The Role Of LncRNA HOXA11-AS In Exercise-enhanced Osteogenic Differentiation And Bone Angiogenesis

Objective:The occurrence of osteoporosis is closely related to the decrease of osteogenesis and the decline of bone angiogenesis ability.In the bone microenvironment,osteogenesis and angiogenesis of the bone microenvironment are coupled.Osteoblasts can secrete relevant angiogenic factors to promote bone angiogenesis.Bone blood vessels provide necessary nutrients for osteogenesis and also pass on bone formation Signal stimulation.Exercise can effectively prevent osteoporosis.At present,exercise-induced mechanical stress stimulation and its role in promoting osteogenesis and angiogenesis in the bone microenvironment are considered to be one of the possible mechanisms for preventing and treating osteoporosis.In recent years,studies have reported that Lnc RNA HOXA11-AS can participate in the regulation of angiogenesis and bone formation,but whether exercise can promote bone formation and bone microenvironment angiogenesis by regulating the expression of Lnc RNA HOXA11-AS has not been reported.Therefore,this study explored the regulation of exercise and mechanical stress stimulation on the expression of Lnc RNA HOXA11-AS and its effect on the expression of related osteogenic and angiogenic factors,and provided a further study on the mechanism of exercise promoting bone formation and angiogenesis in the bone microenvironment.Theoretical basis.Methods:1 animal experiment Eighteen-week-old female C57 BL / 6 mice were selected and randomly divided into an ovary-removed group(Ovx)and an ovary-removed + exercise group(Ovx + Exe).Ovariectomized mice were subjected to ovarian removal surgery after anesthesia.Four weeks after the operation,the ovariectomized + exercise group mice were trained for 9 weeks on the treadmill,five days a day,one hour a day.The first week of pre-adapted running platform training: Monday to Friday,training 30 minutes a day,the speed of 6m / min;the next eight weeks is the formal running platform training phase,the starting speed is 8m / min,and the weekly growth rate is weekly.1m / min,one hour of training per day,including 5min warm-up exercise at a speed of 6m / min and 55 min of formal treadmill training;the ovarian group was routinely raised without any intervention.After the exercise intervention,each group randomly selected three mice's femur and tibia,and extracted total RNA for Lnc RNA gene chip detection.Six mice were taken from the left leg tibia bone marrow to extract RNA for detection of related gene expression.Lnc RNAs with higher expression differences were screened and mapped using Multiple Array Viewer.All animal proce dures satisfied the requirements of the ARRIVE guidelines and the standards for ethics in sport science research,and the study was approved by the Ethics Committee of Shanghai University of Sport.2 Cell Experiment2.1 21-days differentiation test of osteoblasts Primary osteoblasts passaged to the third generation were seeded into a six-well cell culture plate at 1 × 105 / ml,and were divided into Day0 group,Day3 group,Day7 group,and Day21 group for a total of 4 groups.After the cells were completely adhered to the plate for 12 hours,the four-component osteocytes were cultured for osteogenic induction and differentiation for 0 days,3 days,7 days,and 21 days,respectively.After 21 days of osteogenic induction and differentiation culture was completed in the Day21 group,the RNA of each group was extracted,and the expression of HOXA11-AS,OCN,Osterix and EGFL-6 m RNA was detected by Real-time PCR.2.2 7-days stretch test of osteoblasts C57 BL / 6 mouse primary osteoblasts were passaged to the third passage at a density of 1 × 105 / ml and inoculated into Bio Flex 6-well plates.After the cells reached 70%,the cells were stretched using Flexcell-5000 cell stretcher..In the distraction group,the primary osteoblasts were mechanically stretched for 7 days with a 3% stretch amplitude,0.5 Hz intervention frequency,and a 4 h intervention duration.After 7days of intervention,the control group was not stretched,and it only needed to stand for 7 days.During the stretch experiment,complete cell culture medium was changed every 48 hours.At the same time,the post-stretching medium was collected for osteoblast culture in ALP staining and alizarin red staining experiments.Cells were collected after mechanical stretch intervention,and RNA and protein of corresponding cells were extracted.Real-time PCR was used to detect the expression of HOXA11-AS,VEGF,EGFL-6,FGFR1 and Osterix's m RNA;Western blot to detect the expression of ?-catenin and Osterix.All results were expressed as mean standard deviation(X SD)and analyzed using SPSS 20.0 software.Univariate anova was used within the group,and Bonferrori method was used between the groups for comparison.Results:1 Animal experiment1.1 Gene chip detection of Ovx and Ovx + Exe mice showed that after ovarian-removed mice had undergone a certain intensity of treadmill training,the expression of four Lnc RNA genes in the lower limb bones Gm31712,LOC102641308,Gm31126,and HOXA11-AS was obvious.Up-regulated;Vmnlr190-ps,Gm31202,LOC105247265,Gm26535 Lnc RNA gene expression was significantly down-regulated.1.2 PCR analysis of mouse bone marrow RNA showed that the expression of Lnc RNA HOXA11-AS in mouse bone marrow was significantly up-regulated in the Ovx + Exe group compared with the Ovx group(P <0.05),suggesting that a certain intensity of treadmill training can promote mouse bone Lnc RNA HOXA11-AS gene expression.2 Cell experiment2.1 Changes in the expression of Lnc RNA HOXA11-AS and related osteogenic and angiogenic factors during 21-day differentiation of osteoblasts Real-time PCR results showed that during the osteogenic differentiation of osteoblasts,the m RNA expression of osteoblasts such as OCN and Osterix was gradually increased,and the expression of Lnc RNA HOXA11-AS m RNA was gradually increased.On the 7th day of osteogenic differentiation,OCN expression was significantly up-regulated(P<0.01),and on the 21 st day,the expression was significantly increased(P <0.001);on the 7th day of osteogenic differentiation,Osterix expression was also significantly up-regulated(P <0.01),it's expression was significantly increased on the 21 st day(P<0.01);the expression of Lnc RNA HOXA11-AS was significantly increased on the7 th day of osteogenesis(P <0.05),it was significantly increased on the 21 st day(P<0.01).The angiogenesis factor EGFL-6 did not change significantly in the early stage of osteogenic differentiation,and the expression of angiogenic factor in the late stage of osteogenesis began to be up-regulated,and its expression was significantly up-regulated on the 21 st day of osteogenesis(P <0.001).2.2 Effects of mechanically stretched primary osteoblasts on Lnc RNAHOXA11-AS gene expression and related osteogenic and angiogenic factors(1)ALP staining showed that proper mechanical stretch significantly increased the secretion of ALP in osteoblasts(P <0.01).(2)Alizarin red staining showed that compared with the blank control group,the osteocyte calcification nodules were significantly increased after appropriate mechanical distraction(P <0.01).(3)Western blot showed that proper mechanical stretch could promote the expression of ?-catenin and Osterix,the expression of ?-catenin was significantly up-regulated(P<0.01),and the expression of Osterix was significantly up-regulated(P <0.05).(4)Real-time PCR results show that during osteoblast differentiation,mechanical stretch can increase the m RNA expression of Osterix osteogenic factor.Among them,the mechanical stretch stimulation effect on the 5th and 7th days is the most obvious,with significant differences.(P <0.05).Under the stimulation of mechanical tension,the m RNA expression of angiogenic factors such as EGFL-6,VEGF and FGFR1 tended to be gradually increased.On the seventh day of mechanical distraction,the m RNA expression of EGFL-6 and VEGF were significantly increased(P < 0.001).However,FGFR1 m RNA expression was significantly up-regulated at 7 days of mechanical distraction(P <0.05).The expression of Lnc RNA HOXA11-AS m RNA was not significantly changed after 5 days of mechanical distraction,and the expression of Lnc RNA HOXA11-AS m RNA was significantly up-regulated after 7days of mechanical distraction(P <0.001).Conclusion:Exercise can induce differential expression of Lnc RNA s in the bones of ovariectomized osteoporotic mice and up-regulate the expression of Lnc RNA HOXA11-AS in bone marrow.The expression of Lnc RNA HOXA11-AS is significantly up-regulated during osteogenic differentiation,and the expression of related osteogenic factors OCN and Osterix Significantly up-regulated;appropriate mechanical stretch stress stimulation can promote osteoblast differentiation and up-regulate the expression of Lnc RNA HOXA11-AS,related osteogenic factors and related angiogenic factors.