MiR-185 Downregulate TGF-β1 Inhibits The Osteoblast Differentiation Of HMSCs

The research backgroundBone defect is common in trauma, bone tumor and other reasons, defect repair is one of the most common bone surgical problems at present, especially the large bone defect, is a major clinical problem facing the bone repair. The current repair are autologous bone, allograft to repair the bone repair and bone repairing in several ways, to repair because of insufficient source and to once again facing the risk defect and infection in the clinical application of autologous, Co.; the main problem is the immune rejection of allogeneic bone is difficult to survive than less; while avoiding the above two kinds of artificial bone the disadvantage, by a large number of clinical application. At present, artificial bone is mainly made of some metal materials, is also faced with many shortcomings, in addition to rejection oxidation defects, the most important is the lack of biological activity and other functions.Now many scientists are trying to find out a new method, the cultivation of artificial biological bone material, with activity in the lab and if successful, will provide a rich bone substitute material for clinical bone defects, the patient is a gospel.At present, research on biological bone material is mainly composed of cells and cell attachment. Cells have embryonic stem cells, mesenchymal stem cells and somatic cells, and cell attachment is generally have bone properties of substances, such as hydroxyl apatite, gel, also has a titanium sheet of our common. Mainly these cells attached to these objects, and then to culture in vitro, let these cells into a tissue and organ cells, in order to achieve organ deletion or functional repair. I often hear about the artificial ear or artificial certain organs, are using this method to.As far as the cultured cells, embryonic stem cells because of the inconvenience, the culture conditions of high, multi-directional differentiation powerful bad control (common laboratory will automatically be differentiated into different cells, such as myocardial cells), coupled with ethical and other reasons, in fact, is rarely used as induction of cell, and adult because of its highly differentiated cells, easy to aging in the culture environment, also rarely used action research. And mesenchymal stem cells, because of its capability of differentiation, taking is convenient (easy own extraction), no ethical reason of constraint, studies and was widely used as artificial material is easy to culture.How in the lab with mesenchymal stem cells into our objective to induce cell, and has the certain function, more and more researchers are interested, and gradually in-depth study, and some have achieved certain results.As mesenchymal stem cells into osteoblasts induced, many researchers have made a lot of work, they found that some genes in the stem cells play a role in bone differentiation in the process of research, such as vascular endothelial growth factor (VEGF), transforming growth factor (TGF) and a few factors, play a very important role in the differentiation process. However, when the researchers further found that, in these factors influence cell at the same time, there is a class of small molecule in the regulation of these genes, these small molecule is miRNA.MicroRNA (miRNA) is a kind of length 19～25 nucleotide noncoding single stranded small RNA molecules involved in regulation of gene transcription, post, generally through the target gene 3’untranslated region of partially complementary combination method to regulate the regulation of gene pair. In the life process, are widely involved in cell proliferation, growth and development, cell differentiation into functional organ formation etc..Play an important role in the regulation of growth and development of organism. Especially in the regulation of stem cell multilineage differentiation, there is a very significant change, and more and more attention.MiRNA is composed of Lee and other developmental processes in 1993 of C.elegans is among the first discovered, they are a class of non coding small RNA molecules, which exist widely in plant and animal body, length of 19～25 nucleotides. The length is about 20nt RNA is a mature body is the final cut into. At first they transcribed in the nucleus of a cell into the primary products, these products are cutting a called RNase, Drosha enzyme precursor, their precursors is a hairpin structure composed of small RNA molecules of approximately 90nt, and the function of exPortin-5 protein, translocation from the nucleus to the cytoplasm was inside, was finally RNase, Dicer further cutting, and then turned into a mature body, mature body combined with other proteins, forming the RISC complex,3’end, and then with the effect on the target gene combination, to inhibit target gene expression, but the new study also found, some of the miRNA can also be combined with target gene promoter District, to promote the objective gene expression, but more than one thousand miRNA have been found so far, most in the purpose of gene 3’combination, to regulate target gene expression.So far, the research on miRNA has made great progress, found that miRNA not only increasing the number, but also in the known a lot of miRNA function has been introduced in detail, in spite of this, but there are still a lot of the function of miRNA is unknown, waiting for the researchers to explore. In the study of miRNA, many researchers have found an important role of some miRNA, such as miR-185 not only on the differentiation of impact, but also on the cells resistant to radiation has a certain role. There are such as miR-21 on the apoptosis of cells have obvious effect, many people assume to be used in the treatment of cancer. There are some small molecules on organ development has a certain function. Because the miRNA under different conditions have different levels of expression, such as cell carcinogenesis, the expression of high and low expression in normal cells, there is now clinical on some miRNA as an index to judge the rehabilitation after healing disease.On the transforming growth factor TGF, experiments have proved that it plays an important role in the process of stem cell differentiation, not only in the liver cells, cardiomyogenic cells play an important role, while in the osteogenic induction also reported. This shows that in the process of transforming growth factor in the life, to the important role of differentiation. However, we know, life is ultimately to the aging process, this Is it right? Shows that in the process of differentiation, transformation factor role has waned, it reminds us of Is it right? What in controlling these factors, and the new miRNA can regulate gene results prompted us, will not be because miRNA the gene function abate? With doubt, we use the miBASE query, found that miR-185 and TGF have a regulatory site, and then query the latest literature found that at present no such reports, more significantly, miR-185 and anti radiation are selected, then we want, if can resist radiation, can promote or inhibit bone cell differentiation Is it right?, as we in the treatment of bone cancer provides a new idea, in view of the above the curious, so we choose miR-185 study.the research contentFirst of all, we cloned from the genomic miR-185, cloned fragments attached to slow virus carrierOn the other, and then together with the packaging plasmid together into the lentiviral packaging cell line 293T cells, packaging into slow virus particles, and then go to the infection of human bone marrow mesenchymal stem cells, obtained through screening stable cell line, proliferation and standby.Then we synthesized interference fragment, miR-185 will be silenced, into human bone marrow mesenchymal stem cells as a reverse control, see the miR-185 into the bone’s role in cell differentiation.At the same time, we pack not containing only miR-185 containing GFP virus as control group, the experiment started.1. Observation of miR-185 in the osteogenesis of HMSCs.Objective:To investigate the result of over expression or inhibition of miR-185 in the osteogenesis of HMSCs.Methods:The construction and transduction of miR185 over expression or inhibition lentivirusplasima. The HMSC were allocated into 3 group and cultured for 20 days.The cell was stained with alizarin red to observe and quantify the osteogenesis.(Figure3-5、Figure3-6)Result:Overexpression of miR-185 inhibits the osteogenesis of HMSCs. Repressing the expression of miR-185 enchanced the osteogenesis of HMSCs. Conclusion:Upregulation of miR-185 inhibits the osteogenesis of HMSCs, dowregulation of miR-185 significantly enhance the osteogenesis of HMSCs.2. Investigation of effection of miR-185 on the activity of ALP of HMSCs.Objective:To investigate over expression or inhibition of miR-185 to the activity of ALP.Method:HMSCs transducted with over-expression or inhibition of miR-185 and the control group were cultured for 14 days. Cells were ALP stained and quantified the OD value.Results:The stain is obvious in inhibition group than overexpression group. OD value is higher in inhibiton group than overexpression group.Conclusion:Overexpression of miR-185 inhibits the activity of ALP. Inhibition of miR-185 enhances the activity of ALP. (Figure3-7、Figure3-8)3.Investigation of effection of miR-185 on the OCN, OSsterix, OPN activity of HMSCs.Objective:To investigate over expression or inhibition of miR-185 to OCN, OSsterix, OPN activity of HMSCs.Method:HMSC transducted with over-expression or inhibition of miR-185 and the control group were cultured for 20 days. RT-PCR quantified the expression of OCN, OSsterix, OPN.Results:the expression of OCN, OSsterix, OPNwere lower in overexpression group than the control group. the expression of OCN, OSsterix, OPNwere higer in inhibition group than the control group. (Figure3-9、Figure3-)Conclusion:Overexpression of miR-185 inhibits the expression of OCN, OSsterix, OPN. Inhibition of miR-185 enhances the expression of OCN, OSsterix, OPN.4. Investigation of effection of miR-185 on the OCN、DMP1, OPN protein expression of HMSCs.Objective:To investigate over expression or inhibition of miR-185 to OCN-. DMP1, OPN protein expressionof HMSCs.Method:HMSC transducted with over-expression or inhibition of miR-185 and the control group were cultured for 20 days. Western-blot quantified the expression ofOCN、DMP1,OPN.Results:the expression OCN、DMP1, OPN protein were lower in overexpression group than the control group. the expression of OCN、DMP1, OPN protein expressionwere higer in inhibition group than the control group. (Figure3-11、 Figure3-12)Conclusion:Overexpression of miR-185 inhibits the expression ofOCN、DMP1, OPN protein e. Inhibition of miR-185 enhances the expression of OCN、DMP1, OPN protein.5. Investigation the relation of miR-185 and TGF-β1Objective:5.Investigation the relation of miR-185 and TGF-β1Method:Targetscan was employed to predict the target gene.TGF-β1 was the target gene.To confirm the prediction,we cloned the TGF-β1 3’UTR, PGL3 including miR-185 binding site, andTGF-β1 3’UTR-mutant. (Figure4-2、 Figure4-3)Results:luciferase assay displayed the significant difference in miR-185 group Conclusion:TGF-β1 is the target gene of miR-185. Statistical analysisStatistical analyses were performed using SPSS version 13.0 (SPSS, Chicago, IL, USA), and values expressed as mean values ± SD. One-way ANOVA or Student’s t-test was used to determine significant differences between groups. Data were considered statistically significant at p< 0.05.