), 1 up-regulated and 4 down-regulated miRNAs (Figure 5A), and 8 up-regulated and 2 down-regulated circRNAs (Figure 5B). The detailed differentially-expressed mRNAs, lncRNAs, miRNAs, and circRNAs were showed in Supplementary Tables 2–5. Moreover, RT-qPCR showed that the expression level of 12 selected differentially-expressed genes were basically consistent with the results of RNA-seq (Figure 6).

Figure 4. Differentially expressed mRNAs and lncRNAs between miR-10a-5p overexpression group (exp) and control group (ctrl). (A) Heat map (left) and volcano plot (right) of differentially expressed mRNAs (red color denotes upregulated mRNAs and green color denotes downregulated mRNAs). (B) Heat map (left) and volcano plot (right) of differentially expressed lncRNAs (red color denotes upregulated lncRNAs and green color denotes downregulated lncRNAs).

Figure 5. Differentially expressed miRNAs and circRNAs between miR-10a-5p overexpression group (exp) and control group (ctrl). (A) Heat map (left) and volcano plot (right) of differentially expressed miRNAs (red color denotes upregulated miRNAs and green color denotes downregulated miRNAs). (B) Heat map (left) and volcano plot (right) of differentially expressed circRNAs (red color denotes upregulated circRNAs and green color denotes downregulated circRNAs).

Figure 6. Validation of 12 selected downregulated genes after miR-10a-5p overexpression through RT-qPCR. (A) SORCS2; (B) NDUFA4L2; (C) SULT1B1; (D) MKX; (E) CA9; (F) NLGN4X; (G) CPXM2; (H) HCAR1; (I) ADM2; (J) CAPN6; (K) OTUD6B-AS1; (L) hsa_circ_0010029.(n=3; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Functional enrichment analyses for differentially-expressed mRNAs, circRNAs, and lncRNAs

Furthermore, we undertook functional enrichment analyses for these dysregulated genes. GO enrichment analysis for dysfunctional mRNAs indicated that they were mainly enriched in chromatin assembly (GO:0031497), extracellular structure organization (GO:0043062), protein-DNA Complex (GO:0032993), extracellular matrix (GO:0031012), and glycosaminoglycan binding (GO:0005539) (Figure 7A, Supplementary Tables 6–8). KEGG enrichment analysis for dysfunctional mRNAs revealed that they mainly enriched in systemic lupus erythematosus (HSA05322), alcoholism (HSA05034), complement and coagulation cascades (HSA04610), and cholesterol metabolism (HSA04979) (Figure 7B, Supplementary Table 9). Functional enrichment analyses for cis-genes of differentially expressed lncRNAs showed that they were enriched in positive regulation of mesenchymal cell proliferation (GO:0002053), peptidyl-proline modification (GO:0018208), Alcoholism (HSA05034), Arachidonic acid metabolism (HSA00590) (Figure 7C, 7D, Supplementary Tables 10–13). Functional enrichment analyses for parental genes of differentially expressed circRNAs showed that they mainly participated in somatic diversification of T cell receptor genes (GO:0002568), positive regulation of brown fat cell differentiation (GO:0090336), Adherens junction (HSA04520) (Figure 7E, 7F, Supplementary Tables 14–17). Also, GSEA for differentially expressed mRNAs verified that they were mainly associated with organ regeneration (GO:0031100), nucleosome assembly (GO:0006334), negative regulation of phosphorylation (GO:0042326), complement and coagulation cascades (HSA04610), tyrosine metabolism (HSA00350), PPAR signaling pathway (HSA03320), PI3K-Akt signaling pathway (HSA04151), p53 signaling pathway (HSA04115), FoxO signaling pathway (HSA04068) (Figure 8A–8F, Supplementary Tables 18–19).

Figure 7. Functional enrichment analyses for differentially-expressed mRNAs, lncRNAs, and circRNAs. (A) GOBubble plot shows GO terms enriched in differentially-expressed mRNAs. (B) KEGG terms enriched in differentially-expressed mRNAs. (C) GOBubble plot shows GO terms enriched in ci-genes of differentially-expressed lncRNAs. (D) KEGG terms enriched in ci-genes of differentially-expressed lncRNAs. (E) GOBubble plot shows GO terms enriched in parent genes of differentially-expressed circRNAs. (F) KEGG terms enriched in parent genes of differentially-expressed circRNAs.

Figure 8. Gene set enrichment analysis (GSEA) for differentially-expressed mRNAs using NGSEA. Representative three significantly GO enrichment plot in GSEA: (A) organ regeneration (GO:0031100); (B) nucleosome assembly (GO:0006334); (C) negative regulation of phosphorylation. Representative three significantly KEGG enrichment plot in GSEA: (D) complement and coagulation cascades (HSA04610); (E) tyrosine metabolism (HSA00350); (F) PPAR signaling pathway (HSA03320).

PPI network analysis

The protein-protein regulatory network contained 365 nodes and 2210 edges (Figure 9A). MCODE analysis indicated that thirteen modules were extract from the PPI network, but the 42 genes from module 1 had the highest node degree according to CytoHubba (Supplementary Table 20). Therefore, these genes from module 1 were identified as hub genes. Subsequently, we conducted functional enrichment analyses for these hub genes using Metascape [30]. The results revealed that they were mainly enriched in blood microparticle (GO:0072562), endoplasmic reticulum lumen (GO:0005788), complement and coagulation cascades (HSA04610) (Figure 9B).

Figure 9. PPI network analysis for differentially-expressed mRNAs. (A) PPI network of differentially-expressed mRNAs analyzed by STRING database and Module (1-13) were selected from PPI network using MCODE analysis. (B) Functional enrichment analysis of hub genes (Module 1) were performed using Metascape.

Construction of ceRNA regulatory networks

We also constructed the network regulatory interactions across coding and noncoding RNAs triggered by miR-10a-5p. Considering that miRNAs play critical pivotal roles in ceRNA interactions, we constructed ceRNA regulatory networks centralized on significant upregulated and downregulated miRNAs. Downregulated miRNAs-associated ceRNA networks contained 1 circRNA, 42 lncRNA, 5 miRNA and 112 mRNA (Figure 10). Especially, miR-10a-5p was the only upregulated miRNA after miR-10a-5p overexpression. The ceRNA network centered on miR-10a-5p consisted of 27 lncRNA, 1 miRNA, and 10 mRNA (Figure 10). The complicated regulatory networks across coding and noncoding RNAs revealed the powerful regulating effects of miR-10a-5p.

Figure 10. ceRNA regulation network centered on downregulated and upregulated miRNAs after overexpressing miR-10a-5p in Hc-a. The circle represents circRNA, square represents mRNA and triangle represents miRNA, and inverted triangle represents lncRNA.

HOXA3 acts as a targeted gene of miR-10a-5p

To seek for the potential target of miR-10a-5p, we firstly identify the downregulated mRNAs in OA. A total of 767 downregulated mRNAs were identified in IL-1β induced primary chondrocytes after processing the data of GSE74220 using BioJupies [31] (Figure 10A). Furthermore, we investigated that HOXA3 was the only overlapping mRNA across the downregulated mRNAs in GSE74220, the predicted targets of miR-10a-5p via Targetscan, and the downregulated mRNAs in our RNA-seq results (Figure 11B). Subsequently, we found that HOXA3 was downregulated in OA cartilage after analyzing the results of GSE114007 (Figure 11C). The results of Targetscan showed that the seed region of miR-10a-5p was well-paired with 3’UTR of HOXA3, which was conservative among vertebrates (Figure 11D). According to TarBase v.8 [32], high-throughput experiments including PAR-CLIP and HITS-CLIP indicated that miR-10a-5p was the direct inhibition to HOXA3 in HEK293, TZMBL, and 293S (Figure 11E). Furthermore, dual luciferase reporter assay confirmed that luciferase activity of HOXA3-wt was obviously inhibited by miR-10a-5p mimics as compared with that in HOXA3-mut group (Figure 11F–11G). RT-qPCR and western blot results showed that HOXA3 was significantly inhibited in IL-1β induced HC-a (Figure 11H–11I) and miR-10a-5p overexpression (Figure 11J–11K). Taken together, these results indicated that HOXA3 was downregulated in OA and acted as the targeted genes of miR-10a-5p.

Figure 11. HOXA3 acts as a targeted gene of miR-10a-5p. (A) Volcano plot of differentially expressed mRNAs between control group and IL-1β induced primary chondrocyte group from GSE74220. (B) Venn plot of the overlapping mRNA across the downregulated mRNAs in GSE74220, the predicted targets of miR-10a-5p in Targetscan, and the downregulated mRNAs in our RNA-seq. (C) The expression level of HOXA3 in OA cartilage from GSE114007, **P < 0.01. (D) Targetscan shows that the seed region of miR-10a-5p is well-paired with 3’UTR of HOXA3, which was conservative among vertebrates. (E) High-throughput experiments verified that HOXA3 was the direct target of miR-10a-5p in HEK293, TZMBL, and 293S from TarBase v.8. (F) The sequencing results of cloned fragments in luciferase reporter vectors and the yellow highlighted sequence is the target test site. (G) Interaction between miR-10a-5p and HOXA3 was verified by luciferase report assay in 293T cells. (H, I) The relative expression of HOXA3 in control and IL-1β induced Hc-a analyzed by RT-qPCR and western blot (n=3, *P < 0.05). (J, K) The relative expression of HOXA3 after transfecting miR-10a-5p mimics analyzed by RT-qPCR and western blot (n=3, **P < 0.01).

miR-10a-5p exerts biological functions in OA cell model by targeting HOXA3

Based on the above results, we found that miR-10a-5p was a significant contributor to the pathogenesis of OA. RNA-sequencing together with integrated bioinformatics analyses identified that miR-10a-5p promoted the progression of OA through triggering the alterations of the whole transcriptome. Subsequently, one of the most representative downstream genes, HOXA3 (the targeted gene of miR-10a-5p) was chosen to verify the predicted mechanisms from bioinformatics analyses. Three siRNAs were designed to knockdown the expression of HOXA3. RT-qPCR results showed that si-HOXA3-1 had the highest silence efficiency, which was further confirmed by western blot (Figure 12A, 12B). Therefore, si-HOXA3-1 was chosen to perform subsequent experiments. CCK-8 assay, EDU assay, flow cytometry assay, and western blot verified that silence of HOXA3 significantly inhibited chondrocyte proliferation (Figure 12C, 12D, 12F), promoted chondrocyte apoptosis (Figure 12E, 12G) and cartilage matrix degradation (Figure 12H). Next, we further investigated whether miR-10a-5p functioned in IL-1β induced HC-a through targeting HOXA3. We treated HC-a with IL-1β and then co-transfected miR-10a-5p inhibitor and si-HOXA3. Western blot also indicated that si-HOXA3 partly reversed the protective effect of miR-10a-5p inhibitor on IL-1β treated HC-a (Figure 13A). CCK-8 and EDU assay indicated that miR-10a-5p inhibitor partly restored IL-1β-induced inhibition of HC-a proliferation, while si-HOXA3 attenuated the effect of miR-10a-5p inhibitor on IL-1β treated HC-a (Figure 13B, 13D, 13F). Flow cytometry assay showed that miR-10a-5p inhibitor partly reversed IL-1β-induced chondrocyte apoptosis, while si-HOXA3 antagonized the effect of miR-10a-5p inhibitor on IL-1β treated chondrocyte (Figure 13C, 13E). Collectively, these finding indicated that miR-10a-5p exerts biological functions in OA cell model by targeting HOXA3.

Figure 12. Silence of HOXA3 inhibited chondrocyte proliferation and promoted chondrocyte apoptosis. (A) The relative expression of HOXA3 after transfecting si-HOXA3 analyzed by RT-qPCR (n=3; ***P < 0.001). (B) The relative expression of HOXA3 after transfecting si-HOXA3 analyzed by western blot. (C) The effect of HOXA3 knockdown on cell proliferation detected by CCK8 assay (n=3; *P < 0.05, **P < 0.01). (D, F) The effect of HOXA3 knockdown on cell proliferation detected by EDU assay (n=3; *P < 0.05). (E, G) The effect of HOXA3 knockdown on cell apoptosis detected by flow cytometry assay (n=3; *P < 0.05). (H) Effects of HOXA3 knockdown on Col2a1, MMP13, BAX, and GAPDH protein levels detected by western blot.

Figure 13. miR-10a-5p functioned in IL-1β induced Hc-a through targeting HOXA3. Hc-a was treated with IL-1β and then co-transfected miR-10a-5p inhibitor and si-HOXA3. (A) HOXA3, Col2a1, MMP13, BAX, and GAPDH protein levels were detected by western blot. (B, D, F) cell proliferation detected by CCK8 (B; n=3; *P < 0.05, ***P < 0.001) and EDU assay (D, F; n=3, *P < 0.05, **P < 0.01, ***P < 0.001). (C, E) cell apoptosis detected by flow cytometry assay (n=3, **P < 0.01, ***P < 0.001).

Abbreviations

OA: Osteoarthritis; ncRNAs: Non-coding RNAs; miRNAs: microRNAs; lncRNAs: long non-coding RNAs; circRNAs: circular RNAs; MREs: microRNA response elements.

Author Contributions

Conceptualization, Hui-Zi Li, Xiang-He Xu, Zhong-Zhen Su and Hua-Ding Lu; Data curation, Hui-Zi Li and Da-Wei Wang; Formal analysis, Hui-Zi Li, Xiang-He Xu, Yi-Ming Lin, Zhong-Zhen Su and Hua-Ding Lu; Investigation, Hui-Zi Li; Methodology, Hui-Zi Li, Xiang-He Xu, Da-Wei Wang, Nan Lin, Zhong-Zhen Su and Hua-Ding Lu; Project administration, Xiang-He Xu and Hua-Ding Lu; Software, Da-Wei Wang, Yi-Ming Lin and Nan Lin; Visualization, Xiang-He Xu and Hua-Ding Lu; Writing – original draft, Hui-Zi Li, Xiang-He Xu, Zhong-Zhen Su and Hua-Ding Lu; Writing – review and editing, Hui-Zi Li, Xiang-He Xu, Zhong-Zhen Su and Hua-Ding Lu.

Acknowledgments

We thank MS. Lei Xiao and Xiao-Yi Li for their help in manuscript preparation and spiritual assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

This study was supported by grants from the National Natural Science Foundation of China (nos. 81772384, 81572174, and 81902242). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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