[Kcnq1ot1 promotes osteogenic differentiation and suppresses osteoclast differentiation]

Abstract

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.

Keywords: Kcnq1ot1; osteoclastogenesis; osteogenesis; osteoporosis.

1

长链非编码RNA Kcnq1ot1在小鼠疏松股骨组织中的表达 Expression of lnc-Kcnq1ot1 in different mouse models of osteoporosis. A: OVX and shamoperated mice (n=5). B: TS and control mice (n=5). C: 8- and 18-month-old mice (n=5). The data are presented as Mean ± SD from at least triplicate experiments. ^*P < 0.05 vs Sham or 8 months.

2

长链非编码RNA Kcnq1ot1在MC3T3-E1细胞向成骨分化过程中的表达 Expression of lnc-Kcnq1ot1 during osteoblast differentiation of MC3T3-E1 cells. The data are presented as Mean±SD from at least triplicate experiments. ^*P < 0.05 vs 0 d.

3

长链非编码RNAKcnq1ot1在破骨细胞分化过程中的表达 Expression of lnc-Kcnq1ot1 during osteoclast differentiation of BMMs and RAW264.7 cells. The data are presented as Mean±SD from at least triplicate experiments. ^*P < 0.05 vs 0 d.

4

沉默lnc-Kcnq1ot1对成骨细胞分化的影响 Knockdown of lnc-Kcnq1ot1 inhibits osteoblast differentiation. MC3T3-E1 cells were infected with lentivirus-mediated sh-lncKcnq1ot1 or sh-control, and subsequently stimulated with osteogenic differentiation medium for 7 days. The RNA levels of lncKcnq1ot1 (A), Alp (B) and Bglap (C) were determined by qRT-PCR. D: Representative images of ALP staining. The data are presented as Mean±SD from at least triplicate experiments. ^*P < 0.05 vs shRNA-Ctrl; ^#P < 0.05 vs shRNA-Ctrl+7 d.

5

沉默lnc-Kcnq1ot1对小鼠BMMs向破骨细胞分化的影响 Knockdown of lnc-Kcnq1ot1 promotes osteoclast differentiation of BMMs. BMMs were transfected with si-lnc-Kcnq1ot1 or siNC every three days, and simultaneously stimulated with or without M-CSF and/or RANKL for 8 days. The expression of lncKcnq1ot1 (A), Ctsk (B) and Oscar (C) was determined by qRT-PCR. D: Representative images of TRAP staining (Original magnification: ×100). The data are presented as Mean±SD from at least triplicate experiments. ^*P < 10.05 vs NC; ^#P < 0.05 vs NC+8 d.

6

沉默lnc-Kcnq1ot1对RAW264.7细胞向破骨细胞分化的影响 Knockdown of lnc-Kcnq1ot1 promotes osteoclast differentiation of RAW264.7 cells. RAW264.7 cells were transfected with silnc-Kcnq1ot1 or si-control every two days, and simultaneously stimulated with RANKL for 4 days. The expression of lnc-Kcnq1ot1 (A), Ctsk (B) and Oscar (C) was determined by qRT-PCR. D: Representative images of TRAP staining (×100). The data are presented as Mean±SD from at least triplicate experiments. ^*P < 0.05 vs NC; ^#P < 0.05 vs NC+4 d.

7

核浆分离确定lnc-Kcnq1ot1亚细胞定位 Determination of the subcellular localization of lnc-Kcnq1ot1. RNA was extracted from the nuclei or cytoplasm of MC3T3-E1 (A) and RAW264.7 (B) cells. The RNA level of lnc-Kcnq1ot1, Neat1 (nuclear retained), and Gapdh (cytoplasm retained) was determined by qRT-PCR. The data are presented as Mean±SD from at least triplicate experiments.