| Objective1.In the clinical samples,the relationship between the expression of JMJD3 in bone tissue and the expression of RUNX2,the key factor of osteogenesis,was determined.2.In cell research,to determine whether Xianlinggubao promotes osteoblast differentiation by activating JMJD3 / RUNX2 pathway.3.To further explore the molecular mechanism of Xianlinggubao in regulating JMJD3 / RUNX2 pathway.Methods1.Clinical research(1)According to the strict inclusion and exclusion criteria,20 postmenopausal women aged 50-70 years who were scheduled for total hip arthroplasty due to hip osteoarthritis were selected,including 10 cases of osteoporosis group and 10 cases of normal bone mass group.The cancellous bone of femoral neck was taken with sterile bone biting forceps.(2)RNA and protein were extracted from bone tissue samples,and the expression levels of JMJD3,H3K27me3 and RUNX2 were detected by Q-PCR andWB.(3)The correlation between the expression level of JMJD3 in bone tissue and the clinical characteristics and RUNX2 expression level was statistically analyzed.2.Cell research(1)MC3T3-E1 cells were treated with Xianlinggubao for 14 days.CCK-8was used to detect the cell proliferation rate.ELISA and ALP staining were used to detect the expression and activity of ALP,and WB was used to detect the expression of jmjd3 and Runx2.(2)Three si RNA sequences targeting JMJD3 were designed and synthesized.After transfection(NC group,si-JMJD3-1 group,si-JMJD3-2group and si-JMJD3-3 group),RNA was extracted 48 hours later.The relative level of JMJD3 was detected by Q-PCR.(3)According to the results of Q-PCR,the si RNA with the best interference efficiency was selected and transfected into MC3T3-E1 cells(Mock group,XLGB group,OM group,XLGB + si-NC group,XLGB + si-JMJD3group).The expression of ALP was detected by ELISA,and the m RNA and protein expression of RUNX2 were detected by Q-PCR and WB respectively.3.Mechanism research(1)Bioinformatics was used to predict the targeted binding of mi R-100-5p with JMJD3.(2)The target regulation of mi R-100-5p and JMJD3 was verified by double luciferase reporter gene system.(3)The mimic of mi R-100-5p was transfected into MC3T3-E1 treated with Xianlinggubao.The expression of ALP and JMJD3 was detected.Results1.Clinical research(1)The expression level of JMJD3 in osteoporotic bone tissue was significantly lower than that in normal bone tissue.Q-PCR analysis showed that the m RNA expression level of JMJD3 in osteoporotic group was significantly lower than that in control group(P< 0.05),the m RNA expression level of RUNX2 was also significantly lower than that of control group(P < 0.01).Western blot analysis showed that the protein expression level of JMJD3 in osteoporotic group was significantly lower than that in control group(P < 0.05),the protein expression level of RUNX2 was significantly lower than that of control group(P < 0.05),and the expression level of H3K27me3 was significantly higher than that of control group(P < 0.05).(2)There was a positive correlation between the expression of JMJD3 and RUNX2.The m RNA expression levels of JMJD3 and RUNX2 in bone tissue were significantly positively correlated(Pearson r = 0.8227,R2=0.0.6768).(3)The expression of JM3 was positively correlated with BMD.The m RNA expression of JMJD3 was positively correlated with BMD(Pearson r = 0.7102,R2= 0.5044,P < 0.001),and Runx2 was positively correlated with BMD(Pearson r = 0.7383,R2= 0.5451,P < 0.001).2.Cell research(1)Xianlinggubao had no toxicity at the concentration of 0.125 mg/m L-1.0 mg/m L.Xianlinggubao capsule(0.125 mg/m L,0.25 mg/m L,0.5 mg/m L,1.0 mg/m L)could promote the proliferation of MC3T3-E1 cells from the first day to the fifth day,especially when the concentration was 1.0 mg/m L(P < 0.001).The results showed that Xianlinggubao had no toxicity to MC3T3-E1 cellsat the concentration of 0.125 mg/m L-1.0 mg/m L.(2)Xianlinggubao capsule promotes MC3T3-E1 to differentiate into osteoblasts.0.125mg/m L Xianlinggubao capsule(XLGB)can significantly induce the production of ALP,a marker protein of osteogenesis.After the concentration of XLGB reached 0.5mg/m L,it continued to increase.When the concentration reached 1mg/m L,the growth did not increase significantly.Moreover,the expression level of ALP in 0.125 mg/ml Xianlinggubao reached the level of ALP induced by OM.Alkaline phosphatase staining results showed that when the concentration of XLGB reached 0.25 mg/ml,the average optical density of ALP staining signal increased significantly(P < 0.05).With the increase of XLGB concentration,ALP staining signal intensity continued to increase.(3)Xianlinggubao capsule promotes the activation of JMJD3/RUNX2 pathway.The results of Q-PCR and WB showed that compared with the control group,the m RNA expression of JMJD3,RUNX2,JMJD3 and RUNX2 in the Xianlinggubao treatment group were significantly higher than those in the control group(P < 0.05),RUNX2(P < 0.001),JMJD3(P < 0.001)and RUNX2(P < 0.001).(4)Inhibition of JMJD3/RUNX2 pathway can alleviate the promoting effect of Xianlinggubao Capsule on osteoblast differentiation.The expression of ALP in XLGB and OM groups was significantly higher than that in control group(P < 0.001).There was no significant difference between XLGB and XLGB + si-NC groups.However,the expression of ALP in cells treated with XLGB + si-JMJD3 was significantly lower than that in XLGB and XLGB + si-NC groups(P < 0.01).The m RNA and protein expression levels of RUNX2 in XLGB and OM groupswere significantly higher than those in the blank control group(P < 0.05).There was no significant difference in RUNX2 m RNA and protein expression between XLGB and XLGB + si-NC groups,but RUNX2 m RNA expression level in XLGB + si-JMJD3 treatment group was higher than that in control group(P< 0.05)Compared with the XLGB group and the XLGB + si-NC group,the protein expression levels of the two groups were significantly decreased(P <0.05).3.Mechanism research(1)Xianlinggubao capsule inhibited the expression of mi R-100-5p in MC3T3-E1 cells.Targetscan predicted that mi R-100-5p could combine with 3'UTR region of JMJD3.The relative expression level of mi R-100-5p in XLGB MC3T3-E1 cells was significantly lower than that in mock group(P < 0.0001).The expression level of mi R-100-5p in MC3T3-E1 cells in XLGB group was higher than that in OM group(P < 0.0001).(2)Luciferase reporter gene system was used to detect the binding of mi R-100-5p with JMJD3.Compared with the luciferase reporter plasmid with mi R-100-5p mimetic negative sequence and luciferase reporter plasmid with wild-type JMJD3 m RNA 3'UTR(JMJD3-wt),the luciferase activity detected by co-transfection of mi R-100-5p mimic and luciferase reporter plasmid with JMJD3 m RNA 3'UTR was significantly decreased(P < 0.001).However,there was no significant difference in luciferase activity between mi R-100-5p mimic negative sequence and mutant JMJD3 m RNA 3'UTR(JMJD3 MUT)cells.(3)Transfection of mi R-100-5p mimicry could alleviate the promotion effect of Xianlinggubao Capsule on JMJD3 expression.The relative level of JMJD3 in XLGB group was significantly higher than that in mock group(P < 0.01).There was no significant differencein JMJD3 expression between OM and XLGB.There was no significant difference in the expression level of JMJD3 between Xianlinggubao group and mi RNA negative control group(XLGB + mimic NC).The expression level of JMJD3 in MC3T3-E1 cells transfected with mi R-100-5p mimics(XLGB +mi R-100-5p)was significantly lower than that of MC3T3-E1 cells transfected with mi RNA negative control mimics(XLGB + mimic NC group)(P < 0.001).(4)Transfection of mi R-100-5p mimics can alleviate the promoting effect of Xianlinggubao Capsule on osteoblast differentiation.Compared with the blank control group(mock),the expression of ALP in the Xianlinggubao capsule treated group(XLGB)was significantly increased(P < 0.01);there was no significant difference in the ALP expression level between the osteogenic induction solution group(OM)and Xianlinggubao group(XLGB);the ALP expression level in the Xianlinggubao capsule combined with mi RNA negative control group(XLGB + mimic NC)was not The expression level of ALP in MC3T3-E1 cells transfected with mi R-100-5p mimics(XLGB + mi R-100-5p)was significantly lower than that of MC3T3-E1 cells transfected with mi RNA negative control mimics(XLGB+ mimic NC group)(P < 0.01).Conclusion(1)Clinical studies have shown that the expression of JMJD3 in bone tissue of patients with osteoporosis is significantly lower than that of normal people,and the expression level of JMJD3 is positively correlated with RUNX2,the key factor of osteogenic differentiation.It is suggested that JMJD3 may participate in the occurrence and development of osteoporosis through RUNX2.(2)In vitro cell research,this study firstly identified the promotion effect of Xianlinggubao on osteoblast differentiation and the activation of JMJD3/RUNX2 pathway,and compared with the promotion effect of osteoblast induction medium,0.125mg/ml Xianlinggubao can promote the differentiation of osteoblasts.At the same time,we used gene interference technology to transfect the effective si RNA sequence of JMJD3 into MC3T3-E1 cells treated with Xianlinggubao,and found that JMJD3 si RNA can significantly inhibit the promotion of Xianlinggubao on the osteogenic differentiation of MC3T3-E1 cells.It is suggested that Xianlinggubao capsule may promote osteoblast differentiation by activating JMJD3/RUNX2 pathway.(3)In the mechanism study,bioinformatics software was used to predict that mi R-100-5p and 3'UTR of JMJD3 m RNA had targeted binding sites in the peripheral blood of osteoporosis patients.Cell experiment showed that Xianlinggubao capsule and osteogenic induction solution could significantly inhibit the expression of mi R-100-5p in MC3T3-E1.It was found that overexpression of mi R-100-5p could inhibit the expression of JMJD3 through double luciferase reporter gene system.When the binding site of mi R-100-5p on 3'UTR of JMJD3 m RNA was mutated,the expression of JMJD3 was no longer inhibited by mi R-100-5p.At the same time,transfection of mi R-100-5p mimicry could inhibit the promoting effect of Xianlinggubao Capsule on MC3T3-E1 osteogenic differentiation.In conclusion,Xianlinggubao capsule can promote the activation of JMJD3/RUNX2 pathway and promote bone formation by inhibiting the expression of mi R-100-5p,so as to play a role in the treatment of osteoporosis. |
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