RNA Synthesis by DNA Methyltransferase 1 siRNA Transfection with Cationic Liposomes into Osteoblastic Progenitor Cells Based on SiO² Nanomagnetic Beads for Proliferation and Differentiation.

Abstract

DNA methylation regulated gene expression is important for osteoblast proliferation and differentiation during bone remodeling and its deregulation leads to the development of osteoporosis. DNA methyltransferase 1 (DNMT1) is an important regulator of DNA methylation. To explore the effect and mechanism of differential expression of DNMT1 in osteoblast precursor cells, DNMT1 siRNAs were designed and synthesized to interfere with DNMT1 expression in the osteoblast precursor cells, MC3T3E1 (Clone 24; MC3T3E1-24). The expression of the target gene, DNMT1, and osteogenic differentiation indicators osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB). MTT assay was used to detect the effect on cell proliferation. Alkaline phosphatase (ALP) activity and alizarin red staining were used to detect the effect of DNMT1 on osteogenic differentiation. Hematoxylin and eosin (H&E) staining was used to detect the morphological changes in MC3T3E1-24 cells. Twenty-four hours following the transfection of MC3T3E1-24 cells with DNMT1 siRNA using cationic liposomes, DNMT1 mRNA and protein levels decreased significantly (P <0.001 for both). The reduced expression of DNMT1 promoted the OPG mRNA and protein expression (P <0.05), increased the ratio of OPG to RANKL (P <0.05), inhibited the expression of RANKL (P <0.01) without affecting the RANKL gene expression (not significant, P >0.05). The reduced expression of DNMT1 also promoted the proliferation of osteoblast precursor cells. In addition, ALP activity test and alizarin red staining showed that reduced expression of DNMT1 resulted in an increase in OPG/RANKL ratio and promoted the differentiation of the precursor cells. The cultured cells were found to have fibroblast-like appearance, and calcium nodules were observed after 7 days of conventional culture. In addition, to improve the efficiency of RNA extraction and save time, a type of silica nanomagnetic beads was used in the early stage of this study to extract RNA and assist qPCR detection of the target genes. The results showed that the magnetic beads could effectively extract RNA from the cells. In conclusion, low expression of DNMT1 affects proliferation and maturation of osteoblasts by upregulating OPG and OPG/RANKL ratio.