长链非编码RNA Kcnq1ot1促进成骨细胞分化和抑制破骨细胞分化

Kcnq1ot1 promotes osteogenic differentiation and suppresses osteoclast differentiation

目的探究长链非编码RNA Kcnq1ot1对成骨细胞分化和破骨细胞分化的调控作用。方法运用荧光实时定量PCR技术分别在卵巢去势(双侧卵巢切除,n=8)、鼠尾悬吊(后肢悬空,n=14)以及自然衰老(正常饲养至18月龄,n=8)引起的骨质疏松小鼠以及各自对照小鼠(n=6)股骨组织中,小鼠前成骨细胞系MC3T3-E1向成骨细胞分化过程中以及小鼠骨髓源巨噬细胞BMMs和小鼠单核巨噬细胞系RAW264.7向破骨细胞分化过程中检测lnc-Kcnq1ot1、骨钙素(Bglap)、Runt相关转录因子2(Runx2)、碱性磷酸酶基因(Alp)、骨涎蛋白(Bsp)、活化T-细胞核因子c1(Nfatc1)、基质金属蛋白酶9(Mmp9)、组织蛋白酶K(Ctsk)和破骨细胞相关受体(Oscar)的表达水平;运用两对特异性以慢病毒为载体的短发夹RNAs(Lv-shRNAs)或小干扰RNAs(siRNAs)在MC3T3-E1、BMMs和RAW264.7细胞诱导分化过程中沉默lnc-Kcnq1ot1,随后运用荧光实时定量PCR技术检测lnc-Kcnq1ot1、成骨相关基因(Bglap、Alp)和破骨相关基因(Ctsk、Oscar)的表达;运用ALP染色检测碱性磷酸酶活性;运用抗酒石酸磷酸酶染色检测抗酒石酸磷酸酶活性。运用核浆分离联合荧光实时定量PCR技术检测lnc-Kcnq1ot1亚细胞定位。结果与对照相比,lnc-Kcnq1ot1在卵巢去势、鼠尾悬吊以及自然衰老引起的疏松股骨组织中表达显著降低(P<0.05);在MC3T3-E1向成骨细胞分化过程中表达增多(P<0.05);在小鼠BMMs和RAW264.7向破骨细胞分化过程中表达显著降低(P<0.05)。在MC3T3-E1细胞中,沉默lnc-Kcnq1ot1抑制Bglap和Alp的表达(P<0.05),并减弱成骨诱导剂引起的成骨细胞分化;在小鼠BMMs和RAW264.7细胞中,沉默lnc-Kcnq1ot1促进Ctsk和Oscar的表达(P<0.05),并加重RANKL诱导的破骨细胞分化。核浆定位显示lnc-Kcnq1ot1主要定位在MC3T3-E1和RAW264.7细胞核内。结论lnc-Kcnq1ot1促进成骨细胞分化和抑制破骨细胞分化,可能成为骨质疏松症的一个潜在治疗靶点。

Objective To investigate the regulatory role of long non-coding RNA Kcnq1ot1 in osteoclast differentiation,osteogenic differentiation and osteoporosis.Methods The expression of lnc-Kcnq1ot1,Bglap,Runx2,Alp,Bsp,Nfatc1,Mmp9,Ctsk and Oscar were detected by real-time quantitative PCR(qRT-PCR)in the femoral bones from mouse models of postmenopausal osteoporosis(ovariectomized mice,n=8),disuse osteoporosis(induced by tail suspension,n=14)and agerelated osteoporosis(18-month-old mice,n=8),and also in MC3T3-E1 cells during osteoblast differentiation and in murine bone marrow-derived macrophages(BMMs)and RAW264.7 cells during osteoclast differentiation.MC3T3-E1 cells with lnc-Kcnq1ot1 knockdown by lentivirus infection were induced to differentiate into osteoblasts using osteogenic induction medium,and the expression of lnc-Kcnq1ot1,Alp and Bglap was detected with qRT-PCR and ALP activity was assessed with ALP staining.BMMs and RAW264.7 cells were transfected with siRNAs targeting lnc-Kcnq1ot1 and stimulated with RANKL and/or M-CSF,and the expression of lnc-Kcnq1ot1,Ctsk and Oscar was detected by qRT-PCR,and TRAP activity was assessed by TRAP staining.The subcellular localization of lnc-Kcnq1ot1 in MC3T3-E1 and RAW264.7 cells was determined using cell fractionation followed by qRT-PCR.Results The expression of lnc-Kcnq1ot1 was significantly upregulated during osteoblast differentiation but downregulated in the bone tissues of osteoporotic mice and during osteoclast differentiation(P<0.05).Silencing lnc-Kcnq1ot1 obviously decreased the expression of Bglap and Alp(P<0.05)and attenuated osteogenic medium-induced osteoblast differentiation.Knockdown of lnc-Kcnq1ot1 also promoted the expression of Ctsk and Oscar(P<0.05)and aggravated RANKL-induced osteoclast differentiation.The results of cell fractionation and qRT-PCR demonstrated that lnc-Kcnq1ot1 was located mainly in the nuclei of MC3T3-E1 and RAW264.7 cells.Conclusion Our data demonstrate that lnc-Kcnq1ot1 promotes osteogenic differentiation and alleviates osteoclast differentiation,suggesting the potential of lnc-Kcnq1ot1 as a therapeutic target against osteoporosis.