A Study About Differential Expression Of MiR-422A In Peripheral Blood Monocytes From Patients With Postmenopausal Osteoporosis And Also Its Function

Objective:MicroRNA play an important role in the regulation of the genes expression level, but it is still not very clear in etiology of osteoporosis. Given the close relationship between peripheral blood monocytes with osteoporosis, this study focused on the differential expression of miRNA in peripheral blood monocytes form patients with postmenopausal osteoporosis, further on the potential targets of that miRNA, and also its potential biological function.Materials and Methods:1.20postmenopausal Caucasian women recruited were characteristically matched, divided into low bone mineral density group and high bone mineral density group,10cases for each group. Peripheral blood mononuclear cells were seperated by density gradient centrifugation method form extracted peripheral venous whole blood. Then, monocytes were collected by filtering the unwanted cells by means of immunomagnetic cell sorting. The purity were tested by flow cytometry. Following, we used miRNA array chip to analyse the expression profile and obtained miRNAs with differential expression. To verify the virtual expression level of those miRNAs. We, further, carried out RT-qPCR test on the same RNA samples, and fianally confirmed miR-422a. Using similar methods, we also tested miR-422a expression levels of B lymphocytes from the same samples used to extract peripheral blood monocytes including low BMD and high BMD groups.2. Five potential targets of miR-422a were found with bioinformatics methods. For the five potential targets CBL, CD226, IGF-1, PAG1, TOB2, we again made RT-qPCR to detect the actual expression level of them and also analyze the correlation between the expression levels of miR-422a and targets.3. We transfected mimic or inhibitors of mature miR-422a to normal human peripheral blood mononuclear cells by lipofectamine, and hava a preliminary observation on function of miR-422a during the process of peripheral blood monocytes induced to differentiate into osteoclast-like cells.Result:1. For high BMD group of postmenopausal women, lumbar bone mineral density value was (1.128±0.058) g/cm2and lumbar Z value (2.24±0.59), hip bone mineral density value was (1.057±0.101) g/cm2and hip Z value (1.94±0.99).While, for low BMD group, lumbar spine bone mineral density value was (0.826±0.069) g/cm2and lumbar Z value (-0.63±0.64), hip bone density value was (0.725±0.045) g/cm2and hip Z value (-1.04±0.45). Bone mineral density in the hip and spine had significant difference between high BMD and low BMD groups.(P< 0.001) During microRNA array analysis, it tested the expression levels of365miRNAs in RNA of20monocytes samples. While miR-133a and miR-382were both differentially expressed between low BMD and high BMD groups, miR-422a, mir-27b, miR-151and miR-152were in marginal difference between low BMD and high BMD groups. Comparing low BMD with high BMD groups, it was (4.21±2.15vs0.65±0.75, P=0.007) for miR-133a with6.48fold increase, and (2.74±2.18Comparative0.75±0.63, P=0.027) for miR-382with3.65-fold increase. In addition, the marginal ones were (2.91±2.55vs1.24±0.79, P=0.065) for miR-422a,(2.48±2.05vs1.02±0.86, P=0.054) for miR-27b,(1.44±0.87vs.0.86±0.42, P=0.076) for miR-151and (1.32±0.49vs.0.91±0.47, P=0.076) for miR-152. On the foundation of the array analysis, we further verified the four marginal miRNs expression level, since miR-133a and miR-382had been tested in our previous research, by reverse transcription reaction and real-time qPCR in low BMD and high BMD groups. The results showed that:only the expression level of miR-422a has statistical difference between low BMD and high BMD group(1.31±0.54vs0.85±0.27, P=0.029). The other there miRNAs expression levels all had no statistical difference. Specifically,(1.26±0.66vs0.99±0.43, P=0.29) for miR-27b,(1.37±1.08vs0.98±0.41, P=0.30) for miR-151and (1.50±1.11vs0.95.±0.54, P=0.17) for miR-152beween low BMD and high BMD groups. And. the expression level of miR-422a in peripheral blood B lymphocytes had no significant difference between low BMD and high BMD groups (P =0.31).2. With bioinformatic tools, we selected the following five potential targets:CBL, CD226, IGF1, PAG1and TOB2. However, the expression level of those five gene in postmenopausal women peripheral blood monocytes had no statistical difference between low BMD and high BMD groups. Specifically, CBL (4.82±5.43vs2.62±3.27, P=0.29), CD226(2.79±5.40vs.2.29±3.72, P=0.81), IGF1(1.22±1.39vs.4.92±.78, P=0.11), PAG1(2.65±3.78vs.2.88±5.43, P=0.92) and TOB2(1.37±2.44vs.4.96±7.99, P=0.19). What’s more, there had no statistical difference, although all they were in negative correlation between the expression of miR-422a and that of each potential traget gene.(P>0.05)3. On the foundation of peripheral blood monocytes differentiated into osteoclast-like cells, we transfected mimic and inhibitor of mature miR-422a into monocytes. Depending on the interventions, subjects were divided into six groups, specifically, group A(control group), group B(differentiation group), group C (differentiation+miRNA mimic negative control group), group D(differentiation+miRNA mimic group), group E (differentiation+miRNA inhibitor negative control group), and group F (differentiation+miRNA inhibitor group). Then culture cells until8th day, to investige under microscope the differentiation status. The results for each group were as follows. For group A(control group), it had no significant osteoclast-like cells; group B(differentiation group), a certain number of osteoclast-like cells. For group C (differentiation+ miRNA mimic negative control group), it also had a certain number of osteoclast-like cells, but no statistical difference compared with group B. For group D(differentiation+miRNA mimic group), it had a larger number of osteoclast-like cells generated, also having statistically significant difference compared with group C. For group E (differentiation+miRNA inhibitor negative control group), it had a certain number of osteoclast-like cells, also no statistical difference compared with group B. For group F (differentiation+miRNA inhibitor group), it had a smaller number of osteoclast-like cells, having statistically significant difference compared with group E.Conclusion:1. miR-422a is a potential monocyte-specific biomarker for postmenopausal osteoporosis, which may play an important role in etiolgy of postmenopausal osteoporosis.2. CBL, CD226, PAG1, IGF-1and TOB2are potential tragets of miR-422a in postmenopausal osteoporosis peripheral blood monocytes, which provides a reference for further study on control mechanism of miR-422a.3. MiR-422a may play a positve role in regulating the process of peripheral blood mononuclear cells differentiated into osteoclast-like cells.