Study On The Mechanism Of Differentiation Of Osteoclasts By Polygonatum Polysaccharide Based On The Regulation Of MiR-1224

Background and Aims: Osteoporosis is characterized by the degeneration of bone microstructure,the decrease of bone mass and the increase of bone brittleness.Osteoporosis is a systemic bone disease that is prone to fracture.The incidence of osteoporosis is high in the elderly,especially in postmenopausal women.Nowadays,our country is stepping into the aging of population.Osteoporosis has become a severe social problem,which poses a great challenge to our health care system.At present,the drugs(bisphosphates,calcitonin,estrogen and so on)for the prevention and treatment of osteoporosis have serious side effects and are difficult to be adopted and accepted by the majority of patients.Therefore,it will be a great challenge for medical workers to find a new drug with small side effects to prevent the development of osteoporosis.Traditional Chinese medicine and its natural active ingredients have become a hot spot in the research of anti-osteoporosis drugs because of their wide range of materials and small side effects.Polygonatum flavescens polysaccharide is an important active component of medicine,has the functions of anti-aging,anti-inflammation,regulating blood lipids,regulating immune function and anti-tumor and so on.The research on the anti-osteoporosis of Polygonatum sibiricum polysaccharide is only reported by our research group in the world.Our previous study found that Polygonatum sibiricum polysaccharide can prevent and cure osteoporosis in ovariectomized rats by inhibiting osteoclast differentiation and promoting osteoblast differentiation.However,the specific molecular mechanisms of Polygonatum sibiricum polysaccharide in osteoclasts and osteoblasts,especially the detailed molecular mechanisms of inhibiting osteoclast differentiation,need to be further explored.miRNA is a non-coding small molecule,which plays an important role in gene expression and regulation,has become a new research hotspot in the field of disease and prevention in recent years.In previous studies,we found that Polygonatum sibiricum polysaccharide could induce differential expression of miRNAs in osteoclasts when inhibiting the differentiation of osteoclasts,especially miR-1224.Therefore,based on previous studies,we will further silence and over express miR-1224 in osteoclast precursors,and observe the effect of osteoclast differentiation.In addition,the effects of miR-1224 and Polygonatum sibiricum polysaccharide on osteoclast differentiation and its molecular mechanism were observed.Content: 1.We constructed the overexpression and silencing vector of mir-1224 and observed its effect on the activity of osteoclast precursor cells;2.We overexpressed and silenced miR-1224 in osteoclasts and observed its effect on osteoclast differentiation;3.We over-expressed and silenced mir-1224 in osteoclasts and observed its effect on the expression of specific genes in osteoclasts;4.The effect of Polygonatum sibiricum polysaccharide on the differentiation of osteoclasts and the expression of specific genes in osteoclasts expressing miR-1224 was observed;5.The effect of Polygonatum sibiricum polysaccharide on Hippo signaling pathway in osteoclasts expressing mir-1224 was observed.Methods: 1.Raw264.7 cells were transfected with lentivirus to find the best MOI(Multiplicity of Infection)value.The cells were grouped as follows: control group,negative transfection group,pre-miR-1224 overexpression group,anti-miR-1224 group;2.The Raw264.7 cell proliferation and expression of miR-1224 were detected;3.After inducing osteoclast differentiation,TRAP staining and the expression of osteoclast related genes were detected;4.Western blot was used to detect the expression of Limd1 protein and related protein in Hippo signaling pathway.Results: 1.CCK-8 analysis showed that lentivirus transfection into Raw264.7 cells had no effect on cell proliferation;2.After 6 days of osteoclast induction,TRAP staining and qRT-PCR analysis showed that miR-1224 could promote osteoclast differentiation and osteoclast related gene expression;3.CON group,pre-miR-1224 group and pre-miR-1224 group were induced to osteoclasts for 6 days.TRAP staining and qRT-PCR analysis showed that Polygonatum sibiricum polysaccharide could inhibit osteoclasts differentiation and the expression of osteoclast-associated genes by suppressing the function of miR-1224;4.WB detection showed that Polygonatum sibiricum polysaccharide could inhibit the expression of Limd1 protein and other related proteins in Hippo signaling pathway suppressing the function of miR-1224.