| PurposeBased on the microarray microarray results obtained in previous studies-lnc RNAs differentially expressed in bone marrow-derived mesenchymal stem cells(BMSCs)of patients with steroid-induced osteonecrosis of the femoral head(SONFH)and femoral neck fracture,the targeting relationship between LINC-STXBP5-AS1 and mi R-378a-5p is predicted through bioinformatics analysis.It was determined that the possible downstream target gene of mi R-378a-5p is Su Fu.Because Su Fu is an important component of Hedgehog signal pathway,there may be LINC-STXBP5-AS1/ mi R-378a-5p/ Su Fu/ Hedgehog signal pathway axis.Therefore,the purpose of this study is to verify that LINC-STXBP5-AS1 can interact with mi R-378a-5p to inhibit dexamethasone induced apoptosis of BMSCs by activating Hedgehog signaling pathway in vitro.This study is divided into:Part I: Verification of differentially expressed lnc RNA in BMSCs of SONFH patientsMethod:(1)After informed consent of patients with SONFH and femoral neck fracture,femoral bone marrow was collected during total hip arthroplasty(THA),and then BMSCs were isolated by whole bone marrow adhesion method.BMSCs were purified by cell passage culture.The expression of surface antigens CD34,CD45,CD73 and CD90 was detected by flow cytometry.P3 cells were cultured in medium containing osteogenic and adipogenic inducing components respectively to observe their osteogenic and adipogenic differentiation ability.(2)Through bioinformatics method,mi RNA with binding site with 1nc RNA was selected,and its expression in BMSCs of patients with SONFH and femoral neck fracture was detected by q RT-PCR technology,so as to further verify the results of gene chip.(3)The experimental results were statistically analyzed by SPSS 26.0 software.Result:(1)The results of flow cytometry showed that the cells of P3 generation expressed mesenchymal stem cell specific surface antigens CD73(95.50%)and CD90(92.00%),but almost did not express hematopoietic stem cell specific surface antigens CD34(0.22%)and CD45(0.24%),indicating that the cells cultured by whole bone marrow adhesion method have high purification and can be used in subsequent experiments.(2)qRT-PCR showed that the expression of LINC-STXBP5-AS1 was significantly higher and the expression of mi R-378a-5p was significantly lower in BMSCs of patients with steroid induced femoral head necrosis than that of patients with femoral neck fracture(P < 0.05).Conclusion:Compared with normal subjects,the expression of LINC-STXBP5-AS1 in BMSCs of SONFH patients was significantly increased,and there was a structural matching relationship with mi R-378a-5p.Part II: LINC-STXBP5-AS1 regulates dexamethasone-induced apoptosis of BMSCs through mi R-378a-5p / Su Fu / Hedgehog signaling pathway axisMethod:(1)The apoptosis model of BMSCs was established by 10-6 mol / L dexamethasone.Combined with q RT-PCR,the dynamic expression of LINC-STXBP5-AS1 during dexamethasone induced apoptosis of BMSCs was detected.(2)The lentiviral vector sh-STXBP5-AS1 was constructed.The effect of LINC-STXBP5-AS1 on dexamethasone induced apoptosis of BMSCs was observed by CCK-8 detection,Hoechst 33342 staining and flow cytometry analysis.(3)The lentiviral vector of sh-STXBP5-AS1 was constructed by pathway blocking experiment.After adding Hedgehog signal pathway blocker Cyclopamide,the role of Hedgehog signal pathway in LINC-STXBP5-AS1 regulates dexamethasone induced apoptosis of BMSCs was observed by flow cytometry analysis.(4)The lentiviral vector of sh-STXBP5-AS1 was constructed by cell transfection technology.After low expression of mi R-378a-5p and overexpression of Su Fu,the effects of mi R-378a-5p and Su Fu on LINC-STXBP5-AS1 regulating Hedgehog signal pathway and regulating dexamethasone induced apoptosis of BMSCs were observed by Western Blot.Result:(1)q RT-PCR showed that after 10-6 mol / L dexamethasone treatment,the expression level of LINC-STXBP5-AS1 in BMSCs increased(P < 0.05),accompanied by apoptosis.(2)CCK-8 analysis showed that low expression of LINC-STXBP5-AS1 could significantly resist the inhibitory effect of 10-6 mol / L dexamethasone on the proliferation of BMSCs,but LINC-STXBP5-AS1 did not affect the proliferation of BMSCs without dexamethasone treatment.In addition,Hoechst 33342 staining and flow cytometry analysis showed that shSTXBP5-AS1 could significantly resist BMSCs apoptosis induced by 10-6 mol / L dexamethasone.(3)In the process of antagonizing dexamethasone induced apoptosis of BMSCs,shSTXBP5-AS1 can significantly reduce the level of Su Fu(P < 0.05)to activate Hedgehog signaling pathway,so as to reduce the proportion of Cleaved Caspase-3 and inhibit the apoptosis of BMSCs.Cycloparamide,a blocker of this pathway,can significantly increase the proportion of apoptotic cells and weaken the anti-apoptotic effect of sh-STXBP5-AS1.(4)Down regulation of mi R-378a-5p and up regulation of Su Fu expression can increase the proportion of fragmented Cleaved Caspase-3,promote BMSCs apoptosis and weaken the anti-apoptotic effect of sh-STXBP5-AS1.Conclusion:LINC-STXBP5-AS1 can regulate the apoptosis of BMSCs induced by dexamethasone through mi R-378a-5p / Su Fu / Hedgehog signal pathway axis,but it does not affect the proliferation of BMSCsPart III: Verification of targeting relationship between LINC-STXBP5-AS1 and mi R-378a-5p,mi R-378a-5p and Su FuMethod:(1)To observe the interaction between LINC-STXBP5-AS1 and mi R-378a-5p in BMSCs: the expression level of mi R-378a-5p in BMSCs was observed by transfecting cells with shSTXBP5-AS1 lentivirus;(2)The double luciferase reporter gene system was constructed.The structural matching between LINC-STXBP5-AS1 and mi R-378a-5p,mi R-378a-5p and Su Fu was verified,and the targeted binding sites between LINC-STXBP5-AS1 and mi R-378a-5p,mi R-378a-5p and Su Fu were identified.Result:(1)BMSCs were transfected with sh-STXBP5-AS1,and the expression level of mi R-378a-5p was significantly increased(P < 0.05).The above results showed that LINC-STXBP5-AS1 and mi R-378a-5p could regulate with each other,and their expression levels were negatively correlated.(2)The analysis of double luciferase reporter gene system showed that the relative fluorescence value of mi R-378a-5p mimics transfected group was significantly lower than that of other groups(P < 0.05),suggesting that there was targeted binding between LINCSTXBP5-AS1 and mi R-378a-5p,mi R-378a-5p and Su Fu.Conclusion:There is an interaction between LINC-STXBP5-AS1 and mi R-378a-5p in BMSCs,and their expression levels are negatively correlated.In addition,LINC-STXBP5-AS1 has targeted binding sites with mi R-378a-5p;mi R-378a-5p has targeted binding sites with SuFu. |
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