starBase database (starbase.sysu.edu.cn/index.php/) was used to predict the binding site of Kcnq1ot1 and miR-98-5p. miR-98-5p and Tbx5 target site was predicted using TargetScan (targetscan.org/vert_72/).

The MC3T3-E1 mouse pre-osteoblast cell line was sourced from the American Type Culture Collection (cat. no. CRL-2594) and cultured in α-Minimum Essential Medium (cat. no. A1049001) supplemented with ribonucleosides, deoxyribonucleosides, 2 mM L-glutamine and 1 mM sodium pyruvate and 10% FBS (all Gibco; Thermo Fisher Scientific, Inc.) in the absence of ascorbic acid. Cells were cultured at 37˚C in a humidified incubator with 5% CO₂ and the medium was replaced every 2-3 days.

Osteogenic differentiation of MC3T3-E1 cells was induced by incubation with osteogenesis-inducing medium at 80% confluence for 14 days at 37˚C, as previously described (18). The medium consisted of 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 5 mM L-glycerophosphate (Sigma-Aldrich; Merck KGaA), 100 nM dexamethasone (AmyJet Scientific, Inc.) and 50 mg/ml ascorbic acid (Shanghai Aladdin Biochemical Technology Co., Ltd.).

Short hairpin (sh)RNA plasmid for Kcnq1ot1 (sh-Kcnq1ot1; 5'-GCAGAACCAUCGAUGGUGCGU-3'), shRNA targeting Tbx5 (sh-Tbx5; 5'-CGGCUGCUAGUGUCUAUGUUU-3'), shRNA-negative control (sh-NC; 5'-AGUGCUGCGCACGUGUCUCAU-3'), pcDNA3.1(+)/Kcnq1ot1 plasmid (pc-Kcnq1ot1; 5'-GGGGTACCCCAGGTGACAAGGTGCAGGCGC-3'), pcDNA3.1 (5'-AUCUCCGGGGUUUACGUAUAC-3'), miR-98-5p antagomir (antagomiR-98-5p; 5'-AACAAUACAACUUACUACCUCA-3'), antagomiR-NC (5'-UCACAACCUCCUAGAAAGAGUAGA-3'), miR-98-5p agomir (agomiR-98-5p; 5'-UGAGGUAGUAAGUUGUAUUGUU-3') and agomiR-NC (5'-UCGCUUGGUGCAGGUCGGG-3') were constructed by Shanghai GenePharma, Co., Ltd. A final concentration of 100 nM shRNA, pcDNA, antagomir or agomir of miR-98-5p and NC was transfected into MC3T3-E1 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 48 h. At transfection, cells were harvested, and reverse transcription-quantitative PCR (RT-qPCR) was performed to assess the transfection efficiency. On the 7th day of osteogenic differentiation, transfection as aforementioned was performed to maintain the altered gene expression.

ALP activity was measured to assess differentiation of MC3T3-E1 cells. Briefly, cells were seeded into 12-well plates at a density of 4x10⁴ cells/ml 37˚C for 7 days. Subsequently, the cells were fixed in 4% paraformaldehyde for 30 min at room temperature and treated with 0.3 nitro-blue tetrazolium and 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich; Merck KGaA) at room temperature for 2 h. Cells were then washed with deionized water and observed under an inverted light microscope (Nikon Corporation; magnification, x200) at a wavelength of 405 nm.

Total protein was extracted from MC3T3-E1 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was determined using a BCA assay kit (Beyotime Institute of Biotechnology). A total of 30 µg protein/well was resolved using 10% SDS-PAGE and transferred to a PVDF membrane. Subsequently, 5% non-fat milk was used to block the membrane at 37˚C for 1 h, followed by incubation at room temperature for 1 h with primary antibodies as follows: Runt-related transcription factor 2 (RUNX2; 1:1,000; cat. no. 12556; Cell Signaling Technology, Inc.), collagen type I α 1 (COL1A1; 1:1,000; cat. no. ab34710; Abcam), osteopontin (1:1,000, cat. no. ab214050; Abcam), osteocalcin (1:1,000, cat. no. ab133612; Abcam), Tbx5 (cat. no. ab259980; Abcam) and GAPDH (1:2,500, cat. no. ab9485; Abcam). The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,000, cat. no. #5127; Cell Signaling Technology, Inc.) at room temperature for 2 h. The bands were visualized by using an enhanced chemiluminescence (ECL) reagent kit (Shanghai Yeasen Biotechnology Co., Ltd.) and semi-quantified with Image J software (Version 1.49; National Institutes of Health).

Total RNA was extracted from MC3T3-E1 cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). RT was performed to synthesize cDNA from total RNA, using a PrimeScript RT Reagent kit, according to the manufacturer's protocol (Takara Bio, Inc.). A SYBR green PCR Master Mix kit (cat. no. SR1110; Beijing Solarbio Science & Technology Co., Ltd.) was used for cDNA amplification by qPCR in accordance with the manufacturer's instructions on an AFD9600 PCR system (Hangzhou AGS BioTech Co., Ltd.). The primer sequences for PCR were as follows: Kcnq1ot1 forward, 5'-ACTCACTCACTCACTCACT-3' and reverse, 5'-CTGGCTCCTTCTATCACATT-3'; miR-98-5p forward, 5'-ATCCAGTGCGTGTCGTG-3' and reverse, 5'-TGCTTGAGGTAGTAAGTTG-3'; Tbx5 forward, 5'-AAGTAAAGAATATCCCGTGGTC-3' and reverse, 5'-AGACTCGCTGCTGAAAGG-3'; GAPDH forward, 5'-GGGAAACTGTGGCGTGAT-3' and reverse, 5'-GAGTGGGTGTCGCTGTTGA-3' and U6 forward, 5'-CTCGCTTCGGCAGCACATATA-3' and reverse, 5'-ACGCTTCACGAATTTGAGTGTC-3'. The thermocycling conditions were as follows: 94˚C for 60 sec, followed by 40 cycles of 94˚C for 30 sec, 60˚C for 30 sec and 72˚C for 60 sec. The 2^-ΔΔCq method (21) was used to calculate relative gene expression. GAPDH was used as an internal reference for Kcnq1ot1 and Tbx5, while U6 was used as the control gene for miR-98-5p.

The wild-type (Kcnq1ot1-WT or Tbx5-WT) and mutant (Kcnq1ot1-MUT or Tbx5-MUT) 3'-untranslated regions were cloned into a pmirGLO vector (Shanghai GenePharma Co., Ltd.). For luciferase reporter analysis, MC3T3-E1 cells were co-transfected with luciferase reporter vectors, agomiR-98-5p (5'-UGAGGUAGUAAGUUGUAUUGUU-3') and agomiR-NC (5'-UCGCUUGGUGCAGGUCGGG-3') using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h after transfection, the relative luciferase activities were measured by using a Dual-Luciferase Reporter Assay (Promega Corporation) and normalized to Renilla luciferase reporter activity according to the manufacturer's protocol.

An ARS staining kit (GuideChem) was used to detect the formation of mineralized nodules in MC3T3-E1 osteoblasts. Following induction of osteogenesis and cell fixation with 95% ethanol for 20 min at room temperature, the cells were washed with PBS (Shanghai Aladdin Biochemical Technology Co., Ltd.) three times and stained with ARS staining solution for 30 min at room temperature. The cells were observed and images were obtained using a light microscope (magnification, x200).

All experiments were performed independently three times. GraphPad Prism version 6.0 (GraphPad Software, Inc.) was used to analyze the data. Data are presented as the mean ± SD. One-way ANOVA followed by Tukey's post hoc test was used for comparisons between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

A time-dependent increase in the relative activity of ALP was observed during the incubation of MC3T3-E1 osteoblasts in osteogenesis-inducing medium, indicating the occurrence of osteogenic differentiation (Fig. 1A). The protein expression levels of osteogenic differentiation-associated proteins, RUNX2, COL1A1, osteopontin and osteocalcin, were also increased in a time-dependent manner following incubation of MC3T3-E1 cells in osteogenesis-inducing medium (Fig. 1B and C). The expression of Kcnq1ot1 was elevated, and that of miR-98-5p was decreased as the duration of osteogenesis induction increased (Fig. 1D). These results suggested that Kcnq1ot1 expression increased and miR-98-5p expression decreased during osteogenic differentiation.

According to starBase, Kcnq1ot1 was predicted to bind to miR-98-5p (Fig. 2A). A dual luciferase reporter assay showed decreased luciferase activity in MC3T3-E1 cells co-transfected with agomiR-98-5p and Kcnq1ot1-WT compared with that in cells co-transfected with agomiR-98-5p and Kcnq1ot1-MUT, which verified the binding between Kcnq1ot1 and miR-98-5p (Fig. 2B). Furthermore, following successful knockdown of Kcnq1ot1 (Fig. 2C), the expression of miR-98-5p was significantly upregulated compared with the control group (Fig. 2D). These results suggest that Kcnq1ot1 may target and inhibit miR-98-5p in MC3T3-E1 cells.

To determine the role of the interaction between miR-98-5p and Kcnq1ot1 in osteogenic differentiation, MC3T3-E1 cells were transfected with sh-Kcnq1ot1 or co-transfected with sh-Kcnq1ot1 and antagomiR-98-5p. The knockdown effect of antagomiR-98-5p was detected by RT-qPCR (Fig. 3A). miR-98-5p expression was increased by sh-Kcnq1ot1; this was rescued following co-transfection of sh-Kcnq1ot1 and antagomiR-98-5p (Fig. 3B). Kcnq1ot1 knockdown also decreased ALP activity, whereas inhibition of miR-98-5p restored ALP activity (Fig. 3C). Kcnq1ot1 knockdown significantly inhibited the formation of mineralized nodules, which was rescued following co-transfection of sh-Kcnq1ot1 and antagomiR-98-5p in MC3T3-E1 cells (Fig. 3D). Kcnq1ot1 knockdown also significantly inhibited the expression of RUNX2, COL1A1, osteopontin and osteocalcin, whereas antagonizing miR-98-5p in the presence of Kcnq1ot1 knockdown significantly upregulated expression of these proteins (Fig. 3E). These results indicated that antagonizing miR-98-5p may reverse the inhibitory effect of Kcnq1ot1 knockdown on osteogenic differentiation.

TargetScan software was used to predict the binding sites between Tbx5 and miR-98-5p (Fig. 4A). The transfection efficiency of pc-Kcnq1ot1 and agomiR-98-5p was detected by RT-qPCR (Fig. 4B and C). A dual luciferase reporter assay confirmed the results of the TargetScan prediction as significantly decreased relative luciferase activity was observed in MC3T3-E1 cells co-transfected with Tbx5-WT and agomiR-98-5p. Furthermore, MC3T3-E1 cells transfected with Tbx5-WT, agomiR-98-5p and pc-Kcnq1ot1 exhibited increased luciferase activity compared with the Tbx5-WT + agomiR-98-5p group (Fig. 4D). The results of RT-qPCR and western blotting both showed that Tbx5 expression was significantly downregulated by agomiR-98-5p but upregulated following Kcnq1ot1 overexpression (Fig. 4E and F). These results indicated that Kcnq1ot1 may exert a regulatory effect on Tbx5 expression via modulating miR-98-5p.

To determine the role of the interaction between miR-98-5p and Kcnq1ot1, as well as that between Tbx5 and Kcnq1ot1, in osteogenic differentiation and mineralization, agomiR-98-5p or sh-Tbx5 were transfected into MC3T3-E1 cells overexpressing Kcnq1ot1. The transfection efficiency of sh-Tbx5 was detected by RT-qPCR (Fig. 5A). Tbx5 expression was decreased in both the pc-Kcnq1ot1 + agomiR-98-5p and pc-Kcnq1ot1 + sh-Tbx5 groups (Fig. 5B). Meanwhile, the relative increase in ALP activity following Kcnq1ot1 overexpression was decreased following miR-98-5p overexpression or Tbx5 knockdown (Fig. 5C). Kcnq1ot1 overexpression significantly increased mineralization of MC3T3-E1 cells, and this was decreased following transfection with agomiR-98-5p or sh-Tbx5 (Fig. 5D and E). These results suggested that overexpressing miR-98-5p or knocking down Tbx5 may reverse the promotive effect of Kcnq1ot1 overexpression on osteogenic differentiation and mineralization.

The expression levels of osteogenic differentiation-associated proteins, RUNX2, COL1A1, osteopontin and osteocalcin, were detected following co-transfection of pc-Kcnq1ot1 with agomiR-98-5p or sh-Tbx5. The expression levels of differentiation-associated proteins were increased by Kcnq1ot1 knockdown and inhibited by co-transfection of pc-Kcnq1ot1 and agomiR-98-5p or pc-Kcnq1ot1 and sh-Tbx5 (Fig. 6). These findings suggested that miR-98-5p overexpression and Tbx5 knockdown may reverse the promotive effect of Kcnq1ot1 overexpression on osteogenic differentiation-associated protein expression levels.