Study On The Mechanism Of MALAT1 And MiRNA-200b Regulating High Concentration Of Melatonin To Induce Autophagy In Osteoblasts

Objective: Melatonin is an amine hormone.It involves many physiological processes,including sleep,anti-inflammatory function,gastrointestinal physiology,core body temperature regulation,blood pressure,cardiovascular function,immunomodulatory properties,antioxidant defense,detoxification,reproduction,apoptosis inhibition,and bone physiology.Melatonin is also produced in other organs besides the pineal gland,such as the gastrointestinal tract,retina and bone marrow.Mouse and human bone marrow cells have the ability to synthesize melatonin from scratch,and the concentration of melatonin in the bone marrow of mice is about twice that of peripheral blood.Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may affect bone metabolism.Some authoritative reports have pointed out that the prospect of using melatonin in orthopedic treatment lies in osteoporosis and idiopathic scoliosis(IS).IS refers to the 3D rotation of the spine without potential vertebral abnormalities or obvious physical defects.Although it affects about 4% of the population,the etiology and pathogenesis of IS remains poorly understood,mainly due to genetic heterogeneity of the disease and a lack of historical research.Some studies have found that high concentrations of melatonin can inhibit the development of experimental IS.Clinical application of melatonin can also prevent some patients with IS with low melatonin levels from developing malformation.Experiment has proved that low concentration of melatonin on osteoblast of melatonin receptor,promote the proliferation of osteoblast.With the increase of concentration of melatonin,when achieves a high concentration,these pathways are suppressed.The extracellular calcium ions into the cell,causes the intracellular calcium overload,endoplasmic reticulum stress response,by inhibiting the endoplasmic reticulum Bcl-2 pathways,inducing osteoblast apoptosis.Apoptosis is closely related to autophagy.Autophagy is a major metabolic process of eukaryotic cells to repair and degrade damaged macromolecular proteins and organelles.Moreover,autophagy plays a housekeeping role in the elimination of old organelles,misfolded proteins and damaged molecules,and in the recovery of limited nutrients and oxygen.Sometimes,autophagy ACTS as an emergency protection against apoptosis.In both cases,autophagic death constitutes an alternative route to programmed cell death.Methods: Part i: Western blot and immunofluorescence technique were used to detect the effect of high concentration of melatonin on autophagy in human osteoblast cell line(h FOB1.19).Cck-8 technique was used to detect the effect of high concentration of melatonin on the activity of osteoblasts.The expression levels of MALAT1 and mir-200 b in osteoblasts induced by high concentration of melatonin were detected by rt-qpcr.Part ii: bioinformatics was used to predict the target relationship between mir-200 b and MALAT1.Double luciferase reporter genes were used to verify the binding sites.Part iii:transient transfection,rt-qpcr and western blot were used to verify the regulatory relationship between mir-200 b and MALAT1,and its influence on the autophagy function of osteoblasts.Results: Cck-8 experiment shows that high concentration of melatonin can inhibit the proliferation of osteoblasts.High concentration of melatonin increases the level of autophagy in osteoblasts.Immunofluorescence revealed an increased number of autophagosomes in osteoblasts induced by high concentration of melatonin.In osteoblasts induced by high concentration of melatonin,MALAT1 was highly expressed while mir-200 b was poorly expressed,showing an inverse relationship.Bioinformatics analysis showed that the 3 'UTR of MALAT1 had binding sites of mir-200 b,and the results of dual luciferase reporter gene confirmed that mir-200 b could inhibit the expression of MALAT1 through binding sites.MALAT1 is a direct target gene of mir-200 b.Osteoblasts transfected with mir-200 b mimics and mir-200 b inhibitor were used for rt-qpcr detection,and MALAT1 expression was low and high.The autophagy of osteoblasts was enhanced and decreased by western blot.Conclusion: 1.High concentration of melatonin can activate autophagy in osteoblasts and reduce the expression of mir-200 b.2.Mir-200 b directly targets MALAT1 and regulates melatonin mediated autophagy in osteoblasts.