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 lncKcnq1ot1 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.
Conclusions: 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.
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lncKcnq1ot1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。
Keywords: Kcnq1ot1; osteoclastogenesis; osteogenesis; osteoporosis.