The Study On MiR-17-5p Modulates Osteogenic Differentiation And Cell Proliferation Of MSCs In Non-traumatic Osteonecrosis Of Femoral Head

Objective:To observe the expression profile differences of major miRNAs in bone mesenchymal stem cells (MSCs) between non-traumatic osteonecrosis (ON) patients and osteoarthritis (OA) patients. Identify key miRNAs that play important role in non-traumatic osteonecrosis pathological process.Methods:After review a large number of research articles, we found10miRNAs play important roles in osteoblastic differentiation, adipose differentiation and cell proliferation: miR-20a, miR-17-5p, miR-27a-3p, miR-96-5p, miR-124-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p. With approval from the ethics committee of Union Hospital and written informed consents, Bone marrow samples were obtained from non-traumatic osteonecrosis patients (n=20) and OA patients (n=10) undergoing surgery between April2008and July2010at Wuhan Union Hospital, Huazhong University of Science and Technology. MSCs from the bone marrow were separated by centrifugation. MSCs samples of two groups were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).Results:Expression levels of miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p in non-traumatic osteonecrosis patients’ MSCs were significantly lower than that of OA patients (p<0.05). Expression levels differences of miR-96-5p, miR-124-3p between non-traumatic osteonecrosis patients’ MSCs samples and OA samples were not significant (p>0.05).Conclusion:miR-20a, miR-17-5p, miR-27a-3p, miR-125b-5p, miR-133a, miR-143, miR-155-5p, miR-199a-5p might modulate key physiological processes like osteoblastic differentiation, adipose differentiation and cell proliferation play important roles in non-traumatic osteonecrosis pathological process. Objective:The expression level of miR-17-5p in non-traumatic osteonecrosis patients’ MSC is significant lower than that in the control group. The aim of this part of study is to predict the target of miR-17-5p through computational target prediction and then verify it by using luciferase reporter assay. Beside that the expression levels of the target mRNA in non-traumatic osteonecrosis patients’MSC samples and in HMSC-bm osteogenic differentiation will be observed.Methods:Targetscan version6.2and Pictar were used to predict the miR-17-5p targets. Real-time PCR was used to detected the expression level of SMAD7in non-traumatic osteonecrosis patients’MSC samples. We set up a experimental group and a control group, HMSC-bm in experimental group was treated by BMP-2in order to promotes osteogenic differentiation while the HMSC-bm in control group received no treatment. At0,1,2,4,6days after the treatment real-time PCR was used to detected the expression levels of miR-17-5p and SMAD7in both groups. miR-17-5p mimics and miR-17-5p inhibitor were used to regulate miR-17-5p expression level. We use real-time PCR and western blot to observe the SMAD7expression level and Smad7protein concentration when miR-17-5p expression level changed. At last the luciferase reporter assay was carried out to decide if SMAD7a target of miR-17-5p.Result:SMAD7was found to be a potential target of miR-17-5p by using computational target prediction, combined the physiological function of which, we tend to believe miR-17-5p modulates non-traumatic osteonecrosis patients’ MSCs friction by targeting SMAD7. Real-time PCR showed the expression levels of SMAD7and miR-17-5p in non-traumatic osteonecrosis patients’MSC samples are negative correlated (R2=0.5440, p=0.0002). The expression levels of miR-17-5p in HMSC-bm of experimental group were significant higher than that in the control group since the first day after BMP-2treatment, the expression levels of SMAD7in HMSC-bm of experimental group were significant lower than that in the control group since the second day after BMP-2treatment (p<0.05). The expression level of SMAD7and Smad7protein concentration in miR-17-5p mimics group is remarkable lower than that in the negative control group while they are remarkable higher in miR-17-5p inhibitor(p<0.05). Luciferase reporter assay showed miR-17-5p direct target on SMAD7and inhibit its expression.Conclusion:miR-17-5p bind to partially complementary sites in the3’UTR of SMAD7, down-regulates its expression level, though this miR-17-5p modulates the hMSCs function in non-traumatic osteonecrosis pathological process and in HMSC-bm osteogenic differentiation. Objective:To investigate the mechanism of miR-17-5p upregulates proliferation and osteoblastic differentiation of HMSC-bm.Methods:Using miR-17-5p mimics and miR-17-5p inhibitor regulates miR-17-5p expression levels in HMSC-bm, set up negative control as well. After4h of transfection, we use BMP-2(100ng/ml) induce osteoblastic differentiation.6days later, using qRT-RCR to measure the expression levels of RUNX2and COL1A1, using ALP stain to observe ALP expression, using CCK-8kit to assess cell number to observe cell proliferation. To prove the up-regulate of osteoblastic differentiation and cell proliferation of HMSC-bm was caused by low expression level of SMAD7, we transfected pcDNA3.1-SMAD7to up-regulate the expression level of SMAD7to see if the effects reduced or overturned.Result:During HMSC-bm osteoblastic differentiation induced by BMP-2, expression levels of RUNX2and COL1A1, expression of ALP, cell proliferation in miR-17-5p mimics group were significant higher than that in the control group (p<0.05). Expression levels of RUNX2and COL1A1, expression of ALP, cell proliferation in miR-17-5p inhibitor group were significant lower than that in the control group (p<0.05). After transfected pcDNA3.1-SMAD7to increase the expression of SMAD7, the effects above were partly reduced.Conclusion:miR-17-5p can up-regulate osteoblastic differentiation and cell proliferation of HMSC-bm trough down-regulating SMAD7.