Identication of miR-338-5p and miR-4776-3p as mediators of articular cartilage degeneration

Osteoarthritis is one of the most common chronic diseases nowadays, and can cause serious physical illness and economic burden. Cartilage degeneration is one of primary reasons for osteoarthritis, but there were not thorough studies on cartilage lesions. miR-338-5p and miR-4776-3p were overexpressed and knocked down in SW1353 cells. Dual luciferase assay is used to detect the binding of miR-338-5p, miR-4776-3p and SOX6. The expression of MMP13, TNFA, IL6, MCP1, PARP, Runx2, ALP, OPN, WNT4, WNT16, TRACP and CTSK was quantied by qPCR. And the expression of WNT4 at the protein level was quantied by WB. study, found miR-4776-3p high through miR-338-5p improve catabolism, inammation and apoptosis. Meanwhile we validated miR-338-5p and miR-4776-3p may play negative regulation in the process of bone biology. miR-4776-3p reporter gene assay, of lesion the of in


Introduction
With growth of aging people, osteoarthritis is increasingly become main source of pain, disability and socioeconomic consumption worldwide [1]. During osteoarthritis period, cartilage tissue in the joints will corrode, thereby causing pain and serious loss of patients' joint function [2]. Cartilage degeneration is one of main reasons in osteoarthritis, but we have not fully understood the cellular and molecular mechanisms of its occurrence and development [3]. At present, there is no better method which can treat osteoarthritis caused by cartilage degeneration. Therefor disease prevention and early diagnosis appear to be necessary.
Micro-RNA (miRNA) is a kind of endogenous, small RNA with a length of about 20-24 nucleotide, and it has multiple important regulation role in cells. It enables to target most protein-coding transcripts, nearly participating in animals' various development and pathological processes, such as cell proliferation, migration, apoptosis and autophagy [4,5]. miRNA expression also causes many diseases, such as osteoporosis, diabetes, obesity and cancers [6][7][8][9], while some miRNA directly participate in in ammatory reaction and apoptosis process [10][11][12][13], which provides good reference for studies on osteoarthritis.
Recent studies have showed that miR-140 played an important role in cartilage degeneration [14], however, similar studies on miRNA are rarely seen.
In this study, we tried to explore and verify new miRNA which plays an important role in cartilage degeneration. First, we downloaded 2,100 miRNA expression data of patients with lumbar disc herniation (LDH) and patients with osteoarthritis of spine (FJOA) from NCBI GEO database, and made bioinformatics analysis, which found miR-4776-3p and miR-338-5p showed signi cantly high expression in FJOA. Then, we explored functions of miR-4776-3p and miR-338-5p by utilizing miRNA mimics and miRNA inhibitors, which obtained these two miRNAs participated in in ammatory reaction, catabolism and apoptosis process. Finally, we predicted target genes of miR-4776-3p and miR-338-5p via Target scan website, and veri ed target binding of miR-4776-3p with SOX2 and miR-338-5p to SOX9 through experiments. This study, through bioinformatics analysis and experimental veri cation, proved initially the role of miR-4776-3p and miR-338-5p in cartilage degeneration, providing new target and treatment regimen for early diagnosis of osteoarthritis.

Data download
At initial stage, we searched miRNA expression database related to cartilage in GEO database of NCBI (https://www.ncbi.nlm.nih.gov/geo/). We only selected samples of patients with cartilage disease and healthy persons, and ruled out databases derived from animals and human cells, nally only GSE79258 was found to conform to our requirements and used as subsequent analysis and veri cation. GSE79258 is GPL21599 (miRCURY LNA micro-RNA Array, 7th generation, miRBase 20) sequencing platform based data, including 2 cartilage samples from LDH patients and 2 cartilage samples from FJOA patients.
Identi cation of differently expressed miRNA (DE-miRNA) We made differential analysis for data downloaded by using "Limma" package in R language, so as to obtain differently expressed miRNA in LDH and FJOA. We set | log2FC | > 1 and P 0.05 as threshold of identifying DE-miRNA. Heat Map was created by "pheatmap" package in R language, and Volcano Plot was created by Graph Pad Prism 7.

Prediction of potential transcription factors
The upstream transcription factors of DE-miRNAs were predicted by FunRich software, which is a standalone software tool mainly used for functional enrichment and interaction network analysis of genes and proteins. We DE-miRNAs to obtain their upstream transcription factors, and present the top ten transcription factors according to P value.

GO annotation and KEGG pathway enrichment analysis
The GO functional annotation and KEGG pathway enrichment analysis for the DE-genes were conducted by Enrichr, which is a comprehensive resource for curated miRNA sets and a search engine that accumulates biological knowledge for further biological discoveries (http://amp.pharm.mssm.edu/Enrichr/). We input DE-miRNAs, and conducted biological process (BP), cellular component (CC), and molecular function (MF) functional annotation and KEGG pathway enrichment analysis. The top ten results are presented according to P value, with P < 0.05 considered as statistically signi cant.

Predication of potential target gene of DE-miRNA
We predicated downstream target gene of DE-miRNA by using TargetScanHuman (http://www.targetscan.org/vert_72/). TargetScanHuman is a website which predicts miRNA target gene, including miRNA target gene results of 10 species such as human, mouse, rats, opossum, chimpanzees, rhesus monkeys, bovine, dogs, chicken and frog, and it can clearly see binding of miRNA and target gene. TargetScan predicted biological target of miRNA by searching conserved 8mer, 7mer and 6mer loci which match with each miRNA seed region [15].
Cell culture Human chondrosarcoma cell line SW1353 was purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China). SW1353 cell was cultured in L-15 medium (HyClone, Waltham, MA, USA), in which added 10% high-quality fetal bovine serum (Invitrogen, Carlsbad, California, USA), 100U/ml penicillin and 100μg/ml streptomycin (Invitrogen). All cells were cultured in a wet incubator containing 5% CO 2 at 37℃.

Primer design and vector construction
Through whole-genome synthesis method, SOX6 complete coding sequence (63~2688bp) was synthesized and constructed into pIRES2-EGFP carrier, SOX6 3'UTR region 1000bp (4300~5300bp) fragment was synthesized and constructed into psi-check-2 carrier and marked as wild type carrier (Wild type). After successful construction of wild type carrier, mutant type carrier (Mutant) was constructed by using point mutation method. All miRNA mimics and inhibitors were synthesized by Genepharma. See Table 1 for sequence: Cell transfection and luciferase reporter gene assay Wild type and mutant type carriers were co-transfected into SW1353 cells together with miRNA mimics and mimic NC in a concentration of 20nM by Lipofectamine 3000 transfection reagents (Invitrogen, Carlsbad, CA, USA), to complete miRNA over expression cotransfection. Wild type and mutant type carriers were co-transfected into SW1353 cells together with miRNA inhibitors and inhibitor NC in a concentration of 20nM to complete miRNA over expression cotransfection. Cells were lysed after 48h of culturing, and activities of re y luciferase and ranilla luciferase in cell lysis buffer were measured by dual-luciferase reporter gene assay kit (E1910) (Promega, Madison, WI, USA).
RNA extraction and real-time polymerase chain reaction (qPCR) miRNA mimics, mimic NC, miRNA inhibitors and inhibitor NC were transfected into SW1353 cells by Lipofectamine 3000 transfection reagents with a concentration of 20 nM, and then cells were lysed after 48h of culturing; total RNA was extracted by TRIzol reagent (Invitrogen), then transcribed as c DNA by ReverAid (Thermo Scienti c). Real-time PCR was conducted by using qPCR SYBR Green Master Mix (High Rox Plus) (Thermo Scienti c). qPCR system was 20ul, including 1ul upstream primer, 1ul downstream primer, 0.4ul cDNA (1000ng/ul), 10ul Green Master Mix and 7.6ul ultra-pure water. This system was reacted for 60s at 95°C, then 30s at 95°C; 30s at annealing temperature and 30s at 72°C; 40 cycles were done. Finally DNA was detected for 30s at 95°C and 35s at 5°C. 2-ΔΔCt method was used to determine relative gene expression level. See Table 2 for primer sequence:

Western blot analysis (WB)
After treating the cells with miRNA mimics, miRNA inhibitors, SOX6 overexpression vector and SOX6 siRNA for 48 h, the cells were collected. 1× (10 6 ) cells were cracked by 1 ml of RIPA and 10 μl of PMSF, then centrifuged at 12,000 r/min at 4°C for 4 min. The intermediate protein layer solution was removed, and the BCA protein quanti cation kit was used for protein quanti cation. Samples of each group were diluted to 50 μg/ml, and the diluted protein was mixed with Sample Buffer at the ratio of 4:1 and heated at 100°C for 5 min. Then Mixing Acrylamide, Resolving Buffer, Starcking Buffer, distilled water, 10% APS, and TEMED were mixed in proportion to make SDS-PAGE separation gel and stacking gel, and poured into the gel plate. The Prestained Protein Ladder and the sample were separately added into the sample wells of the gel plate, and the proteinloaded SDS-PAGE gel was subjected to vertical gel electrophoresis for 50 min. The polyvinylidene di uoride (PVDF) membrane was activated by methanol for 1 min and then transmembrane was performed. After that, the PVDF membrane was blocked by 5% fat-free milk containing TBST solution for 1 h. After blocking, the PVDF membrane was washed by TBST. The WNT antibody (Thermo Fisher Scienti c, rabbit polyclonal antibody-Catalog Number PA5-27321) was incubated at 25°C for 2 h at a dilution of 1:500, and the second antibody was incubated at 25°C for 1 h.
Finally, Supersignal West Pico PLUS was used to ll the PVDF membrane and was placed in the iBright FL1000 (Thermo Fisher Scienti c) for observation.

Statistical analysis
The data were presented as mean value ±SD. Statistical difference between groups was determined by two-tailed T test. Statistical difference between groups was analyzed by one-way variance, then Student-Neuman-Keuls multiple comparative test was used to make analysis. All experiments were conducted at least 3 times independently, with similar results; also, typical experiments were shown. It was statistically signi cant if P <0.05. NS> 0.05 * P <0.05 ** P <0.01 *** P <0.001.

Results
Differently expressed miRNA By data analysis, we found 55 differently expressed miRNA in LDH and FJOA, including 21 highly expressed miRNAs in FJOA, 29 lowly expressed miRNAs in FJOA. miRNA details are presented in attached table 1. Expression quantity of miR-338-5p and miR-4776-3p in FJOA was nearly 4 times of that in LDH, which was selected as target miRNA for subsequent experiments ( Figure 1A Figure   3. The biological process analysis results show that the biological process of DE-miRNAs is mainly concentrated in regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism ( Figure   3A), the cellular component is mainly concentrated in nucleus and cytoplasm ( Figure 3B), the molecular function is mainly concentrated in transcription factor activity and transcription regulator activity ( Figure  3C). The biological process of DE-miRNAs is mainly concentrated in S1P, VEGFP, ErbB signaling network and pathway ( Figure 3D). miRNA target gene prediction miR-338-5p and miR-4776-3p target genes were predicted by TargetScanHuman website, which found miR-338-5p binds 4813 genes naturally and miR-4776-3p binds 4175 genes naturally; the details are presented in attached table 2. Various references reported transcription factors SOX5/SOX6 and SOX9 together promoted chondrogenesis in whole genome by super-enhancers [16,17], miR-338-5p and miR-4776-3p made target binding with SOX6, so SOX6 was chosen as study target (Figure 4 A, B). miR-338-5p and miR-4776-3p bind with SOX6 Through dual-luciferase reporter gene assay, we found exogenous miR-338-5p mimics can bind with 3'UTR of SOX6, allowing its activity to reduce by about 30%, while miR-4776-3p mimics can bind with 3'UTR of SOX6, allowing its activity to reduce by about 30%. However, it was found SOX6 activity would rise by about 40% after transfection of miR-338-5p inhibitors and also about 40% after transfection of miR-4776-3p inhibitors ( Figure 5A-D).
By bioinformatics method, it was found expression quantity of miR-338-5p and miR-4776-3p in FJOA was about 4 times of that in control group, and then we further explored how these two miRNAs worked in cartilage degeneration. After miR-338-5p mimics and miR-4776-3p mimics were transfected to SW1353 cells, it was found expression level of catabolism genes (MMP13), in ammation marker gene (TNFA, IL6, and MCP1) and apoptosis marker genes (PARP) was signi cantly improved. This indicated miR-338-5p and miR-4776-3p can promote catabolism, in ammation and apoptosis, thereby promoting chondrocyte degeneration ( Figure 6A, B).

SOX6 mediates the expression of osteogenic genes and osteoclast genes
To verify whether the changes in the expression levels of osteogenic and osteoclast markers upon overexpression or inhibition of miR-338-5p and miR-4776-3p are directly caused by changes in SOX6, we overexpressed and knockdown SOX6 in human SW1353 cells. By qRT-PCR, we found that the expression of Runx2, ALP, OPN, WNT4 and WNT16 were signi cantly upregulated and the expression of TRACP and CTSK was signi cantly downregulated when SOX6 overexpression (Fig. 9A). The expression of Runx2, ALP, OPN, WNT4 and WNT16 were signi cantly downregulated and the expression of TRACP and CTSK was signi cantly upregulated when SOX6 knockdown (Fig. 9B). Overexpression of miR-338-5p and miR-4776-3p can reduce the expression of WNT4 at the protein level, while knockdown of miR-338-5p and miR-4776-3p can increase the expression of WNT4 (Fig. 9C).

Discussion
The most challenging aspect in the treatment of articular cartilage injury mainly includes determination of matrix changes and cells and molecular mechanism of chondrocyte differentiation and of dedifferentiation behavior, and understanding of how to affect structural integrity and tissue remodeling of articular cartilage [18]. Several studies have showed that miRNA in tissues played a vital role in cellular processes (such as apoptosis, cell proliferation, differentiation, metabolism and cell cycle control) [19]. Several existing studies have shown that miRNA may be related to premature degradation of cartilage [14,20], and some studies also have indicated multiple miRNA had higher expression quantity in cartilage with arthritis [21][22][23]. However, most studies involved compassion of miRNA expression quantity between samples of osteoarticular cartilage and normal samples, and did not go into the molecular mechanism of cartilage degeneration caused by miRNA. In this paper, we preliminarily studied the molecular mechanism of cartilage degeneration caused by miR-338-5p and miR-4776-3p, providing new ideas for further studying cartilage injury and degeneration.
Throughout the study, we intended to nd miRNA with signi cantly high expression of cartilage in osteoarthritis, and study the role of miRNA in cartilage lesions. We downloaded 2100 miRNAs expression data of LDH patients and FJOA patients from NCBI GEO database, and made bioinformatics analysis, which found miR-4776-3p and miR-338-5p showed signi cantly high expression in FJOA. To determine miR-338-5p and miR-4776-3p have physiological and pathological actions action in articular cartilage degeneration or not, miR-338-5p and miR-4776-3p were over expressed and knocked down in chondrocyte SW1353, so as to determine their impact on cartilage. Through detection of expression quantity of catabolism, in ammatory reaction and apoptosis marker genes, we found miR-338-5p and miR-4776-3p can improve catabolism and trigger apoptosis, thereby promoting cartilage degeneration. Through dualluciferase reporter gene assay, miR-338-5p and miR-4776-3p can bind with cartilage-related gene SOX6 naturally, but further study on the molecular mechanism of cartilage lesion and the role of SOX6 in in vivo is required.
Related studies have shown that cartilage in osteoarthritis usually accompanies bone lesions. Joint integrity comes from balance of physiological interaction between skeletons and cartilages. Accumulated evidences in the bodies also indicated the increase of bone absorption occurred at early stage of osteoarthritis progression, and cytokines blocking bone absorption can prevent cartilage injury, thereby con rming the role of bone factors in crosstalk of the two tissues [24]. In this study, we also preliminarily studied the role of miR-338-5p and miR-4776-3p in bone formation and absorption, which found they can inhibit osteogenesis and promote osteoclastogenesis. However, we did not study correlation and molecular mechanism of miR-338-5p and miR-4776-3 between bone formation and absorption in cartilage lesions, which will be the focus in future studies.

Conclusions
To sum up, we, by using bioinformatics methods in the whole study, found miR-338-5p and miR-4776-3p had signi cantly different high expression quantity of cartilage in osteoarthritis, and veri ed miR-338-5p and miR-4776-3p can improve catabolism and trigger in ammation and apoptosis by experiments. At the same time, they can promote osteoclastogenesis and inhibit osteogenesis. These study results showed that miR-338-5p and miR-4776-3p played an important role in cartilage lesion, and can be used as diagnostic marks of early cartilage lesions.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.   The top ten upstream transcription factors of upregulated DE-miRNAs.