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, finally only GSE79258 was found to conform to our requirements and used as subsequent analysis and verification. 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.
Identification 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 stand-alone 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 significant.
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 .
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% CO2 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 firefly 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 Scientific). Real-time PCR was conducted by using qPCR SYBR Green Master Mix (High Rox Plus) (Thermo Scientific). 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× (106) 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 quantification kit was used for protein quantification. 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 difluoride (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 Scientific, 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 fill the PVDF membrane and was placed in the iBright FL1000 (Thermo Fisher Scientific) for observation.
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 significant if P <0.05. NS> 0.05，* P <0.05，** P <0.01，*** P <0.001.