Effect Of LncRNA H19 On Osteogenic Differentiation Of Mechanically Stretched Bone Marrow Mesenchymal Stem Cells

Objective: Osteoporosis is a metabolic disease,mainly due to osteoclast dominated bone absorption than osteoblast dominated bone formation.Studies have shown that exercise can effectively promote bone formation and inhibit bone absorption,and thus play a role in combating osteoporosis,but the mechanism is not yet completely clear.The osteoblasts in the body are derived from BMSCs,which are stem cells with multipotent differentiation potential and can be divided into osteoblasts under suitable conditions in vitro.The structure composed of cells,cell membranes,and cell matrices can carry out the transmission of forces,and the extracellular signal(mechanical force)is transferred to the intracellular signal.BMSCs can sense a variety of mechanical stimuli in the body and regulate the osteogenesis process of BMSCs.The coding gene in the human genome is only 2 %,and 91 % of them are non-coding RNAs(ncRNAs).A kind of ncRNAs with a length of more than 200 nt,accounting for more than 80 % of the total ncRNAs,they do not directly participate in the coding of proteins,called long-stranded non-coding RNAs(long n-coding RNAs,lcnRNAs).LCNAs can regulate the proliferation and differentiation of cells by regulating chromatin reconstruction,DNA methylation,histone modification,and RNA polymerase activity.Among them,lncRNA H19(long non-coding RNA H19,lcnRNA H19)is a lncRNA with a molecular weight of 2.3 KB and a long-chainnon-coding RNA located in the human telomere region of 11p15.5.The study found that lcnRNAs can regulate the transcription of osteogenic genes such as RUNX2,OCN,and ALP through bone metabolic signaling pathways such as Wnt / ?-Catenin and BMP,and thus affect osteogenesis.Therefore,this paper provides a theoretical basis for further research on the mechanism of exercise prevention and control of osteoporosis by mechanical stretching of BMSCs to explore the effects of mechanical stretching stress on lncRNA H19 and its downstream pathways.Methods:1.Extraction and culture of primary bone marrow mesenchymal stem cells from mice Growth stage C57BL/6 mice were selected.After anesthesia,the mice were placed in75% alcohol and soaked for 2 minutes.The muscles,ligaments and tendons were separated from the femur,tibia and humerus using surgical scissors.The stripped clean bones were transferred to a 100 mm aseptic culture dish and 10 ml of a complete-mem culture medium was added.All bones were processed within 30 minutes.With1% / penicillin streptomycin PBS flushing of the bone,and then move to a new 100 mm sterile petri dish containing complete medium,to use tweezers clip bones in the middle part,using anatomical cut the ends of the bone marrow cavity resection,use 1ml syringe,after absorbing medium,insert in the marrow cavity,the bone marrow developed slowly,until the bones turned white,put a dish in 37 ? and 5% co2 incubator culture;Cell fusion was 70~90%,and passaged according to the ratio of1:2--1:3.2.Mechanical Drawing Scheme The primary BMSCs of C57BL/6 mice were inoculated into BioFlex 6 orifice plate with 1 *105/ml.The cells grew up to about 80% and fused.The cells were stretched by Flexcell-5000 cell stretcher.(1)One-day traction group,using 3 % traction strength,0.5 Hz,mechanical pull 1 day,intervention time is 2H and 4h,control group static culture,and collect cells after one-day of mechanical traction intervention..(2)The traction group was used for the next day of 7 days.The traction strength was3 %,0.5 Hz,and the mechanical pull was 7 days.The intervention time was 2 H and 4H,that is,the intervention time was 1,3,5,and 7 days.The control group was statically cultivated.Replace the culture fluid every 48 hours,and collect cells after the 7th day of mechanical traction intervention.3.Indicator detection Real-time PCR was used to detect the expression of lncRNA H19 and the expression of microRNAs related to lncRNA H19(microRNA-675-3P,microRNA-675-5P,microRNA-22 and microRNA-141).Real-time PCR and Western blot were used to detect the expression of osteogenesis-related genes(ALP,RUNX2,Osterix,OCN)and protein(wnt1,beta-Catenin).Results:1.Stimulation every other day for 7 consecutive days was more effective than one-off mechanical stretch in promoting the expression of lncRNA H19 mrna.In addition,the expression of lncRNA H19 was significantly increased in the mechanical stretchgroup for 4 hours every other day for 7 consecutive days compared with the control group(P < 0.01).2.Stimulation every other day for 7 consecutive days was more effective than mechanical traction in promoting the expression of lncRNA H19-related microRNA.The mechanical stretching stress for 7 consecutive days after 2 h and 4 h increased the content of microRNA-675-5P,while the expression of microRNA-22 and microRNA-141 decreased after 4 h of stretching stimulation(P < 0.05).3.Stimulation every other day for 7 consecutive days is more effective than mechanical stretch in promoting the expression of osteogenic differentiation-related genes and proteins.The expression of ALP,Osterix and RUNX2 was increased by mechanical stretching stress for 4 hours on 7 consecutive days(P < 0.01),and the expression of Osterix and RUNX2 was increased by mechanical stretching stress for 2hours(P < 0.01).Western blot results showed that the expression of beta-Catenin protein increased significantly in 4H group and 2H group,but the expression of Wnt1 in mechanical stretch group was not significant.Conclusions:1.Mechanical traction promoted the osteogenic differentiation of BMSCs,and the effect of 7 days was better than that of 1 day2.LcnRNA H19 and its downstream signaling pathway were changed in the 7-day traction group,suggesting that mechanical stretch may regulate the osteogenic differentiation of bone marrow mesenchymal stem cells through "lcnRNA H19--microRNA--target gene".