GEO bioinformatics analysis
GSE226245 was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo) and applied the DESeq2 software to obtain differentially expressed genes (DEGs) in sunitinib-resistant RCC cells 786-O(Sun-7R) with control or FUS knockdown. The threshold for screening DEGs was: log2 Fold Change |log2FC |> 1 and an adjusted p-value < 0.05.
Cell culture and osteogenic differentiation
MC3T3-E1 cells, a well-characterized mouse pre-osteoblast cell line, are obtained from the American Type Culture Collection (ATCC CRL-2594) and typically maintained in an α-MEM culture medium (Gibco BRL Life Technologies, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (Gibco BRL Life Technologies), and incubated in 5% CO2 humidified incubator at 37°C. MC3T3-E1 cells were authenticated by STR DNA profiling analysis and tested for mycoplasma contamination. For osteoblast differentiation, MC3T3-E1 cells are exposed to a medium containing with 50 µg/mL l-ascorbic acid and 10 mM β-glycerophosphate. For hypoxia treatment, cells were cultured with serum-free medium under hypoxic conditions (5% CO2, 1% O2) for 6 h in Heracell™ VIOS 160i Tri-Gas CO2 Incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then cultured with complete medium for reperfusion (5% CO2, 21% O2) for 18 h in a normal incubator.
Plasmids construction
To overexpress LINC00323 in MC3T3-E1 cells, the full length of cDNA was cloned into pCDH-CMV-MCS-EF1α-Puro Cloning and Expression Lentivector (pCDH-CMV-MCS-EF1α-Puro (System Biosciences, USA). Lentiviruses particles were produced using the ViaFect™ transfection reagent (Promega, Madison, WI, USA) by co-transfection HEK293T cells with the packaging plasmids psPAX2 and pMD2.G. Lentiviruses particles were collected and infected MC3T3-E1 cells 48 h after transfection. 1 mg/ml puromycin (Sigma) were used to select. The efficiency of infection was assessed by qRT-PCR analysis.
For gene knockdown, the small interfering RNA (siRNAs) targeting FUS (siFUS), PDGFB (siPDGFB), and negative control shRNA (siNC) were designed and provided by GenePharma (Shanghai, China). MC3T3-E1 cells were transfected with control or specific siRNAs 50 nM using the ViaFect™ transfection reagent (Promega). The efficiency of gene knockdown was evaluated using western blot 48 h after transfection.
Reverse Transcription-PCR
Total RNA was isolated from the cells with TRIzol Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. A total of 500 ng RNA was subsequently transcribed into cDNA using PrimeScript™ RT reagent Kit (Takara Biotechnology co., LTD., Dalin, China). Real time PCRs were performed with TB Green® Fast qPCR Mix (Takara Biotechnology co.). Amplification by PCR was performed using the ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, USA). The primers used as follows: β-actin (ACTB), 5′-GGCTGTATTCCCCTCCATCG-3′ and 5′-CCAGTTGGTAACAATGCCATGT-3′; Runt-related transcription factor 2 (Runx2), 5′-TTCAACGATCTGAGATTTGTGGG-3′ and 5′-GGATGAGGAATGCGCCCTA-3′; collagen type I alpha 1 chain (Col1A1), 5′-GCTCCTCTTAGGGGCCACT-3′ and 5′-ATTGGGGACCCTTAGGCCAT-3′; osteocalcin (OCN), 5′-CAGGAGGGCAATAAGGTAGT-3′ and 5′-TCTGCTACAGGGAAAACAGT-3′; osteopontin (OPN), 5′-AGTTTCGCAGACCTGACATCC-3′ and 5′-TTCCTGACTATCAATCACATCGG-3′; The 2−ΔΔCt method was employed to analyze gene expression.
Cell Viability Assay
For Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) assays, cells were seeded in a 96-well plate at a density of 8 × 103 cells per well. The absorbance was measured at 450 nm after 72h days with a microplate reader (Bio-Rad, CA, USA).
ALP activity
MC3T3-E1 cells were seeded into 6-well plate followed by differentiation for 21 days. For the ALP activity assay, the cells were washed with ice-cold PBS. After lysing, the supernatant was collected for measurement of the ALP activity and protein concentration. The ALP activity assay was assessed using Alkaline Phosphatase Assay Kit (Abcam, Cambridge, MA, USA) following the manufacturer’s protocol.
ARS Staining
For ARS staining, cells were washed with PBS three times and fixed in 70% ice-cold ethanol for 1 h at RT. The mineralized matrix was stained with 40 mM Alizarin red staining (Sigma Aldrich, St. Louis, MO, USA) under gentle agitation for 15 min at RT. After staining, the cells were washed with PBS 5 times. The red stain was destained with 10% (w/v) cetylpyridinium chloride (Sigma Aldrich) for 1 h, the absorbance at OD 570 nm was collected to assess the degree of mineralization.
RNA pull-down assay
The RNA-pulldown analysis was conducted as previously described using Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer’s instructions [20]. In brief, LINC00323 or PDGFB was labeled using Biotin RNA Labeling Mix (Roche, Basel, Switzerland), then digested with DNase I, protease inhibitor and RNase inhibitor. The supernatant of cell lysate was incubated with an equal amount of streptavidin magnetic beads at 37°C for 1 h. The protein level of FUS in the LINC00323-protein complexes was analyzed by western blot.
RNA immunoprecipitation
RNA immunoprecipitation (RIP) was conducted with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore Corp, Billerica, MA, USA) following the manufacturer’s protocol. In brief, cells were lysate and incubated with RIP immunoprecipitation buffer supplemented with magnetic beads conjugated with negative IgG or anti-FUS antibody. After digesting with Proteinase K, Immunoprecipitated RNAs were reversely transcribed into cDNA and following quantitative real-time PCR analysis.
RNA stability assay
For detection of mRNA stability of PDGFB, cells were treated with 5 µg/mL actinomycin D (Sigma-Aldrich) for 0, 20, 40, and 60 minutes, respectively. Then RNA was extracted and analyzed by RT-qPCR analysis.
Hematoxylin and eosin (H&E) stain and Masson trichrome stain
The fractured femur and calluses were collected on day 28 days after the bone fracture. After undergoing decalcification in 10% formic acid for a duration of one week, the samples were subsequently encased in paraffin wax. They were then cut longitudinally into sections with a thickness of 5 micrometers and carefully placed onto glass microscope slides. For the H&E staining of bone tissue, sections are first dewaxed in xylene and rehydrated through a graded alcohol series before being stained with hematoxylin to label nuclei. After rinsing, eosin is applied to stain cytoplasmic elements. In Masson's Trichrome staining, similarly prepared sections undergo sequential staining with Weigert's hematoxylin for nuclear definition, followed by Biebrich scarlet-acid fuchsin solution, and then differentiated in phosphomolybdic/phosphotungstic acid solution. Collagen is stained blue with aniline blue, providing contrast to red muscle fibers. The stained sections are dehydrated, cleared, and mounted. The slides were examined and photographed under an BX53 microscope light microscope (Olympus, Tokyo, Japan).
Western blot
Total cell proteins were extracted from cells using RIPA lysis buffer (Cell Signaling Technology, Berkeley, CA, USA) containing the protease inhibitors (Roche). After the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific), Western blotting was performed using standard protocols. The primary antibodies used in the study were as follows: anti-FUS Polyclonal Antibody (Thermo Fischer Scientific), anti-PDGFB Polyclonal antibody (Proteintech, Wuhan, China), anti-PDGFR beta Polyclonal antibody (Proteintech), and anti-β-actin (Proteintech). β-Actin was used as the loading control.
Radiographic analysis and micro-computed tomography examination
At 28 days after the bone fracture, fracture healing of mouse tibial was examined with radiographs (X-ray) with MX-20 Specimen Radiography System (Faxitron Bioptics, LLC, Tucson, AZ, USA). Micro-computed tomography (µCT) scanning was performed to aseess the microstructure of the bone callus as our previous described [19]. Meanwhile, the bone structural indices, including including the bone volume fraction (BV/TV, %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm) and trabecular separation (Tb.Sp, mm) were calculated.
Tibial Fracture Model and treatment
An in vivo mouse tibial fracture model was constructed using 10-weeks age male C57BL/6 mice as described previously [19]. The mice were housed in sterilized cages at the Experimental Animal Center of Capital Medical University (Beijing, China). All animal care and experimental procedures were performed in adherence to the National Institute of Health guidelines for Care and Use of Laboratory Animals and were approved by Institutional Animal Care and Use Committee of SHZY. (Permission No: SHZY-202106AF). In all, 3 groups of animals (n = 8 each group) were used, including model group (model), model group with lentiviruses particles with control vector (vector), and model group with lentiviruses particles with LINC00323 (LINC00323). Finally, callus tissue around the fracture site was harvested for subsequent detection.
Statistical analysis
All data was presented as mean ± standard deviation (SD) from at least three independent experiments. GraphPad Prism 5 Demo (GraphPad, Inc., La Jolla, CA, USA) was used to statistical analysis. Student's t test was used to analyze the differences between two groups. Ordinary one-way ANOVA combined with Tukey's multiple comparisons test was used to compare differences among three or more groups. A p value less than 0.05 was considered statistically significant.