Suppression of osteogenic-like differentiation in human renal interstitial fibroblasts by miRNA-410-3p through MSX2

Abstract

Background: The aim of this stay was to determine the effect of calcium ions in promoting osteogenic-like differentiation in human renal interstitial fibroblasts (hRIFs). The role of miRNA-410-3p in upregulating Msh homeobox 2 (MSX2) level in hRIFs was also investigated.Methods: Quantitative polymerase chain reaction (qPCR) analysis was used to assess the expression levels of miRNA-410-3p in Randall's plaque (RP) and normal renal papillary (nRP) tissues. Furthermore, the expression levels of osteogenesis-related protein in the RP and nRP tissues were assessed with qPCR and immunohistochemistry (IHC). hRIFs were cultured from isolated human kidney papilla before treatment with calcium chloride or osteogenic medium, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed at 1, 5, 9, and 14 days post-treatment. Alizarin red staining was used to estimate the deposits of calcium aggregates. After the overexpression or knockdown of miRNA-410-3p, we evaluated the changes in the osteogenic-like differentiation and osteogenesis-related protein by alizarin red staining and qPCR, respectively. A binding relationship between miRNA-410-3p and MSX2 was established through a dual-luciferase reporter gene assay. Rescue experiments demonstrated that miRNA-410-3p regulated the osteogenic-like differentiation by targeting MSX2.Results: miRNA-410-3p levels were lower in RP tissue than in control nRP tissues. qPCR and IHC showed that the level of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN) were higher in RP tissues. The calcium deposition of hRIFs showed a time-dependent trend when treated with osteogenic medium or calcium chloride. The overexpression of miRNA-410-3p downregulated the levels of osteogenesis-related expression and attenuated mineralization. The knockdown of miRNA-410-3p yielded the opposite trend. Dual-luciferase reporter gene assay and rescue experiments indicated that miRNA-410-3p could target MSX2, while the overexpression of MSX2 reversed the effects of miRNA-410-3p on osteogenic-like differentiation. Conclusions:The current findings suggest that calcium ions could promote the osteogenic-like differentiation of hRIFs and miRNA-410-3p regulates hRIFs osteogenic-like differentiation by inhibiting MSX2.

Identification of the core genes in Randall’s plaque of kidney stone and immune infiltration with WGCNA network

“…Previous research into Randall’s plaque has investigated multiple fields, such as microscopic morphology ( Evan et al, 2018 ) and metabolic disturbance ( Coe et al, 2016 ). Recent experiments emphasize d M1/M2 macrophage polarization and osteogenic reaction, which was associated with osteopontin (OPN), runt-related transcription factor 2 (Runx2), and bone osteocalcin (OCN) ( Cui et al, 2020 ) in Randall’s plaque formation. Compared with former findings, our study concludes that DCN, LUM, and P4HA2 are downregulated in Randall’s plaque in coordination with the abnormal distribution of resting mast cells and M2 macrophages.…”