Therapeutic effect and mechanisms of intraarticular injections of microRNA-140-5p on early-stage osteoarthritis in rats

Background MicroRNAs (miRs) have received extensive attention in osteoarthritis (OA) pathogenesis in recent years, and our previous study have confirmed that single intraarticular injection (IAJ) of miR-140-5p alleviates early-stage OA (EOA) progression in rats. This study aims to further investigate the effects of single IAJ of miR-140-5p on different stage OA and multiple IAJs of miR-140-5p on EOA, as well as the potential mechanisms. Methods Firstly, OA model was surgically induced in rats, 9 rats were treated with IAJ of Cy5-miR-140-5p at 1 week after surgery, and fluorescence distribution was measured. Then, 72 rats were treated with single IAJ of miR-140-5p at different time after surgery or multiple IAJs of miR-140-5p at 1 week after surgery, and OA progression were evaluated macroscopically and histologically. Finally, bioinformatics analyses were performed and the potential targets and molecular mechanisms of miR-140-5p were predicted. Results Strong fluorescence was observed in the chondrocytes and joint where Cy5-miR-140-5p was injected. Behavioural scores, chondrocyte numbers and cartilage thickness in cartilage were higher, while pathological scores were lower in the miR-140-5p group than in the control group. Specifically, the earlier a single IAJ of miR-140-5p, the better the therapeutic effect, and multiple IAJs exhibited better therapeutic effect than single IAJ on EOA. Bioinformatics analyses predicted 84 potential target genes of rno-miR-140-5p and revealed that these genes enrich in various biological processes and pathways. Conclusions IAJs of miR-140-5p effectively alleviate EOA progression by modulating various biological processes and pathways, and may be a promising therapeutics for EOA. cytoplasm; then, we found that the earlier a single IAJ of miR-140-5p, the better the therapeutic effect, and multiple IAJs of miR-140-5p exhibit better therapeutic effect than single IAJ on EOA; finally, the bioinformatics analysis predicted the potential targets of miR-140-5p and the possible mechanisms by which miR-140-5p participates in OA pathogenesis. These results suggest that IAJs of miR-140-5p is an effective and promising strategy for the treatment of EOA, and also provide support for further exploration of miR-based OA therapeutics.


Background
Osteoarthritis (OA) is a major cause of disability and a leading source of sociental cost in older people, and is becoming more prevalent than in previous decades as the number of ageing adults increases [1][2][3]. Although the specific mechanisms leading to OA have not been fully elucidated, OA is characterized by various epigenetic changes, which refer to alterations in gene promoter that lead to a reduced expression of a specific protein [4,5].
Unlike genetic inheritance, epigenetic changes can be reversed and easily controlled, and its mechanisms consist mainly of DNA methylation, histone modifications and regulatory microRNAs (miRs) [6]. miRs regulate their target genes (mRNAs) at the post-transcriptional level and have received extensive attention in OA pathogenesis in recent years [7][8][9]. Our previous studies reported that miR-140-5p levels were significantly reduced in OA chondrocytes and synovial fluid, and single intra-articular injection (IAJ) of exogenous miR-140-5p agomir effectively alleviates early-stage OA (EOA) progression in rats [10]. However, the effects of single IAJ of miR-140-5p on different stage OA and multiple IAJs of miR-140-5p on EOA, as well as the related molecular mechanisms were not addressed.
Therefore, this study aims to validate whether the intra-articularly injected miR-140-5p could enter the cartilage and be taken up by chondrocytes first, then to investigate the effect of single IAJ of miR-140-5p on different stage OA and multiple IAJs of miR-140-5p on EOA, and finally to explore the possible mechanistic network of miR-140-5p by bioinformatics methods.

Rats and OA model
Twelve-week-old male and female (1:1) Sprague-Dawley rats were purchased from the Chengdu Dossy Experimental Animals Co.,Ltd, and housed at the Laboratory Animal Centre of Sichuan University under standard diurnal light/dark conditions, fed a standard commercial diet and allowed access to tap water ad libitum. All animals received humane care, and all procedures were carried out according to the Guide for the Care and Use of Laboratory Animals. The rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg; Tocris, Bristol, UK), and euthanized by injection of excessive pentobarbital sodium. OA was induced in the right hind knee by anterior cruciate ligament transection and destabilization of the medial meniscus (ACLT + DMM) as described elsewhere [11]. All surgical procedures were performed under sterile conditions, and the rats were allowed to move, eat and drink freely after surgery.

IAJ of miR-140-5p
The miR-140-5p agomir and miR agomir negative control (scrambled 22nt nucleotides, miR-Scr) were synthesized by Guangzhou RiboBio Co., Ltd (China), and the working solutions were prepared immediately before injection. One week after surgery (EOA), 9 rats were injected with Cy5-labelled miR-140-5p agomir (5 nmol / 100 µl) in the right hind knee and with Normal Saline (NS) in the left hind knee under ultrasonic guidance through a medial patellar approach using an insulin syringe with a 29G needle (BD, USA), and then, the rats were allowed unrestricted weight-bearing and motion. Twenty-four, 48 and 72 hours later, 3 rats were randomly selected and fluorescence was measured using a SpectrumCT in vivo imaging system (PerkinElmer, USA). Then, the bilateral hind knee tissues including femoral condyle, tibial plateau, meniscus and synovium were obtained, washed, coated with OCT and quickly frozen, and 6-µm serial sections were prepared and naturally dried away from light. Fluorescent images of the sections were acquired by a fluorescent microscope (Zeiss, Germany).
Behavioural and cartilage degeneration assessment Behavioural changes were assessed by measuring the number of rears performed by the rats as described previously [10]. Then, the rats were euthanized and the gross morphologic changes in their femoral condyle were photographed and scored using the following 0-4 scale: 0 = surface appears normal; 1 = minimal fibrillation or slight yellowish discoloration of the surface; 2 = erosion extending to the superficial or middle layers; 3 = erosion extending to the deep layer; and 4 = erosion extending to the subchondral bone [10]. Finally, the femoral condyles were routinely fixed with 4% paraformaldehyde at 4 °C for 48 h and then decalcified with 10% EDTA at pH 7.4 for 4-6 weeks. After dehydration, all tissues were embedded in paraffin and sagittally sectioned at 6-µm thickness. The sections were dewaxed and hydrated before being stained with haematoxylin & eosin (HE) and toluidine blue. The chondrocyte numbers in an equal area and the cartilage thickness were measured as reported previously [12], and the severity of the cartilage lesions was graded using the modified Mankin score. All sections were assessed independently by three blinded assessors (SHB, YTM and CY), and the final results were determined via a consensus.
Bioinformatics analysis of the possible mechanism of rno-miR-140-5p The mature sequence of rno-miR-140-5p was obtained in NCBI and UCSC database, and its putative targets were determined using the miRDB, miRmap (the cut-off values was set to 80), PicTar, TargetScan and DIANA microT-CDS (the threshold was set to 0.8) databases.
To make the predicted target genes more convincible, only the target genes predicted by at least three databases were selected for further analysis. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to perform enrichment analyses for the target genes of rno-miR-140-5p, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and p < 0.05 was considered statistically significant. Finally, the interactive relationships among the proteins encoded by the target genes were mapped using the Search Tool for the Retrieval of Interacting Genes (STRING) database, and the interactions with a combined score > 0.7 (high confidence) were considered significant and visualized.

Statistical analysis
Quantitative data were presented as the mean and 95% confidence intervals (95%CI) and assessed for normality of distribution using the Kolmogorov-Smirnov and Shapiro-Wilk tests. The t-test or Mann-Whitney U test was performed to identify the differences between two groups, while one-way analysis of variance (ANOVA) with Tukey's post hoc analysis or Kruskal-Wallis H test with Student-Newman-Keuls (SNK) post hoc analysis was conducted for multiple group comparisons. All statistical analyses were performed using SPSS software (IBM, USA), and P < 0.05 were considered statistically significant.

Results
Intra-articularly injected miR-140-5p enters cartilage and be taken up by Single IAJ of miR-140-5p exhibits better therapeutic effect on early-stage OA Rearing behaviour is an indicator of joint pain and spontaneous activity levels. As shown in Fig. 2B, the number of rears was higher in the SIAJ groups than in the control group at T1 to T3, and the differences between the SIAJ-1W and the control groups at T2 and T3 and the difference between the SIAJ-1W and the SIAJ-8W groups were statistically significant. Gross images of the medial femoral condyle are shown in Fig. 2C. The cartilage surface was smooth in normal rats, while cartilage degeneration characterized by fibrillation, pitting, ulceration, osteophyte formation and even subchondral bone exposure was observed at T1 to T3, and the rats in the SIAJ groups displayed less extensive cartilage lesions than those in the control group. Quantitatively, pathological lesion scores were significantly lower in the SIAJ-1W group than in the control group at T1 to T3, and the score in the SIAJ-1W group was also significantly lower than that in the SIAJ-8W group at T3 (Fig. 2D).
The typical images of HE and toluidine blue staining are shown in Figs. 3A and 3B.
Compared with the control group, the chondrocyte numbers and cartilage thickness in the SIAJ-1W group were significantly higher at T2 and T3 (Figs. 3C and 3D), while the Mankin score was significantly lower in the SIAJ-1W group at T1 to T3 and in the SIAJ-4W group at T2 (Fig. 3E). Moreover, the chondrocyte numbers and cartilage thickness were significantly higher, while the Mankin score was significantly lower in the SIAJ-1W group than in the SIAJ-8W group at T3; meanwhile, the cartilage thickness at T3 was significantly higher, while the Mankin score at T2 was significantly lower in the SIAJ-1W group than in the SIAJ-4W group.
Multiple IAJs of miR-140-5p exhibit better therapeutic effect than single IAJ on early-stage OA As shown in Fig. 2E, the number of rears was higher in the MIAJ groups than in the control group at T1 to T3, and the differences at T2 and T3 were statistically significant. Gross images of the medial femoral condyle are shown in Fig. 2C, the rats in MIAJ groups displayed less extensive cartilage lesions compared with the control group, and pathological lesion scores were significantly lower in the MIAJ groups than in the control group at T1 to T3, and the MIAJ-3 showed the best results (Fig. 2F). Compared with the control group, the chondrocyte numbers and cartilage thickness in the MIAJ groups were significantly higher at T2 and T3 (Figs. 3F and 3G), while the Mankin score was significantly lower at T1 to T3 (Fig. 3H). Moreover, the Mankin score was significantly lower in the MIAJ-3 group than in the MIAJ-1 group at T3. Bioinformatics analyses of the potential mechanisms by which rno-miR-

Discussion
The cartilage lesions mainly affect the superficial layer in EOA, and are gradually worsened and deepened with the progress of OA, suggesting that it is of great clinical significance to begin intervention in the early-stage of OA. In this study, we firstly verified that the intra-articularly injected miR-140-5p could rapidly enter the cartilage and reach the chondrocyte cytoplasm; then, we found that the earlier a single IAJ of miR-140-5p, the better the therapeutic effect, and multiple IAJs of miR-140-5p exhibit better therapeutic effect than single IAJ on EOA; finally, the bioinformatics analysis predicted the potential targets of miR-140-5p and the possible mechanisms by which miR-140-5p participates in OA pathogenesis. These results suggest that IAJs of miR-140-5p is an effective and promising strategy for the treatment of EOA, and also provide support for further exploration of miR-based OA therapeutics.
Intra-articular treatments have emerged in recent years and may offer safe therapeutics that have few extra-articular adverse effects [13][14][15][16], and we firstly proved in this study that the intra-articularly injected miR-140-5p could rapidly enter the cartilage and reach the chondrocyte cytoplasm, while no extra-articular uptake, and exhibit a protective role in OA. Based on these results, we further discovered that the potential target genes of rno-miR-140-5p were enriched in various biological processes and mainly located within 10 pathways, implying that the potential mechanisms by which rno-miR-140 attenuates OA progression may be the regulation of these pathways.
The PI3K-Akt, Notch, Wnt and MAPK signaling pathways are well known to be involved in OA pathogenesis, and regulate a variety of biological processes, including inflammation, cell growth, survival and metabolism [17][18][19]. However, few studies have reported the interactions between these pathways in chondrocytes and OA pathogenesis.
Although the central role of cartilage degeneration in OA has been recognized, OA is a total joint disease, while not just a dysfunction of cartilage [12]. Although we found in this study that the intra-articularly injected miR-140-5p has no extra-articular distribution, it is substantially uptaken by the intra-articular non-cartilage tissues such as synovium and meniscus. Peng et al. reported that synovial fibroblasts (SFs) also respond to miR-140-5p which could inhibits the proliferation and migration of SFs and promotes apoptosis of SFs in autoimmune arthritis mice [26]. Li et al. reported that miR-140 plays an important role in fracture healing [27], and Genemaras et al. found that miR-140-5p can be detected in meniscal cells [28]. However, few studies reported the specific effect of miR-140-5p on osteoblasts, osteoclasts or meniscus cells. Therefore, there is still a need to further investigate the effects of miR-140-5p on intra-articular cells and tissues other than chondrocytes and cartilage.
Previous studies have confirmed that drugs or genes injected directly into the joint cavity will be cleared rapidly due to the action of lymphatic vessels and synovial blood vessels, and non-targeted aggregation further reduces the bioavailability, thus multiple, high-dose injections which might make the administration more cumbersome and increase the risk of infection are often required [29]. In order to enhance the cartilage-targeting delivery   scores between the MIAJ and the control groups, respectively (n = 6 in each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001.  Bioinformatics analyses of the potential mechanisms by which miR-140