MALAT1/miR-320a in bone marrow mesenchymal stem cells function may shed light on mechanisms underlying osteoporosis

Abstract

Introduction: Growing evidence supports the involvement of long noncoding RNAs (lncRNAs) in bone metabolism and diseases. This study aims to investigate the involvement of the lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the pathological process of osteoporosis and the effects of MALAT1 on regulation of BMSC differentiation through competitive endogenous RNA (ceRNA) mechanisms.

Material and methods: The expression of MALAT1 and miR-320a was determined using RT-PCR in bone tissue derived from female SD (Sprague Dawley) rats with osteoporosis. Immunohistochemical (IHC) staining was used to evaluate the expression of neuropilin-1 (NRP-1) and β-catenin. Bone marrow mesenchymal stem cells (BMSCs) were divided into 4 groups: control, NC (negative control), MALAT1 siRNA, and miR-320a mimics. Forty-eight hours later, the effect of MALAT1 on the miR-320a expression, proliferation and osteogenic differentiation of BMSCs was investigated. Two weeks later, the cell activity, alkaline phosphatase (ALP) activity, and mRNA expression of Osterix and Runx2 were evaluated. Three weeks later, alizarin red staining of calcified nodules and Western blot analysis of the expression of β-catenin, NRP-1, osteocalcin (OCN), and osteopontin (OPN) were performed.

Results: Downregulated MALAT1or upregulated miR-320a expression inhibited the activity and osteogenic differentiation of BMSCs, resulting in low ALP activity and NRP-1 expression, fewer calcified nodules, decreased mRNA levels of Osterix and Runx2, and inhibited expression of NRP-1, OCN, and OPN. MALAT1 silencing did not decrease the protein level of β-catenin in the cytoplasm but suppressed that in the nucleus.

Conclusions: Downregulated MALAT1 and upregulated miR-320a expression play an important role in the pathological process of osteoporosis, via inhibition of the osteogenic differentiation of BMSCs.

Keywords: bone marrow mesenchymal stem cells; competitive endogenous RNA; metastasis-associated lung adenocarcinoma transcript; miR-320a; osteoporosis.

Figure 1

A – HE staining results of the tibial metaphyseal bone obtained from ovariectomy and sham surgery rats (n = 24). B – Images from micro-CT scanning of the proximal tibia in osteoporotic and intact SD rats; a significant reduction in the number of trabeculae and a decrease in the continuity of bone trabeculae can be seen in the osteoporosis group. C, D – Expression levels of miR-320a and MALAT1 were determined by qRT-PCR, and significantly reduced expression of MALAT1 and overexpression can be seen in Figure 1 C and 1 D. E – Protein expression of NRP-1 and β-catenin was determined by immunohistochemistry. Scale bar = 20 µm (*p < 0.05, **p < 0.01 compared with control)

Figure 2

A – MALAT1 level in BMSCs was determined by qRT-PCR after transfection with the vector, MALAT1 siRNA-1, MALAT1 siRNA-2 and MALAT1 siRNA-3 for 48 h. MALAT1 siRNA-1 showed the best transfection efficiency and can be used for further study. The MALAT1 level (B) and the miR-320a level (C) in BMSCs were determined by qRT-PCR after transfection with the vector, MALAT1 siRNA-1, and miR-320a mimics for 48 h. D – Activity of BMSCs was observed via a CCK-8 assay after osteogenesis was induced with osteogenic induction medium for 14 days. The groups in the CCK-8 test were divided into the following groups: 1) Control + ordinary medium: blank control + culture media; 2) Control: blank control + osteogenic induction medium; 3) NC: vectors + osteogenic induction medium; 4) MALAT1 siRNA: MALAt1 siRNA1 transfection + osteogenic induction medium; 5) miR-320a mimics: miR-320a mimics transfection + osteogenic induction medium (*p < 0.05, **p < 0.01 compared with control)

Figure 3

A – ALP activity of osteoblasts was determined by alkaline phosphatase kit 14 days after osteogenesis induction in BMSCs (ALP: alkaline phosphatase), and the activity of ALP in the MALAT1 siRNA group and miR-320a mimics group was significantly decreased (p < 0.01). The expression of Osterix (B) and RUNX2 (C) was determined by qRT-PCR after osteogenesis was induced in BMSCs for 14 days (RUNX2: Runt-related transcription factor 2). D – Alizarin red staining was conducted to observe the formation of calcified nodules under an inverted microscope after BMSC osteogenesis was induced for 21 days; a large number of calcified nodules were observed in the control group and NC group compared with the MALAT1siRNA group and miR-320a mimics group, and the number of calcified nodules in the cells of the MALAT1 siRNA group and miR-320a mimics group was significantly decreased. Scale bar = 20 µm (*p < 0.05, **p < 0.01 compared with control)

Figure 4

A – Protein level of β-catenin in the cytoplasm and nucleus was determined by Western blot; β-catenin expression was observed to be significantly decreased in MALAT1 siRNA and miR-320a mimics groups in the nucleus. B – The ratio of β-catenin in the nucleus to that in the cytoplasm was calculated and compared between groups (**p < 0.01 compared with control). C–F – Western blot analysis was used to identify the osteogenic differentiation of BMSCs in different groups, by evaluation of the protein levels of NRP-1, OCN, and OPN (NRP-1 – neuropilin-1; OCN – osteocalcin; OPN – osteopontin) (*p < 0.05, **p < 0.01 compared with control)