Down-regulation of miR-340-5p promoted osteogenic differentiation through regulation of runt-related transcription factor-2 (RUNX2) in MC3T3-E1 cells

Abstract

Diabetic osteoporosis (DOP) is a chronic complication of diabetes in the skeletal system. High level of miR-340-5p may be harmful to the bone formation. In this study, the DOP model of rats was successfully established via streptozotocin (STZ) and ovariectomy (OVX) treatment. It was manifested by reduced body weight, insulin level, alkaline phosphatase (ALP) activity, and osteocalcin (OCN) and collagen-I expressions, as well as increased concentration of fasting blood glucose. Moreover, we found that miR-340-5p expression was increased while runt-related transcription factor-2 (RUNX2) was decreased in femurs. Furthermore, the effects of miR-340-5p on osteogenic differentiation (OD) in high glucose (HG)-treated MC3T3-E1 cells were explored. Exposure to OD and HG contributed to elevated miR-340-5p level. Inhibition of miR-340-5p enhanced ALP level, calcium deposition, and OCN, collagen-I and RUNX2 levels. On the contrary, miR-340-5p overexpression reversed these promotional effects. Luciferase assay indicated that RUNX2 may be a target gene of miR-340-5p. Moreover, RUNX2 deficiency decreased miR-340-5p inhibition-induced ALP activity, calcium accumulation and OCN, collagen-I, RUNX2 levels. In short, the above findings revealed that inhibition of miR-340-5p facilitated osteogenic differentiation through regulating RUNX2 in MC3TC-E1 cells, which provided targeted therapeutic strategies for the treatment of DOP.

Keywords: Mir-340-5p; mc3t3-e1 cells; osteogenic differentiation; runx2.

Figure 1.

The rat model of diabetic osteoporosis was established. (a-c) Diabetes was mediated by STZ in rats. Subsequently, bilateral OVX was carried out. The rats were fed for 8 weeks, and body weight was detected once a week. Then the animals were fasted overnight, and fasting blood glucose and blood insulin contents were assessed using the commercial kits. Finally, all the animals were sacrificed, and rat femur tissues were collected for following experiments. (d) Detection of BMD. (e) ALP staining was performed in femur tissues. Scale bar = 100 μm. (f) Measurement of miR-340-5p expression by qRT-PCR. (g) Evaluation of OCN, collagen-I, and RUNX2 levels with immunoblotting. β-actin was used as the internal reference. STZ, streptozotocin; OVX, ovariectomy; BMD, bone mineral density; ALP, alkaline phosphatase; OCN, osteocalcin. Data were expressed as means ± SD (N = 6 per group). ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus sham group; **P < 0.01 and ***P < 0.001 versus OVX group

Figure 2.

High glucose inhibited osteogenic differentiation in MC3T3-E1 cells. osteoblast differentiation was induced in cells, and cells were treated with HG for 14 days. (a) ALP activity was measured with the kit. (b) Treated cells were stained with Alizarin Red and quantified accordingly. Scale bar = 50 μm. (c) The level of miR-340-5p was detected by qRT-PCR. (d) OCN, collagen-I, and RUNX2 protein levels were assessed with immunoblotting. OD, osteogenic differentiation; HG, high glucose; IOD, integrated optical density. Results were presented as means ± SD (N = 3 in each group). ^$$$P < 0.001 versus control group; ^&&P < 0.01 versus OD group

Figure 3.

Down-regulation of miR-340-5p promoted osteogenic differentiation in MC3T3-E1 cells. (a) Cells were infected with lentivirus-based miR-340-5p inhibitor (anti-miR-340-5p), pre-mir-340 (miR-340-5p) or their negative controls (anti-NC and NC) for 72 h. Subsequently, osteoblast differentiation was induced, and cells were treated with HG for 14 days. (b) ALP activity was measured with the kit. (c) Treated cells were stained with Alizarin Red and quantified accordingly. Scale bar = 50 μm. (d) OCN, collagen-I, and RUNX2 protein levels were assessed with immunoblotting. NC, negative control. Data were expressed as means ± SD (N = 3 per group). ^aaaP < 0.001 versus NC group; ^bbbP < 0.001 versus anti-NC group; ^&P < 0.05 and ^&&P < 0.01 versus OD group; ^!P < 0.05, ^!!P < 0.01 and ^!!!P < 0.001 versus OD+HG+anti-NC group

Figure 4.

Up-regulation of miR-340-5p blocked osteogenic differentiation in MC3T3-E1 cells. MiR-340-5p was overexpressed (miR-340-5p) via lentivirus infection. Subsequently, osteoblast differentiation was induced, and cells were treated with HG for 14 days. (a) ALP activity was measured with the kit. (b) Treated cells were stained with Alizarin Red and quantified accordingly. Scale bar = 50 μm. (c) OCN, collagen-I, and RUNX2 protein levels were assessed with immunoblotting. Data were indicated as means ± SD (N = 3 per group). ⁺P < 0.05, ⁺⁺P < 0.01 and ⁺⁺⁺P < 0.001 versus OD+HG+NC group

Figure 5.

RUNX2 was a possible target gene of miR-340-5p. (a) The binding sequences of miR-340-5p and RUNX2 3ʹ UTR (WT or MT). (b) 293 T cells were co-transfected with miR-340-5p mimic or NC mimic and RUNX2 3ʹ UTR (WT) or RUNX2 3ʹ UTR (MT) for 48 h. After co-transfection, cells were lysed, and the luciferase activity was evaluated by the kit. Results were presented as means ± SD (N = 3 in each group). ^^^P < 0.001 versus miR-340-5p mimic+RUNX2 3ʹ UTR (MT) group. WT, wild type; MT, mutant type; UTR, untranslated region

Figure 6.

MiR-340-5p facilitated osteogenic differentiation through regulation of RUNX2 in MC3T3-E1 cells. (a) Cells were infected with lentivirus-based shRUNX2 or NC shRNA for 72 h. The protein expression of RUNX2 was detected by immunoblotting. (b) Cells were co-infected with anti-miR-340-5p or anti-NC and shRUNX2 or NC shRNA for 72 h. Subsequently, osteoblast differentiation was induced, and cells were treated with HG for 14 days. ALP activity was measured with the kit. (c) Treated cells were stained with Alizarin Red and quantified accordingly. Scale bar = 50 μm. (d) OCN, collagen-I, and RUNX2 protein levels were evaluated with immunoblotting. Data were represented as means ± SD (N = 3 per group). ^cccP < 0.001 versus NC shRNA; ^%%P < 0.01 versus OD+HG+anti-NC+NC shRNA group; ^@P < 0.05 and ^@@P < 0.01 versus OD+HG+anti-miR-340-5p+NC shRNA group