| Background Osteoporosis is the fourth globaly harmful disease for human beings,which is characterized by progressive bone loss and skeleton fragility increase.Osteoporosis always initiates and develops without any obvious symptoms,and is often observed by physical examination or occurance of complications.Fragility fracture is one of the main and most severe complications for osteoporosis,and near half of the osteoporotic patients have at least one fragility fracture throughout their life.Fracture of long bone and central bone always leads to immobility,paralysis and even death,which will bring enormous economic and mental burden to families and society.Therefore,it is very important to diagnose osteoporosis at early stage for prevention of severe bone loss and occurance of fragility fracture.At present,bone density examination based on dual energy X-ray absorptiometry(DXA)is a commonly accepted gold standard for osteoporosis diagnosis in clinic.However,expensive cost,immobility and potential risk of radiative damage limit the usage of DXA in clinic.Thus,it is essential to explore a new diagnostic method with high sensitivity,strong specificity and convenience for diagnosing osteoporosis.Circualting micro RNA is a sort of micro RNA exsiting in body fluid,which is always stably expressing in vivo and maintaining stability for long times in vitro.Compared with traditional serum bone turnover markers,circulating micro RNA has the advantages of higher sensitivity,stronger specificity and better stability.It is reported that dysregulated expression of circulating micro RNA is always closely related with pathological physiology of diseases.Therefore,it is potential for circulating micro RNA to become molecular diagnostic biomarker in clinic.At present,as biomarkers,circulating micro RNAs have shown good performance on diagnosing tumor and other system diseases.Although previous researches reported dysregulated spectrum of circulating micro RNAs in osteoporosis and other skeleton diseases,its role and diagnostic performance estimation on osteoporosis diagnosis is needed to explore.Purpose This research aims at revealing changes of circulating micro RNAs spectrum during osteoporosis development,and determining role and performance of circulating micro RNAs on diagnosing osteoporosis via high throughput micro RNA microarray study,large sample size training cohort study,independent validation cohort study and animal model study.Methods 1.Evaluation of sample representativeness.The basic information,BMD and T score of samples in biobank was analyzed by constituent ratio analysis,box-plot analysis and correlation analysis.The analytic results were compared with investigative results from national epidemiological survey on osteoporosis to determine the sample representativeness.2.Primary screening of differentially expressed circulating micro RNAs.Samples in subgroups were equally pooled for microarray analysis.Differentially expressed circulating micro RNAs among healthy control,osteopenia and osteoporosis were screened by high throughput micro RNA microarray.And criteria of expression,tendency and conservation were used for further screening.3.Role and performance estimation of circulating micro RNAs on diagnosing osteoporosis.Expressions of circulating micro RNAs from healthy control,osteopenia and osteoporosis(288 in total)were analyzed by real-time RT-PCR.The tendency and performance on osteoporosis diagnosis of circulating micro RNAs were determined by box-plot analysis and ROC curve analysis.4.Estimation of combinative diagnostic model.Significantly contributed micro RNAs were screened by Logistic regression model and combinative diagnostic models were developed using Logistic regression formula and simple linear formula.The tendency and performance on osteoporosis diagnosis of circulating micro RNAs were determined by box-plot analysis and ROC curve analysis.The advantages of circulating micro RNA based combinative diagnostic models were determined through comparison with traditional bone turnover markers.5.Independent validation of performance of circulating micro RNAs and their combinative diagnostic markers on osteoporosis diagnosis.Expressions of circulating micro RNAs from healthy control,osteopenia and osteoporosis(160 in total)were verified by real-time RT-PCR.The tendency and performance on osteoporosis diagnosis of circulating micro RNAs were verified by box-plot analysis and ROC curve analysis.The advantages of circulating micro RNA based combinative diagnostic models were verified through comparison with traditional bone turnover markers.6.Estimation of circulating micro RNAs for early-warning of osteoporosis and surveillance of therapeutic effect.Osteoporosis of human beings and anti-osteoporosis therapy were respectively simulated by aging model,ovarietomized model and cytotherapy model.BMD of femurs from mouse with differet ages,rats with different times after ovarietomy and rats received cytotherapy was analyzed by micro CT scanner.Expressions of circulating micro RNAs were analyzed by real-time RT-PCR.The tendency and performance of circulating micro RNAs on osteoporosis warning and therapeutic surveillance were determined by box-plot analysis and ROC curve analysis.Results 1.There were 678 samples included in biobank.The proportions of osteoporosis in full samples,male samples and female samples were similar with the investigative results from national epidemiological survey on osteoporosis.Additionally,there were some relationship between BMD and age,BMD and height,BMD and weight,BMD and BMI,which were in accordance with the results from national epidemiological survey on osteoporosis.2.Twenty-two gradually and differentially expressed circulating micro RNAs among healthy control,osteopenia and osteoporosis were selected via high throughput micro RNA microarray and criteria.Among them,thirteen micro RNAs were up-regulated and nine micro RNAs were down-regulated.3.Ten gradually and differentially expressed circulating micro RNAs among healthy control,osteopenia and osteoporosis were further screened through large sample size training cohort study.These micro RNAs were able to diagnose osteoporosis independently,whereas their AUC,sensitivity,specificity and accuracy were not satisfied.4.Compared with individual circulating micro RNA,combinative diagnostic models significantly improved the AUC,sensitivity,specificity and accuracy.Diagnostic performance of Logistic regression formular based Index1 was much higher than that of simple linear formular based Index2.Compared with serum t PINP and ?-CTx,Index1 significantly improved the diagnostic performance.Additionally,the performance of Index1+P+C was comparable with that of Index1.5.The performance of individual circulating micro RNA on osteoporosis diagnosis was verified by independent validation cohort study,and the AUC,sensitivity,specificity and accuracy were not satisfied.Compared with individual circulating micro RNA,combinative diagnostic models significantly improved the AUC,sensitivity,specificity and accuracy.The diagnostic performance of Index1 was much better than that of Index2,serum t PINP and ?-CTx.Additionally,the performance of Index1+P+C was comparable with that of Index1. 6.The BMD values of femurs from the mouse with 2-month-old,8-month-old,12-month-old and 16-month-old were gradually decreased,meanwhile the serum miR-199a-5p was simultaneously down-regulated.The BMD values of femurs from the sham rats,OVX-1-day rats,OVX-2-weeks rats and OVX-8-weeks rats were gradually decreased,meanwhile the serum miR-199a-5p was simultaneously down-regulated.The bone loss was significantly declined,osteoblastic activity was increased and osteoblastic activity was decreased in OVX rats after BMMSCs administration,meanwhile the serum miR-199a-5p was simultaneously up-regulated.Conclusion 1.The samples included in the biobank,with good clinical representativeness,are able to reflect the epidemiological features of osteoporosis.2.Expressive spectrum of circulating micro RNAs was dysregulated among healthy participants,osteopenia participants and osteoporosis patients.Circulating micro RNA based molecular biological diagnostic method is able to diagnose osteoporosis and shows the potential as substitute of traditional bone turnover markers.3.There is no improvement of performance on osteoporosis diagnosis to bring traditional bone turnover markers in combinative diagnostic model.4.It is potential of circulating micro RNAs for osteoporosis warning and therapeutic surveillance. |
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