目的 探讨miR-214-5p通过靶向调控PTEN的表达对破骨分化和骨质疏松的影响。方法 选取2019-03至2020-05在空军军医大学唐都医院外科进行脊柱手术的骨质疏松患者20例(骨质疏松组),同时选取非骨质疏松症患者20例为对照组,抽取两组骨髓血。将miR-214-5pmimics(miR-214-5pmimics组)、miR-214-5p空质粒(miR-214-5pvector组)、shZFAS1载体(sh PTEN组)、shZFAS1空载体(sh vector组)转染到骨质疏松患者细胞,对照组细胞不进行任何转染处理。采用RT-qPCR 检测骨髓单个核细胞中的miR-214-5p和PTEN mRNA及 Raw264.7单核巨噬细胞中NFaTc1、MMP9、TRAP、CTSK的mRNA水平;通过TRAP染色检测TRAP染色的阳性率;通过Western blot 检测PTEN、NFATc1、AKT和PI3K蛋白水平。结果 骨质疏松组miR-214-5p水平的表达明显高于对照组(P＜0.001),骨质疏松组PTEN的表达显著低于对照组(P=0.009);miR-214-5pmimics组中TRAP表达阳性率明显高于对照组和miR-214-5p vector组(P＜0.001);sh PTEN组中TRAP表达阳性率明显高于对照组和sh vector组(P＜0.001);miR-214-5p mimics组中的NFaTc1、MMP9、TRAP和CTSK水平显著高于对照组(P<0.001);sh PTEN组中的NFaTc1、MMP9、TRAP和CTSK水平显著高于对照组(P<0.001);与miR-214-5p mimics组相比,miR-214-5pmimics+PTEN组中TRAP表达阳性率显著降低(P＜0.001)。结论 骨质疏松症患者的骨髓单个核细胞中miR-214-5p表达上调,miR-214-5p可以调控PTEN表达,过表达miR-214-5p可能通过靶向PTEN促进破骨细胞分化。
Abstract
Objective To investigate the effect of miR-214-5p on osteoclastic differentiation and anti osteoporosis through PTEN expression. Methods Twenty patients with osteoporosis who were admitted in Tangdu Hospital of Air Force Military Medical University from March 2019 to May 2020 were chosen as osteoporosis group (n=20). miR-214-5p mimics (miR-214-5p mimics group), miR-214-5p empty plasmid (miR-214-5p vector), shZFAS1 vector (sh PTEN group) and shZFAS1 empty vector (SHNC group) were transfected into the cells. The control group (n=20)cells were not treated with any transfection. The mRNA levels of miR-214-5p and PTEN in bone marrow mononuclear cells and NFATc1, MMP9, TRAP and ctsk mRNA in RAW264.7 monocyte macrophages were detected by RT qPCR; the positive rate of TRAP staining was detected by TRAP staining; the protein levels of PTEN, NFATc1, Akt and PI3K were detected by Western blot. Results The expression of miR-214-5p in osteoporosis was significantly higher than that in control group (P< 0.001), and the expression of PTEN in osteoporosis was significantly lower than that in control group (P=0.009); the percentage of TRAP positive cells in miR-214-5p mimics group was significantly higher than that in control group and miR-214-5p vector group (P< 0.001); the percentage of TRAP positive cells in SH PTEN group was significantly higher than that in control group and sh PTEN group The levels of NFATc1, MMP9, TRAP and ctsk in miR-214-5p mimics group were significantly higher than those in control group (P<0.001); the levels of NFATc1, MMP9, TRAP and ctsk in SH PTEN group were significantly higher than those in control group (P<0.001); the percentage of TRAP positive cells in miR-214-5pmimics+PTEN group was significantly lower than that in miR-214-5p mimics group (P< 0.001). Conclusions The expression of miR-214-5p in bone marrow mononuclear cells of patients with osteoporosis is up-regulated, miR-214-5p can regulate the expression of PTEN, and overexpression of miR-214-5p may promote osteoclast differentiation by targeting PTEN.
关键词
miR-214-5p /
PTEN /
破骨分化 /
骨质疏松
Key words
miR-214-5p /
PTEN /
osteoclastic differentiation /
osteoporosis
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