The Effect Of MiR-148a On Regulating Osteoclast Differentiation And Abnormal Bone Mass Of Patients With Systemic Lupus Erythematosus

Section One:The expression level and effects of hsa-miR-148a in osteoclast precursor cells of untreated systemic lupus erythematosus (SLE) patientsAims:To separate and purify CD14+peripheral blood mononuclear cells (PBMCs) from untreated systemic lupus erythematosus patients and normal controls respectively; to investigate the expression level of hsa-miR-148a in CD14+PBMCs of untreated systemic lupus erythematosus patients. To study the relationship between hsa-miR-148a expression and bone mineral density (BMD) in untreated systemic lupus erythematosus patients.Methods:We recruited31Chinese young women. Among them,16are diagnosed as SLE without glucocorticoids therapy history and the other15are normal controls.We first separated the CD14+PBMCs by the method of density gradient centrifugation, and selected those with highly purified CD14+PBMCs using immunomagnetic cell sorting. Then we compared the hsa-miR-148a expression level of CD14+PBMCs between subgroups by using real-time quantitative PCR (qRT-PCR) technology. The isolated CD14+PBMCs from each subgroup were differentiated into osteoclasts in the presence of recombinant human-macrophage colony stimulating factor (rh-MCSF) and human receptor activator of nuclear factor-κB ligand (hRANKL).The growth and function of osteoclasts were assessed by TRAP staining, TRAP activity and TRAP/NFATcl mRNA levels detected by real-time quantitative PCR. CD14+PBMCs from SLE patients were transfected with anti-miR-148a. After the transfection of CD14+PBMCs with anti-miR-148a, osteoclast differentiation was induced by the addition of rh-MCSF and hRANKL. The growth and function of osteoclasts were assessed by TRAP staining, TRAP activity and TRAP/NFATcl mRNA levels detected by real-time quantitative PCR.Results:(1)Untreated SLE patients had lower BMD compared with normal controls, while the hsa-miR-148a expression level increased in CD14+PBMCs from untreated SLE patients.(2) The number of TRAP+multinucleated giant cells, TRAP activity and TRAP/NFATcl mRNA expression increased more obviously in cultured CD14+PBMCs from lupus patients compared with those from normal controls.(3) Transfection of anti-miR-148a in CD14+PBMCs from SLE patients inhibited the formation of TRAP+multinucleated giant cells, also reduced TRAP activity and the mRNA levels of TRAP/NFATcl.Conclusion:The increased hsa-miR-148a expression of CD14+PBMCs in untreated SLE patients contributed to the enhanced osteoclastogenesis and lower BMD in lupus patients. Section two:The effects of mmu-miR-148a on the bone metabolism of ovariectomized (OVX) miceAims:To explore the effects of mmu-miR-148a on the bone metabolism of OVX mice by studing the changes of BMD,micro-CT analysis, histomorphometric analysis, osteoblast/osteoclast-related factors expression level and serum biochemical makers of bone turnover detection.Methods:The36six-week-old female C57BL/6mice were randomly divided into six groups:OVX+antagomiR-148a group, OVX+PBS group, OVX+negative control (mut-antagomir) group,sham operation (SHAM)+antagomiR-148a group, SHAM+PBS group and SHAM+negative control (mut-antagomir) group.We used the OVX to mimic menopause. AntagomiR-148a was used to inhibit the expression of mmu-miR-148a in vivo in both OVX and SHAM groups.To explore the effects of antagomiR-148a on the metabolism of OVX mice:Dual-energy X-ray absorptiometry (DXA) was used to detect the BMD in neck of femur, micro-CT was used to detect bone volumn (BV/TV)and Trabecular Thickness (Tb.Th) in proximal tibia diaphyses, histomorphometric analysis was used to assess bone formation and resorption parameters. Meanwhile, the mRNA levels of osteoblast/osteoclast related factors (NFATcl,TRAP and ALP) were measured by real-time quantitative PCR, the serum biochemical makers of bone turnover (TRAP and B-ALP) levels were measured by enzyme-linked immunosorbent assay (ELISA).Results:(1)antagomiR-148a inhibited mmu-miR-148a expression in vivo.(2) The inhibition of mmu-miR-148a expression in SHAM mice increased the bone density, improved trabecular bone volumn (BV/TV) and Trabecular Thickness (Tb.Th), while decreased bone formation and resorption parameters, the mRNA levels of osteoblast/osteoclast related factors and the serum biochemical makers of bone turnover.(3) Compared to SHAM mice, OVX mice had decreased bone density, BV/TV and Tb.Th, while elevated bone formation and resorption parameters, the mRNA level of osteoblast/osteoclast related factors and the serum biochemical makers of bone turnover. AntagomiR-148a treatment in OVX mice may correct these changes.Conclusion:Silencing of mmu-miR-148a in mice decreased bone resorption, resulting in increased BMD and improved bone microstructure, hindering the progress of osteoporosis. Section three:Study on the effect of mmu-miR-148a in osteoclast differentiationAims:To investigate the role of mmu-miR-148a in osteoclast differentiation of RAW264.7cells and mouse bone marrow monocytes.Methods:The mmu-miR-148a expression profile was detected by qRT-PCR during the inductive agents-induced osteoclastogenesis of RAW264.7cells and mouse bone marrow monocytes.We constructed the expression vector of mmu-miR-148a by using the pSilencer4.1-CMV puro vector. The expression vector was named "pre-miR-148a".After the transfection of RAW264.7cells and mouse bone marrow monocytes with pre-miR-148a, osteoclast differentiation was induced by the addition of the inductive agents.The growth and function of osteoclasts were assessed by TRAP staining. The TRAP/NFATcl mRNA levels were detected by real-time quantitative PCR. Then, we transfected RAW264.7cells and mouse bone marrow monocytes with2’-O-methyl antisense inhibitory oligoribonucleotides (anti-miR-148a).The growth and function of osteoclasts were assessed by TRAP staining. The TRAP/NFATcl mRNA levels were measured as described above.Results:(1)The expression of mmu-miR-148a in RAW264.7cells and mouse bone marrow monocytes was detected by qRT-PCR after treatment with the inductive agents and increased progressively with time.(2) High expression of mmu-miR-148a was obtained in RAW264.7cells and mouse bone marrow monocytes after transfection with the expression vector of mmu-miR-148a constructed by using pSilencer4.1-CMV puro vector.(3)Compared with control cells,mmu-miR-148a overexpression in RAW264.7cells and mouse bone marrow monocytes promotes the formation of TRAP+multinucleated giant cells.Transfection of pre-miR-148a promoted the mRNA levels of TRAP/NFATcl.(4) Transfection of anti-miR-148a in RAW264.7cells and mouse bone marrow monocytes inhibited the formation of TRAP+multinucleated giant cells, also reduced the mRNA levels of TRAP/NFATcl.Conclusions:Mmu-miR-148a played a positive regulatory role in osteoclast differentiation.