ABSTRACT
Objective To explore the effect of miR-23b-3p regulation on osteogenic differentiation of renal intersti-tial fibroblasts(hRIFs)on the formation of Randall plaque and its possible mechanism.Methods qRT-PCR was used to detect the expression levels of miR-23b-3p and osteogenic marker:myocyte enhancer factor 2C(MEF2C),osteocalcin(OCN),osteopontin(OPN),runt-related transcription factor 2(Runx2)mRNA in Randall plaque tis-sue of CaOx stone patients(RP)and normal papillary tissue of kidney tumor patients undergoing nephrectomy(nRP).Isolation and culture of human normal hRIFs were isolated and cultured in vitro.The miR-23b-3p overex-pression plasmid pSi-miR-23b-3p and its negative no-load plasmid pSi-NC,the MEF2C lentivirus overexpression plasmid Lv-MEF2C and the no-load plasmid Lv-NC were transfected into hRIFs cells,and the cells were induced to osteogenic differentiation for 14 days.The activity of alkaline phosphatase(ALP)was determined by ELISA.Aliz-arin red staining was used to observe the formation of mineralized nodules.The expression levels of miR-23b-3p and MEF2C,OCN,OPN,Runx2 mRNA were detected by qRT-PCR.The expression level of MEF2C protein was de-tected by Western blot.Dual luciferase reporter gene assay verified the targeting relationship between miR-23b-3p and MEF2C.Results ① Compared with the nRP group,miR-23b-3p was low expressed and MEF2C,OCN,OPN,and Runx2 were highly expressed in the RP group.② 14 days after osteogenic induction of hRIFs cells,the activity of ALP in cells significantly increased,the ability of cells to form mineralized nodules was enhanced,the expression level of miR-23b-3p significantly decreased,the mRNA expression levels of MEF2C,OCN,OPN,and Runx2 significantly increased,and the expression level of MEF2C protein significantly increased.③ Overexpres-sion of miR-23b-3p decreased the activity of ALP in hRIFs cells after osteogenic induction,inhibited the formation of mineralized nodules in cells,and down-regulated the mRNA expression levels of OCN,OPN,and Runx2 in cells.④ Overexpression of MEF2C reversed the inhibitory effect of miR-23b-3p overexpression on osteoblast differ-entiation of hRIFs cells.⑤ MEF2C was the downstream target gene of miR-23b-3p.Conclusion miR-23b-3p is underexpressed in RP tissues and during osteoblastic differentiation of hRIFs cells.Up-regulation of miR-23b-3p in-hibits osteogenic differentiation of hRIFs cells,and its mechanism may be related to targeted silencing MEF2C.
ABSTRACT
AIM:To investigate the effects of osteogenic induction media and the medias containing different concentration of calcium on the induction of osteogenic differentiation of human renal fibroblasts in vitro.METHODS: Culturedhuman renal fibroblasts were divided into 5 groups in this experiment: osteogenic induction group (osteogenic inductionmedia), CaⅠgroup (0.5 mmol/L Ca2 + media), CaⅡgroup (1.5 mmol/L Ca2 + media), Ca Ⅲ group (2.5 mmol/LCa2 + media) and control group (PBS).The cell activity in each groups was measured by MTT assay .At 9th day, the cellcalcium Alizarin red S staining and alkaline phosphatase (ALP) Gomori calcium cobalt staining were performed respectivelyto observe the formation of calcium nidus and the expression of ALP .In addition, the expression of Runt-related transcriptionfactor 2 (Runx2) at mRNA and protein levels was determined by real -time PCR and Western blot, respectively.RE- SULTS: The remarkable positive signs which represented the formation of calcium nidus and the deposit of calcium objectsin all experiment groups were observed .The mRNA and protein expression of Runx2 in osteogenic induction group increasedin accordance with the induction time .Compared with control group, the mRNA and protein expression of Runx2 inthe CaⅠ ~Ⅲ groups increased gradually in a calcium concentration dependent manner at the 9th induction day.CON- CLUSION: Human renal interstitial fibroblasts show the potential activity in osteogenic differentiation induced by osteogen -ic induction media or high level calcium in vitro, which may be account for the cytological formation of the Randall ’splaque in the kidney.