| Background:Degenerative Disc Disease(DDD) has been widely recognized as the main cause of low back pain. More and more evidence has proved that the intervertebral disc degeneration is associated with various proinflammatory cytokines and metabolites.In recent years, large numbers of studies have shown that MicroRNA (miRNA) plays an important role in the occurance and development of most diseases including degenerative disc disease. There is evidence that the disc degeneration is related with inflanmmatory factors, but the exact relationship between them reaches no consistent conclusion. In addition, there are few researches about analysis in the perspective of inflammatory pathways. Although we understand they are related to each other, target genes have not been found to improve intervertebral disc cells in inflammation, as a lot of such researches show, lacking practical significance. MicroRNA-146a is one of the short non-coding RNA, a kind of factor, regulating gene expression after transcription mediation involved in many biological processes, and a growing number of studies have demonstrated that MicroRNA-146a plays an important role in inflammatory response.Based on the previous researches above, our study purposes to study the regulatory mechanism of MicroRNA-146a in degenerative disc diseases in vitro and vivo test, and to identify the exact way that MicroRNA-146 and TRAF/NF-κB pathway interact with each other, expecting to find a new way to cure intervertebral disc degeneration disease which is considered as the incurable disease. Our study proposes that overexpression of MicroRNA-146a may ameliorate inflammation on disc degeneration by inhibition of TRAF6/NF-κB pathway. MicroRNA-146a may be used as a new target for treatment of intervertebral disc degeneration disease.Objectives:The aim of this study is (1) to illustrate the expression level of microRNA-146a in patients with intervertebral disc degeneration and identity the relationship between microRNA-146a and intervertebral disc degeneration; (2) to investigate the correlations among MicroRNA-146a、TRAF6 and NF-κB, analyze the exact mechanism of microRNA-146a in the occurrence and development of intervertebral disc degeneration in vitro and identify whether inflammatory pathway plays a role in it.Materials and methods:1. Sixty-three patients from Shandong Provincial Hospital between May 2013 and March 2014 are divided into three groups. Group one is a group of patients with intervertebral disc degeneration, lumbar degenerative diseases 21 cases (12 males, 9 females; aged 18-30, an average of 25.6years). Group two is a group of patients with spinal disorders, a total of 21 cases (11 males, 10 females; aged 18-31, an average of 26.1 years). Group three is the control group, 21 cases healthy check-up crowd. (13 cases of males, 8 females; aged 18-32,an average of 26.7years). There are no significant differences among the three groups in age, sex ratio (P >0.05), We compared microRNA-146a、NF-κB、TNF-α and the level of IL-1 in patients’peripheral blood mononuclear cells of the control group, the group of patients with intervertebral disc degeneration and other spinal disorders by quantitative RT-PCR.All the results are analyzed by SPSS 18.0 and Excel 2007 software for statistical analysis. The experimental data is expressed as mean±standard deviation,and differences between groups are analyzed by group t test, P < 0.05 defined as statistically significantly different.2. A nucleus pulposus cell line is used in this study (HNPC4800, bought from American Type Culture Collection, ATCC), with containing 10% fetal bovine serum,100 u, 100 u/ml/ml penicillin streptomycin of DMEM (Dulbecco’s minimum essential medium, DMEM) medium, at 37 ℃, 5% C02 incubate cultivation in the box. Carrier system is composed of carrier composition and packaging components:MicroRNA-146a mimic (analog) and MicroRNA-146a blank plasmid. As a carrier of plasmid, in addition to providing exogenous gene insertion site, they still have the important components of integration of viral replication and report gene; PHelper 1.0,pHelper 2.0 for packaging plasmid, provides the structure of the protein and virus packaging need membrane proteins. According to different experimental processing factors, to do the following grouping, NP cells received different intervention treatments. Cells in MicroRNA-146a group were given NP MicroRNA-146a carrier of siRNA sequences intervention; cells in Negative Control group (NC group )were given MicroRNA-146a blank plasmid; cells in the lipopolysaccharide group (LPS group) is intervented with siRNA sequences carrier cells for LPS stimulation; cells in Blank cells group (BL group) were not receive any intervention, only under the condition of the cultivation of the same culture observation; All cells were set at the time of processing experiment after three holes, N = 3. Human nucleus pulposus cells(NP cells) were transfected, and test transfection rate was measured. LPS (10 microns)was used to stimulate inflammation in NP cells qRT-PCR was used to detect mRNA expression level of microRNA-146a in the NP cells. Western blot was used to detect protein expression of TNF-α, TRAF6 and NF-κB gene in NP. Experiment is repeated three times. The computer is used to scan the results of X-ray film and BandScan image analysis software 5.0 is used to read the gray value of each protein bands on the film, to serve as an internal beta actin, purpose strip with ck band ratio as the relative expression of target protein, using SPSS18.0 statistical software analysis and processing, all experimental data expressed as mean±standard deviation. Comparison between multiple groups are analyzed by single factor analysis of variance (One Way ANOVA), and the comparison between the two groups are analyzed by two independent samples t test, with P < 0.05 for the statistically significant difference.Results:1. In patients with intervertebral disc degeneration, microRNA-146a expression was 3.122±2.132 mol/L,significantly higher than the other two groups,and there is obvious difference (P < 0.05). When comparing the control group to the group of patients with spinal disease, there was no significant difference (P > 0.05). In terms of the expression level of NF-κB in three groups, patients with intervertebral disc degeneration expression was 137.52±11.45 mg/L,significantly higher than the other two groups, and there is obvious difference (P < 0.05). While comparing the control group with the group of patients with spinal disease, there was no significant difference (P > 0.05). In terms of the expression level of TNF-a in patients with intervertebral disc degeneration,it is 257.12±10.43 mg/L,significantly higher than the other two groups,and obthere is obvious difference (P < 0.05). While compareing the control group with those of other group of patients with spinal disease, there was no significant difference (P > 0.05). And comparing the expression level of IL-1 in three groups, there is no significant difference (P > 0.05). All the data above indicates that the expression of microRNA-146a is significantly lower in the gourp of patients with intervertebral disc degeneration than the other two, while the expression of NF- κB and TNF-α is obviously higher in the gourp of patients with intervertebral disc degeneration than the other two.2. Linearization miR-146a mimic carrier and oligo synthesis of DNA in the proper reaction system,after connection transformation,extraction with recombinant plasmid of NP-siRNA sequences, the sequencing results as shown in figure, clearly indicates that the constructed recombinant plasmid conforms to requirements. Carrier of MicroRNA-146a mimic with Enhanced Green Fluorescent Protein (Enhanced Green Fluorescent Protein, EGFP) report gene, packaged into carrier particles after infected cells, the cells express EGFP, inverted fluorescence microscope, infected cells with Green fluorescence. Through a series of preliminary screening experiment conditions,the results showed that in 6 orifice, cell number 1 x 105 / hole, when the transfection cell alignment was 30%～50%,plural (MOI) multiplicity is of infection, infection =50, polybrene 5 ug/mL can obtain higher efficiency of infection.RNA displays 3 stripes, with the brightness 28s >18s > 5s, as shown in Figure 4.The total RNA is of great integrity. The results of nucleic acid protein detector test showed that samples’ OD260/OD280 values are all from 1.8 to 2.0, showing that the total RNA are not degradated and conform to the requirements of the experiment.Results of the real time PCR show that the decreased expression levels of miRNA-146a correlates with the increased expression levels of TRAF6 and NF-κB.Beta actin is used as internal reference through western blot analysis between groups of protein expression. To measure expression of TRAF6 and NF-κB of each group, the expression of the molecue from the blank group was defined as the 100%.TRAF6 and NF-κB expression rate (%) = 100% * (intervention group expression quantity/blank group expression), computing the relative expression level among each group. Relatively high protein concentration in the LPS group are shown, illustrating the correlation exists between them. Inhibition of microRNA-146a stimulated the expression of TRAF6 and the NF-κB, showing negative correlation among them.Overexpression of MicroRNA-146a may significantly reduce the level of inflammation cell molecues in NP cells induced by LPS and reduce the level of TRAF6 and NF- kB.Conclusion:1. The expression level of MicroRNA-146a may correlate with the inflammation of NP cells.2. Ovexpression of MicroRNA-146a may ameliorate the inflammation of NP cells.3. The expression level of key molecues in the TRAF6/ NF-κB pathway correlates with the expression level of MicroRNA-146a.4. MicroRNA-146a may regulate the inflammation of NP cells through the key molecues in the TRAF6/ NF-κB pathway.5. Overexpression of MicroRNA-146a may ameliorate inflammation on disc degeneration by inhibition of TRAF6/NF-κB pathway. MicroRNA-146a may be used as a new target for treatment of intervertebral disc degeneration disease. |
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