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H19 Promotes the Proliferation of Osteocytes by Inhibiting P53 During Fracture Healing

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H19 Promotes the Proliferation of Osteocytes by Inhibiting P53 During Fracture Healing European Review for Medical and Pharmacological Sciences 2018; 22: 2226-2232 H19 promotes the proliferation of osteocytes by inhibiting p53 during fracture healing Q.-P. ZHOU1, F. ZHANG1, J. ZHANG2, D. MA1 1Department of Orthopaedics, People’s Hospital of Xuyi, Xuyi, China 2Department of General Surgery, the First Affiliated Hospital with Nanjing Medical University, Nanjing, China Qinpo Zhou, Feng Zhang and Jian Zhang contributed equally to this work Abstract. – OBJECTIVE: We explored the of fractures, which brings physical and psycho- possible mechanism underlying the expression logical distress to patients and their families2. change of H19 during fracture healing. MATERIALS AND METHODS: The best therapy for delayed union or nonunion A total of 18 3,4 male SD mice aged from 6-8 weeks old (18.5-24.6 currently is autologous bone graft . However, g) were selected to establish tibial fracture mod- the suitable implantable bone in the human body els. The left tibia undergoing sham surgery was is very limited, and the incidence of adverse re- considered as the control group, and the right actions in the donor bone will increase after tran- tibia undergoing sawing treatment was consid- splantation5. Currently, the fracture treatment ered as the experimental group. The control tibia with less damage and fewer complications is in and fracture tibia from three mice were harvest- ed at six time points after operation, respective- urgent need. Long non-coding RNA (lncRNA) ly. QRT-PCR was utilized to detect the changes is a class of RNA longer than 200 nt without of H19 and p53 mRNA expression. protein-coding function6. Previous studies were RESULTS: Compared with the control group, mostly focused protein encoding genes, whereas the expression of H19 in the experimental group non-coding RNAs were thought as “junk RNA” was significantly increased at 4, 8, and 12 d. or “noise” in life processes. However, with the However, there was no significant difference in the expression of H19 between the experimental rapid development and application of high-throu- group and the control group at 16, 20, and 24 d. ghput sequencing technologies during the last The proliferation of chondrocytes and osteo- decade, more and more researches have shown blasts from mouse and human was significantly that lncRNAs can regulate protein-coding ge- inhibited, and the apoptosis was significantly in- nes expression at epigenetic, transcriptional and creased after interference of H19. As p19 plays im- post-transcriptional level, thus affecting a series portant roles in diverse biological process, we de- of biological processes7-9. In addition, lncRNAs tected the expression level of p19 after inference can also serve as molecular markers for disease of H19. In addition, knockdown of H19 significantly 10 up-regulated the expression of p53 in osteoblast diagnosis and target of therapy . cell lines, while the down-regulation of p53 expres- H19 (H19, imprinted maternally expressed sion reversed the proliferation of osteoblasts. transcript) is located on the human chromosome CONCLUSIONS: H19, as a molecular marker Chr11p15.5. Recent studies12-14 have shown that for promoting fracture healing, promote the pro- its exon region also encodes a small RNA, miR- liferation of osteocytes by inhibiting the expres- 11 sion of p53. 675 . H19 is abundant in embryonic development, but its expression is decreased in tissues other Key Words: than skeletal muscle after birth. The high expres- Fracture healing, H19, p53. sion of H19 is found to be associated with tumor cell proliferation, apoptosis and metastasis in many oncological diseases15. Meanwhile, H19 is Introduction involved in cell proliferation and tissue repair in several diseases of the skin and skeletal muscle16. According to statistics, about 10% of fracture However, H19 functions in fracture healing have patients have delayed union or nonunion and re- not been elucidated yet. This study aims to eluci- quired further treatment1. Fracture nonunion or date whether and how H19 functions in fracture delayed healing is one of the major complications healing. 2226 Corresponding Author: Danian Ma, BM; e-mail: [email protected] H19 promotes the proliferation of osteocytes by inhibiting p53 during fracture healing Materials and Methods used in transfection were as follows: si-H19-01: 5’-GGAGAGTTAGCAAAGGTGACATCTT-3’; Animals si-H19-03: 5’-AGAGTTAGCAAAGGTGACA- A total of 18 male SD (Sprague Dawley) mice TCTTCT-3’; si-scramble: 5’-GGATGATCGA- (18.5-24.6 g) aged 6-8 weeks old were anesthe- AGATGAGACTAGCTT-3’; Si-p53: 5’-CUA- tized by intraperitoneal injection of 1% sodium CUUCCUGAAAACAACG-3′. pentobarbital at a dose of 1 mL/kg (there was no significant difference in age, sex and weight of Western Blot mice). After that, sham operation was performed Total protein from cells was extracted using a on the left tibia of the mice, and the right tibia of cell lysate (RIPA) containing protease. After the mice in treatment group was sawn off (to cause measurement of protein concentration by Nano- stable fracture) and completely matched with the Drop 2000 micro spectrophotometer, the protein intramedullary fixation needle. The left and right was denatured with 5× loading buffer. The appro- tibia tissues of three mice were collected on the 4, priate amount of protein samples and protein mar- 8, 12, 16, 20, and 24 d after operation, respective- ker was separated by SDS-PAGE (sodium dodecyl ly. The samples were stored in -80°C refrigerator. sulphate-polyacrylamide gel electrophoresis) gel This study was approved by the Animal Ethics electrophoresis. After transferred from the gel to Committee of Nanjing Medical University Ani- the PVDF (polyvinylidene difluoride) membrane, mal Center. the protein samples were incubated with primary and secondary antibodies sequentially. Finally, Cell Culture the membranes were exposed and photographed. Chondrocytes (ATDC5), osteoblasts (MC3T3-E1), human articular chondrocytes Cell Counting Kit-8 (CCK-8) Assay (HC-a) and osteoblasts (hFOB1.19) were purcha- Cells were seeded in 96-well plates and syn- sed from the American Type Culture Collection chronized for 12 h after cell adherence. 6 repli- (ATCC, Manassas, VA, USA). The cells were cate wells were set up for each sample and the cultured in DMEM (Dulbecco’s modified Eagle total reaction volume per well was 200 μL. Each medium) medium containing 10% FBS (fetal bo- well was added with 20 μL of CCK-8 reaction so- vine serum), 100 U/mL penicillin and 100 μg/mL lution and then incubated at 37°C for 2 h in the streptomycin (Invitrogen, Carlsbad, CA, USA), dark. After shaking for 10 min on a micro sample and then placed in an incubator at 37°C with 5% shaker, absorbance of each well at 450 nm was CO2. The medium was changed every 2-3 days measured with a microplate reader. and cells were passaged when fusion degree rea- ched 80-90%. Flow Cytometry After cell supernatants were collected into labe- qRT-PCR led tubes, EDTA-free trypsin was used to digest We used TRIzol to extract total RNA and re- the cells. The cell suspension was centrifuged verse transcription was performed according to and washed twice with phosphate buffered saline the instructions of PrimeScript RT reagent Kit (PBS), and 200 μL of binding buffer containing (TaKaRa, Dalian, China). Diluted cDNA and a calcium ions were added to the tubes after centri- certain amount of primers, premix and ultrapure fugation. 5 min prior to incubation in the dark, 10 water were mixed into a 20 μL reaction system. μL of Annexin V-FITC fluorescent probe and 5 μL ABI 7500 FAST Real-time PCR instrument was of PI (propidium iodide) were added to the tubes. used for cDNA amplification. The expression FL1 and FL3 dual channel wavelength detection level of the target gene was calculated using the was performed to access the apoptosis of cells. 2-ΔΔCT method. Primers used in qRT-PCR were as follows: H19: 5’-GCGGGTCTGTTTCTT- Statistical Analysis TACTTC-3’ (forward), 5’-TTTCATGTTGTGG- We used statistical product and service solu- GTTCTGG-3’ (reverse); GAPDH: 5’-GGAC- tions 17.0 software (SPSS Inc., Chicago, IL, USA) CAATACGACCAAATCCG-3’ (forward), for statistical analysis. All data were expressed 5’-AGCCACATCGCTCAGACAC-3’ (reverse); as mean ± SD, and independent sample t-test p53: 5’-CCTCAGCATCTTATCCGAGTGG-3’ was used to analyze the difference between two (forward), 5’-TGGATGGTGGTACAGTCAGA- groups. p<0.05 was considered statistically signi- GC-3’ (reverse). Small interference sequences ficant. 2227 Q.-P. Zhou, F. Zhang, J. Zhang, D. Ma Results expression in chondrocytes and osteoblasts (Fi- gure 2A, F). Knockdown of H19 in chondrocytes H19 Expression Was Increased in and osteoblasts remarkably inhibited the cell Fracture Tissues in Fractured proliferation and enhanced the apoptosis, while Animal Models overexpression of H19 markedly increased the In order to study the physiological changes proliferation of both chondrocytes and osteobla- and molecular mechanisms during fracture hea- sts from mouse and human (Figure 2 B-E, H-J). ling, we compared the tissues at different stages Above results indicated that H19 expression was of fracture healing with normal bone tissues. We up-regulated early in fracture healing and might performed qRT-PCR to detect the expression of play an essential role in promoting proliferation H19 in the left and right tibia of mice at 4, 8, 12, and inhibiting apoptosis in osteoblasts and chon- 16, 20, and 24 d after operation. The results revea- drocytes. led that the expression of H19 in the experimental group was significantly increased at 4, 8, and 12 d H19 Promoted the Proliferation of compared with the control group, while there was Osteocytes by Inhibiting the p53 Gene no significant difference in the expression of H19 It is generally accepted that lncRNAs are in- between the experimental group and the control volved in various biological activities of cells at group at 16, 20 and 24 d (Figure 1 A).
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