| [Objective]It is very complex of fracture healing,Inadequate blood supply is a significant contributing factor for delayed fracture healing or nonunion.The disrupted blood flow post fracture usually leads to a hypoxic environment at the site of fracture [3], as follows by cell death, delayed chondrocyte and osteoblast differentiation, and impaired fracture healing.Thus, better understanding of the mechanisms that impair fracture healing under ischemic-hypoxic condition facilitates exploring novel therapies to stimulate fracture healing. Oxygen is involved in multiple basic cellular processes which are implicated in fracture healing, e.g. aerobic metabolism such as hydroxylation, oxidation and cyclodehydration. And an adequate oxygen supply is also important for the collagen synthesis process and for the expression of several angiogenic genes through the hypoxia inducible factor (HIF) pathway. In addition, tissue oxygen levels may modulate stem cell maintenance, mobilization, and recruitment to sites of fracture [9-13], and regulate the proliferation, mineralization and differentiation of alveolar osteoblasts. Therefore, the deep exploring of the hypoxic injury to osteoblasts and other cells implicated in fracture healing, and responded adaptation of these cells to the hypoxia lay light on strategies to stimulate fracture healing.To better understand the ischemic-hypoxia-induced fracture healing impairement, we determined in present study the microRNA-210 expression in broken bone specimens and in osteoblasts under hypoxia, and then determined the influence of microRNA-210 overexpression on the osteoblast cell proliferation and apoptosis.To explorehypoxia-inducible factor-1? (HIF-1?)under hypoxic condition training by the former generation of rat embryo specific expression of osteoblast and its relationship with the apoptosis caused by bone nonunion, provide a reference value on the basis of clinical treatment.[Methods]Part OneHuman tissue specimensUtilization of all catagmatic tissues and the normal bone tissues was approved by our hospital Internal Review Board (IRB) in Renmin Hospital of Wuhan University. 34 catagmatic tissues were obtained from femoral comminuted fractures, with size less than 0.5 cm in diameter, and not suitable for reposition, in emergercy from 24 to 36 hours post fracture. And 18 normal bone tissues were from remaining femoral bone graft. All tissue specimens were stored at-80? before utilizing.Cell culture and treatment with reagents.The human osteoblast MG-63 cells were provided by the cell resource center of Wuhan University and were grown in Eagle's Minimum Essential Medium (EMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA) at 37?, with 5% CO2. For hypoxia treatment, cells were placed in a hypoxia incubator with 5% CO2 and 2% oxygen. Oxygen concentration was monitored continuously (Forma 3130; Thermo Scientific, Rockford, IL, USA). MG-63 cells post hypoxia or normoxia treatment for 0,12,24,48 or 72 h, were collected for miRNA or mRNA isolation, for cellular protein extraction, or for apoptosis analysis. RNAi technology was used to knockdown HIF-la expression, with siHIF-la 1 (CUC AAG CAA CUG UCA UAUA), siHIF-la 2 (UGC CAC CAC UGA UGA AUUA) and siRNA control (UAA GGC CAG ACG CGA AUUA), which were synthesized by GenePharma Technology (Shanghai, China). In brief, MG-63 cells were transfected siRNA oligos with 50 nM by lipofectamine 2000,6 hours later, the supernatant was updated and cells were subject to followed treatment. microRNA-210 mimics (CUG UGC GUG UGA CAG CGG CUGA) or mimics control (UCA CAA CCU CCU AGA AAGA) (QIAGEN, Valencia, CA, USA) and were utilized to manipulate the microRNA-210 level.25 or 50 nM microRNA-210 mimics/mimics control was transfected into MG-63 cells with lipofectamine 2000.5-FU (Sigma-Aldrich, St. Louis, MO, USA), which is a thymidylate synthase inhibitor, was utilized to inhibit the MG-63 cell proliferation with a concentration of 5 ?g/mL and to induce MG-63 apoptosis with a concentration of 5 or 40 ?g/mL.RNA Isolation, Reverse Transcription, quantitative real-time PCR Total cellular RNA was isolated with PureLink(?) RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manual, and reverse transcription (RT) was performed with M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). RT-qPCR analysis of the HIF-1? in mRNA level was performed with Takara One Step RT-PCT kit (Takara, Dalian, China). All mRNA expression levels were normalized to ?-actin. miRNA specimens was isolated with the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) and quantitatively examined for microRNA-210 expression with the mirVana qRT-PCR miRNA Detection Kit (Ambion, Austin, TX, USA), with the U6 small nuclear RNA as internal control. ??Ct method was used for relative quantification [31]. Preparation and western blot analysis of protein samples. Whole cellular proteins were extracted with a cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manual and added with protease inhibitor cocktail (Roche, Basel, Switzerland). Polyclonal rabbit antibodies against HIF-1? (Abcam, Cambridge, UK), caspase 3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or caspase 9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were utilized to detect the HIF-1? level, cleaved caspase 3 (CASP 3) or cleaved caspase 9 (CASP 9) level by western blot analysis. ?-actin was used as an internal control with polyclonal rabbit antibodies against ?-actin (Sinobio, Beijing, China). Goat anti-rabbit IgG conjugated to horseradish peroxidase (Pierce, Rockford, IL, USA) and ECL detection systems (Super Signal West Femto; Pierce, Rockford, IL, USA) were used for detection.Cell proliferation, cell colony formation, and cell apoptosis assay. Cell proliferation assay was performed by CCK-8 assay (DOJINDO, Kumamoto, Japan). In brief, MG-63 cells post microRNA-210 mimics or mimics control transfection were inoculated for 24,36 or 48 h, in the presence of 5 ?g/mL 5-FU, and then were incubated with CCK-8. The 450 nm absorbance of cells was detected after visual color occurrence. Cell colony formation assay was performed as usual.103 Cells were incubated in 12-well plates at 37? containing 5% CO2, and were transfected with 0 nM or 50 nM microRNA-210 mimics or mimics control and were inoculated for another 2-5 days. Then cells were stained with crystal violet (0.005%) for 20 minutes and recorded the colony numbers. MG-63 cell apoptosis was examined with an annexin V-FITC apoptosis detection kit (Sigma-Aldrich, St. Louis, Missouri). Briefly, approximate 5x105 cells were stained with annexin V-FITC and propidium iodide and were detected by a FACScan flow cytometer (BD Biosciences). The results were calculated using the CellQuestTM Pro software (BD Biosciences) and evaluated as the percentage of apoptotic cells to total cells.Part two:From 19 d KM mice's skull, improved tissue block method was applied to cultivate the original generation of rat embryo osteoblast, at the same time use to stick to the cell wall of purification processing, again to culture the third-generation cell count last week, at the same time to have cultured osteoblasts identification of 4th generation cells, and use of calcium-cobalt, alkaline phosphatase staining test cells. In 125 moll/L in the concentration of cobalt chloride conventional DMEM medium, train the third generation of osteoblast, then give chemical simulated hypoxic condition, application of inverted phase contrast microscope to observe the cells cultured under low oxygen and count, then different time cultured osteoblasts to 0,1, 3,5, and 7 d hypoxia group, and the conventional oxygen under the condition of cultivation of the third generation of cells to 0,1,3,5, and 7 d conventional oxygen groups. Again to hypoxia group and conventional oxygen at different times of osteoblast, analysis of HIF-1 alpha expression and apoptosis caused by bone nonunion. At the same time using western blot method the Bbl.-2 in the detection of osteoblast and Capsize 3 protein expression.[Result]Part one:microRNA-210 is upregulated in human bone specimens post fracture, in association with HIF-1?To compare the difference of microRNA-210 expression between human bone specimens with fracture and the normal bone tissues, we chose 34 aged patients with fractures in the study, who have a mean (±SD) diagnosis age of 47.2±2.1 years, and we used bone specimens from 18 healthy age-matched volunteers as controls. To investigate the microRNA-210 preferentially expressed in human bone tissues to be involved in bone fracture healing, we assessed the expression of microRNA-210 in catagmatic tissues by real-time PCR. The mean (±SD) AACT value was 1.87±1.23 in the 34 samples from patients with bone fracture and 1.00±0.32 in healthy controls (p < 0.01; Figure 1A). Therefore, we identified that the microRNA-210 in bone specimens significantly increased in patients with bone fracture. It has been well known that hypoxia induced microRNA-210 via HIF-1? [32]. To investigate a possible upregulation of microRNA-210 by HIF-1? in catagmatic tissues, we firstly determined HIF-1? mRNA expression in the bone fracture group and normal specimens by RT-qPCR. Compared to controls, Figure 1C showed a significant high level of HIF-1? mRNA expression in those catagmatic tissues (p<0.01). We then investigated the correlation of microRNA-210 with HIF-1? mRNA expression. In bone fracture patients, microRNA-210 expression showed a positive correlation with HIF-1? mRNA (R2=0.3626, p<0.01) (Figure 1 D). microRNA-210 upregulation is regulated by HIF-?.To further confirm the upregulation of microRNA-210 by hypoxia in catagmatic tissues or in osteoblasts, we determined the microRNA-210 level by real-time qPCR in MG-63 osteoblast cell line under normoxia or hypoxia. Coinciding with results of clinical specimens, the substantial and significant induction of microRNA-210 expression in the osteoblast cell line under hypoxia was confirmed in vitro with a time-dependence, from 24 h post hypoxia, there was a significant upregulation of microRNA-210 (Figure 2A; p<0.05 or p<0.01) by the hypoxia. Moreover, HIF-1? was also upregulated by the hypoxia at both mRNA and protein levels (Figure 2B and 2C; either p<0.05 or p<0.01). To elucidate the correlation between microRNA-210 and HIF-1?, HIF-1? was knockdown by siRNAs targeting HIF-la. Figure 2D showed that the HIF-1? specific siRNA-1 or siRNA-2 significantly downregulated the mRNA level of HIF-1? (p<0.01 for siHIF-1?-1 or siHIF-1?-2) rather HIF-2?. And the siHIF-1?-1 or siHIF-1?-2 could effectively abrogated the hypoxia-promoted miRNA-210 upregulation (Figure 2E). Therefore, the microRNA-210 upregulation was associated with HIF-1? in catagmatic tissues, and its upreuglation in MG-63 cells in response to hypoxia was HIF-1?-dependent. microRNA-210 mimics reduces the 5-FU-caused death and promotes the growth of osteoblast cellsTo investigate the effect of microRNA-210 to the osteoblast cell proliferation, microRNA-210 expression level in MG-63 cell lines was manipulated via transfection with microRNA-210 mimics. As Figure 3 A showed, microRNA-210 mimics induced a significant increasing in microRNA-210 level in MG-63 cells (p<0.001 for 25 or 50 nM), compared to the control group. After that, we used CCK-8 assay to detect the proliferation of MG-63 cells post microRNA-210 mimics or miRNA control transfection and with a treatment with 5 ?g/mL 5-FU. As expected, the microRNA-210 mimics, rather than miRNA control, reduced the 5-FU-caused cell death in time-dependent and dose-dependent manners in MG-63 cells (Figure 3B and 3C). Furthermore, the difference in colony formation of MG-63 cells was investigated with microRNA-210 mimics or miRNA control transfection. Figure 4 showed that higher capability of colony formation for MG-63 cells post transfecting with 50 nM microRNA-210 mimics than post transfecting with 50 nM miRNA control (p<0.05). In conclusion, these results demonstrated that up-regulated microRNA-210 reduced the 5-FU-caused cell death and promoted the colony formation of MG-63 cells in vitro.microRNA-210 mimics ameliorates the hypoxia-induced apoptosis in osteoblast cells in vitro Recent reports showed that hypoxia induces apoptosis in various osteoblast cells. It is the inhibition of the electron transport chain at the inner membrane of the mitochondria which is the most direct induction of hypoxia induced apoptosis. Firstly, the sensitivity of MG-63 osteoblast cells to 5-FU was measured. At normoxia culture conditions, the 5-FU induced low level of apoptosis with a slight time-dependent increasing, while hypoxia costimulates higher level of apoptosis with 5-FU in MG-63 cells (Figure 5A). Next, the activation of caspase 3 (cleaved CASP 3) and of casepase 9 (cleaved CASP 9) was examined by western blot assay, both of which are executive molecules in apoptosis. Results indicated that significant high levels of cleaved CASP 3 and cleaved CASP 9 were induced by a low dose of 5-FU under a hypoxia culture condition rather than under a normoxia condition (Figure 5B and 5C). To recognize the role of microRNA-210 in the hypoxia-induced apoptosis in MG-63 cells, we transfected MG-63 cells with microRNA-210 mimics before subject to hypoxia. Figure 5C demonstrated that from 48 hour post hypoxia, less MG-63 cells post microRNA-210 mimics transfection developed apoptosis (p<0.05 for 48 or 72 hours post treatment). And the western blot assay for cleaved CASP 3 and cleaved CASP 9 reconfirmed the inhibition of microRNA-210 against hypoxia-induced apoptosis; less CASP 3 and CASP 9 were cleaved in the microRNA-210-transfected MG-63 cells (Figure 5E-G). Therefore, the apoptosis promotion and the cleavage of both CASP 3 and CASP 9 was blocked by the microRNA-210 mimics transfection.Part two:Osteoblasts have more projection, more triangular, polygonal or short spindle, outline more clearly, some of the projections and distant cell processes would have a certain connection, the nucleus presents oval or round, can find a clear or 3 nucleoli, under an inverted microscope, amplification rate of the cells is very slow in the first 6d reached a climax, and then continued to show slow growth trend, but the number has decreased in the fourth cell culture alkaline phosphatase staining, we see that occur within a small amount of particulate matter and in the cytoplasm surrounding cells showed dispersed state, in the Economic and cobalt chloride treatment, membrane fuzzy boundaries, and shrunken, also found that more dense cytoplasm, chromatin Margi nation presented morphological of after treatment 7d, can be found the nucleus has been cracking the treated cells were counting process, the number of cells can be found in section 5d to the peak, but still far lower than the conventional oxygen groups, then the number of cells began to rapidly decline. Also found that the cobalt chloride treatment, hypoxia-inducible factor-1? Caspase-3m RNA expression will presented with increasing time of increased (P<0.01), in addition to the initial phase, all other time periods than conventional oxygen group (P<0.01), the conventional oxygen were no significant changes in various time periods, statistically, the difference was not statistically significant (P>0.05); but the expression of Bcl-2m RNA was contrary to the first two, as time progression, will show a downward trend (P<0.01), in addition to the initial stage, the other time periods were lower than conventional oxygen group (P<0.01), while the conventional oxygen group had no significant change (P> 0.05) at each time period. Detected by immunoblotting, cobalt chloride after treatment, the expression of Caspase-3 protein showed a gradual increasing trend (P<0.01), in addition to the initial stage of each time period were higher than the conventional oxygen group (P<0.01), Conventional oxygen was no significant change (P>0.05) at each time period; and Bcl-2 protein expression in contrast with the first two, showing lower (P<0.01), except the initial stage, were significantly lower than conventional oxygen group (P<0.01), the conventional oxygen there was no significant change (P>0.05).[Conclution]1. microRNA-210 is upregulated in a HIF-1?-dependent manner in both human catagmatic tissues and osteoblast cells post hypoxia.2. Throughout microRNA-210 analogue improve microRNA-210 expression protein quantity,and to reduce the function of 50FU on induce cell apoptosis.3. The microRNA-210 upregulation promotes osteoblast cells proliferation in vitro via inhibiting apoptosis.4. MicroRNA-210 analogue suppress caspase-3 and activate caspase 9 and suppress MG-63 cell apoptosis.5. Modified tissue has all the advantages of traditional tissue explant can be obtained in a short time more osteoblasts, the cells were cultured with typical osteoblast function and morphology, in the case of cobalt chloride simulated hypoxia in promotion of apoptosis of bone and produce large amounts of hypoxia inducible factor HIF-1?, this factor can be achieved by Caspase-3 Bcl-2 protein in the regulation and to promote osteoblast apoptosis and cause the formation of nonunion, and this promotion and hypoxia time closely related. |
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