基于竞争性内源性RNA调控轴探讨通痹生骨方对骨髓间充质干细胞成脂分化的影响及其作用机制OA

Objective:To investigate the effects of Tongbi Shenggu Fang(通痹生骨方,TSF)on the adipogenic differentiation of bone mar-row mesenchymal stem cell(BMMSC),and to elucidate its underlying molecular mechanism based on the competing endogenous RNA(ceRNA)regulatory axis.Methods:①Screening for the optimal working concentration of TSF.The passage 3(P3)human bone marrow mes-enchymal stem cells(hBMMSCs)were selected and divided into the blank group and the 50,150,300,and 500 μg/mL groups.The hBMMSCs in the blank group were cultured in Opti-MEM medium,while the ones in the 50,150,300,and 500 μg/mL groups were cultured in Opti-MEM medium containing 50,150,300,and 500 μg/mL TSF lyophilized powder,respectively for 24 hours.After the end of the cul-ture,the hBMMSCs viability was assessed using the CCK-8 assay to identify the optimal working concentration.②Detecting the adipogenic differentiation of hBMMSCs and the expression of molecules in the ceRNA regulatory axis.The P3 hBMMSCs were selected and divided into blank control group,adipogenic induction group,adipogenic induction combined with TSF group,hsa_circ_0028442 knockdown group,hsa_circ_0028442 overexpression group,and hsa_circ_0028442 overexpression combined with TSF group.The P3 hBMMSCs in the blank control group was cultured with hBMMSC-specific medium;the ones in adipogenic induction group with hBMMSC adipogenic induction differentia-tion medium;the ones in adipogenic induction combined with TSF group with hBMMSC adipogenic induction differentiation medium con-taining TSF lyophilized powder;while the ones in the hsa_circ_0028442 knockdown group was transfected with small interfering RNA(siRNA)against hsa_circ_0028442 for 48 hours,then switched to hBMMSC-specific medium;the ones in hsa_circ_0028442 overexpression group was transfected with hsa_circ_0028442 overexpression plasmid for 48 hours,then cultured with hBMMSC-specific medium;the ones in hsa_circ_0028442 overexpression combined with TSF group was transfected with hsa_circ_0028442 overexpression plasmid for 48 hours,then switched to hBMMSC-specific medium containing TSF lyophilized powder.Following 14-day intervention,the adipogenic differentiation of hBMMSCs was evaluated by Oil Red O staining,and the mRNA and protein expression levels of adipogenic differentiation-related genes,in-cluding sterol regulatory element-binding protein 1(SREBP1),fatty acid-binding protein 4(FABP4),and peroxisome proliferator-activated receptor γ(PPARγ),as well as the expression levels of molecules in the ceRNA regulatory axis,including hsa_circ_0028442,hsa-miR-942-5p,and epithelial-stromal interaction 1(EPSTI1)mRNA,were detected by real-time quantitative PCR(RT-qPCR)and Western Blot,respec-tively.③Verification of the circular characteristics of hsa_circ_0028442.The total RNA was extracted from the hBMMSCs in the blank con-trol group and divided into two aliquots.One was treated with ribonuclease(RNase)R and its reaction buffer(RNase R group),and the oth-er with an equal volume of RNase R reaction buffer only(mock treatment group).The expression levels of hsa_circ_0028442 and EPSTI1 mRNA were detected by RT-qPCR.④Verification of the binding of hsa-miR-942-5p to the predicted target sites of hsa_circ_0028442 and EPSTI1.The targeted binding relationships between hsa-miR-942-5p and hsa_circ_0028442 or EPSTI1 were verified by a dual-luciferase re-porter assay.In the hsa-miR-942-5p mimic group,the wild-type(WT)and mutant(MUT)subgroups were co-transfected with the WT and MUT dual-luciferase reporter gene plasmids of hsa_circ_0028442 and EPSTI1,respectively,together with the hsa-miR-942-5p mimics,into hBMMSCs.Similarly,in the negative control(NC)group,the WT and MUT subgroups were co-transfected with the corresponding WT and MUT dual-luciferase reporter gene plasmids and the hsa-miR-942-5p NC mimics.Results:①The optimal working concentration of TSF.Pairwise comparisons of cell viability among the blank group and the 150 and 300 μg/mL groups showed no statistically significant differ-ences.The 50 μg/mL group exhibited higher viability than the blank group(P=0.008),but no significant differences compared to the 150 and 300 μg/mL groups.Moreover,the 500 μg/mL group showed lower viability than the blank group and the 50,150,300 μg/mL groups(P=0.019,P=0.000,P=0.001,P=0.006).Ultimately,150 μg/mL,a concentration without notable cytotoxicity,was selected as the working concentration for subsequent experiments.②The adipogenic differentiation of hBMMSCs.Oil Red O staining was negative in the blank control group and hsa_circ_0028442 knockdown group,but positive in the adipogenic induction group,adipogenic induction combined with TSF group,hsa_circ_0028442 overexpression group,and hsa_circ_0028442 overexpression combined with TSF group.Notably,the lipid droplets were markedly reduced in the adipogenic induction combined with TSF group compared to the adipogenic induction group,as well as in the hsa_circ_0028442 overexpression combined with TSF group compared to the hsa_circ_0028442 overexpression group.③The ex-pression levels of molecules in the ceRNA regulatory axis and the mRNA expression levels of adipogenic differentiation-related genes.Com-pared with the blank control group,the adipogenic induction group and hsa_circ_0028442 overexpression group exhibited significantly up-regulated expression levels of hsa_circ_0028442,EPSTI1 mRNA,SREBP1 mRNA,FABP4 mRNA,and PPARγ mRNA(P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.002;P=0.000,P=0.000;P=0.000,P=0.000),but downregulated hsa-miR-942-5p level(P=0.000,P=0.000);conversely,the hsa_circ_0028442 knockdown group showed significantly downregulated expression levels of hsa_circ_0028442,EPSTI1 mRNA,SREBP1 mRNA,FABP4 mRNA,and PPARγ mRNA(P=0.000,P=0.001,P=0.000,P=0.001,P=0.000),but upregulated hsa-miR-942-5p level(P=0.000).Furthermore,compared with the adipogenic induction group,the adipogen-ic induction combined with TSF group exhibited significantly decreased expression levels of hsa_circ_0028442,EPSTI1 mRNA,SREBP1 mRNA,FABP4 mRNA,and PPARγ mRNA(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000),but increased hsa-miR-942-5p level(P=0.000).Similarly,compared with the hsa_circ_0028442 overexpression group,the hsa_circ_0028442 overexpression combined with TSF group also showed decreased hsa_circ_0028442,EPSTI1 mRNA,SREBP1 mRNA,FABP4 mRNA,and PPARγ mRNA levels(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000),but increased hsa-miR-942-5p level(P=0.000).④Protein expression level of adipogenic differentiation-related genes.Compared with the blank control group,the protein expression levels of SREBP1,FABP4,and PPARγ were higher in the adipogenic induction group and hsa_circ_0028442 overexpression group(P=0.000,P=0.000;P=0.004,P=0.003;P=0.004,P=0.004),while lower in the hsa_circ_0028442 knockdown group(P=0.000,P=0.034,P=0.002).Furthermore,the protein expression levels of all these three markers were lower in the adipogenic induction combined with TSF group compared to the adipogenic induction group(P=0.000,P=0.004,P=0.001),and also lower in the hsa_circ_0028442 overexpression combined with TSF group compared to the hsa_circ_0028442 overexpression group(P=0.000,P=0.004,P=0.000).⑤The circular characteristics of hsa_circ_0028442.No significant difference in the expression level of hsa_circ_0028442 was observed between the RNase R group and the mock treatment group,whereas the mRNA expression level of EPSTI1 was significantly lower in the RNase R group compared to the mock treat-ment group(P=0.000).⑥The binding of hsa-miR-942-5p to the predicted target sites of hsa_circ_0028442 and EPSTI1.In the hsa-miR-942-5p mimic group,the relative luciferase activities of both WT hsa_circ_0028442 subgroup and WT EPSTI1 subgroup were lower than those in the NC group(P=0.005,P=0.005);while,the relative luciferase activities in the MUT hsa_circ_0028442 subgroup and MUT EPSTI1 subgroup showed no statistically significant differences compared to the NC group.Conclusion:TSF can inhibit the adipogenic differentiation of BMMSCs and suppress the expression of adipogenic differentiation-related genes.It may exert the effect via a ceRNA regu-latory axis.Specifically,it may downregulate the expression of hsa_circ_0028442,thereby attenuating its competitive sponging effect on hsa-miR-942-5p,and subsequently enhance the inhibitory effect of hsa-miR-942-5p on its target gene EPSTI1,thereby achieving a negative regulation on adipogenic differentiation.