标题:LncRNA-H19靶向miR-654-5p对人骨髓间充质干细胞成骨分化的促进效应研究
作者:蒋励,许耀,王世龙,汤超亮,陈文钧 复旦大学附属华山医院骨科,上海 200040
卷次:
2021年32卷14期
【摘要】 目的 探讨长链非编码RNA H19 (LncRNA-H19)靶向miR-654-5p对人骨髓间充质干细胞成骨分化的促进效应。方法 选取 20只 SPF级 5~6周龄近交系BALB/c小鼠,分离及培养骨髓间充质干细胞,采用随机数表法分为阴性对照组、LncRNA-H19转染组和LncRNA-H19抑制组,LncRNA-H19转染组转染LncRNA-H19慢病毒载体、LncRNA-H19沉默质粒。采用MTT法检测细胞增殖,测定各组碱性磷酸酶活性(ALP),荧光素报告基因实验验证 LncRNA-H19靶向miR-654-5p作用,Western blot检测 BMP-2、OCN蛋白表达。结果 阴性对照组、LncRNA-H19转染组和LncRNA-H19抑制组细胞培养 0 h、24 h、48 h及 72 h时OD值比较差异无统计学意义(P>0.05);LncRNA-H19转染组ALP活性值为(13.11±1.92) U/gprot,明显高于阴性对照组的(9.65±1.12) U/gprot和LncRNA-H19抑制组的(5.30±1.20) U/gprot,差异均有统计学意义(P<0.05);LncRNA-H19转染组miR-654-5p-3'UTR调节的荧光素酶活性为0.612±0.099,明显低于阴性对照组的1.010±0.100和LncRNA-H19抑制组的1.453±0.102,差异均有统计学意义(P<0.05);各组miR-654-5p-突变型 3'UTR调节的荧光素酶活性比较差异无统计学意义(P>0.05);LncRNA-H19转染组BMP-2、OCN蛋白相对量为 1.102±0.102、1.242±0.088,明显高于阴性对照组的 0.493±0.100、0.372±0.094和 LncRNA-H19抑制组的 0.202±0.082、0.182±0.092 (P<0.05)。结论 LncRNA-H19通过靶向miR-654-5p促进人骨髓间充质干细胞向成骨分化,可能与调控MMP-2、OCN蛋白表达有关。
【关键词】 长链非编码RNA H19;miR-654-5p;靶向作用;人骨髓间充质干细胞;成骨分化
【中图分类号】 R329.2+1 【文献标识码】 A 【文章编号】 1003—6350(2021)14—1769—04
Promoting effect of LncRNA-H19 targeting miR-654-5p on osteogenic differentiation of human bone marrowmesenchymal stem cells.
JIANG Li, XU Yao, WANG Shi-long, TANG Chao-liang, CHEN Wen-jun. Department ofOrthopedic Surgery, Huashan Hospital Affiliated to Fudan University, Shanghai 200040, CHINA
【Abstract】 Objective To investigate the promoting effect of long non-coding RNA H19 (LncRNA-h19) target-ing miR-654-5p on osteogenic differentiation of human bone marrow mesenchymal stem cells. Methods Twenty SPF5 to 6 week old inbred BALB/c mice were selected to isolate and culture bone marrow mesenchymal stem cells. Accord-ing to random number table, the mice were divided into the negative control group, LncRNA-H19 transfection group(transfected with LncRNA-H19 lentiviral vector), LncRNA-H19 inhibition group (transfected with LncRNA-H19 silenc-ing plasmid). MTT assay was used to detect cell proliferation and alkaline phosphatase activity (ALP), and the luciferasereporter gene assay was used to verify the effect of LncRNA-H19 targeting miR-654-5p. Western blot was used to de-tect the protein expression of MMP-2 and OCN. Results There was no significant difference in OD value between neg-ative control group, LncRNA-H19 transfection group and LncRNA-H19 inhibition group at 0 h, 24 h, 48 h and 72 h afterculture (P>0.05). The ALP activity of LncRNA-H19 transfection group was (13.11±1.92) U/gprot, which was significant-ly higher than (9.65±1.12) U/gprot of negative control group and (5.30±1.20) U/gprot of LncRNA-H19 inhibition group(P<0.05). The luciferase activity regulated by miR-654-5p-3'UTR in LncRNA-H19 transfection group was 0.612 ±0.099, which was significantly lower than 1.010±0.100 in negative control group and 1.453±0.102 in LncRNA-H19 inhi-bition group (P<0.05). There was no significant difference in the luciferase activity regulated by miR-654-5p-mutant3'UTR among the groups (P>0.05). The relative amounts of BMP-2 and OCN protein in LncRNA-H19 transfectiongroup were 1.102±0.102 and 1.242±0.088, which were significantly higher than 0.493±0.100 and 0.372±0.094 in nega-tive control group or 0.202 ± 0.082 and 0.182 ± 0.092 in LncRNA-H19 inhibition group (P<0.05). Conclusion Ln-cRNA-H19 promotes the osteogenic differentiation of human bone marrow mesenchymal stem cells by targetingmiR-654-5p, which may be related to the regulation of MMP-2 and OCN protein expression.
【Key words】 Long non-coding RNA H19; miR-654-5p; Targeting; Human bone marrow mesenchymal stemcells; Osteogenic differentiation
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