Circ_0000064 knockdown attenuates high glucose-induced proliferation, inflammation and extracellular matrix deposition of mesangial cells through miR-424-5p-mediated WNT2B inhibition in cell models of diabetic nephropathy.

Circular RNA (circRNA) is widely shown to be associated with the development of diabetic nephropathy (DN). Our study aimed to further explore the role of circ_0000064 and provide a new mechanism for its action in DN. Cell models of DN in vitro were constructed by treating human renal mesangial cells (HRMCs) with high glucose (HG). The expression of circ_0000064, microRNA-424-5p (miR-424-5p) and Wnt family member 2B (WNT2B) mRNA was detected by quantitative real-time PCR (qPCR). Cell proliferation was assessed by CCK-8 assay and EdU assay. Cell cycle was characterized by DNA content using flow cytometry. The releases of pro-inflammatory factors were checked using commercial ELISA kits. The expression of cell cycle- and fibrosis-associated proteins was detected by western blot. The interplays between miR-424-5p and circ_0000064 or WNT2B were verified by dual-luciferase reporter assay and RIP assay. Circ_0000064 and WNT2B were upregulated, while miR-424-5p was downregulated in HG-treated HRMCs. Circ_0000064 knockdown largely attenuated HG-induced proliferation, inflammatory responses and extracellular matrix (ECM) accumulation in HRMCs, and miR-424-5p deficiency reversed the role of circ_0000064 knockdown. MiR-424-5p was a target of circ_0000064, and miR-424-5p directly bound to WNT2B. MiR-424-5p restoration alleviated HG-induced proliferation, inflammatory responses and ECM accumulation in HRMCs, and WNT2B overexpression partially abolished the effects of miR-424-5p. Circ_0000064 knockdown ameliorated HG-induced HRMC dysfunctions through miR-424-5p enrichment-mediated WNT2B inhibition, hinting that circ_0000064 contributed to DN development.

Li J ，Min Y ，Zhao Q 《-》

Circ-Luc7l Absence Attenuates Diabetic Nephropathy Progression by Reducing Mesangial Cell Excessive Proliferation, Inflammation, and Extracellular Matrix Accumulation via Mediating the miR-205-5p/Tgfbr1 Pathway.

Diabetic nephropathy (DN) threatens the survival quality of patients, with complex pathogenesis. Circular RNA (circRNA) dysregulation occurs in DN development. This work aimed to investigate the role of circ-Luc7l in DN cell models and related molecular mechanisms. The expression of circ-Luc7l, microRNA (miR)-205-5p, and transforming growth factor-beta receptor 1 (Tgfbr1) was examined by real-time quantitative PCR (RT-qPCR). Cell viability and proliferation were detected by Cell Counting Kit-8 (CCK-8) assay and EdU assay. The expression of extracellular matrix (ECM)-related markers and Tgrbr1 protein was measured by Western blot. The binding between miR-205-5p and circ-Luc7l or Tgfbr1 was validated by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, or RNA pull-down assay. Experimental animal models were established to elucidate the function of circ-Luc7l in vivo. Circ-Luc7l expression was notably enhanced in high glucose (HG)-treated mesangial cells. Knockdown of circ-Luc7l attenuated HG-induced cell proliferation, inflammation, and ECM accumulation in vitro and relieved inflammation and ECM accumulation of kidneys of diabetic mice in vivo. Circ-Luc7l targeted miR-205-5p, and miR-205-5p inhibition rescued the depletion effects of circ-Luc7l knockdown on cell proliferation, inflammation, and ECM accumulation. MiR-205-5p bound to Tgfbr1 whose expression was negatively regulated by circ-Luc7l. Tgfbr1 overexpression also rescued the depletion effects of circ-Luc7l knockdown on cell proliferation, inflammation, and ECM accumulation. In HG conditions, increased circ-Luc7l upregulated Tgfbr1 expression via targeting miR-205-5p to induce DN progression.

Fang Z ，Wang D ，Sun F ，Chang J ，Yuan D ，Lin S ，Teng J ... -  《-》

Autocrine small extracellular vesicles induce tubular phenotypic transformation in diabetic nephropathy via miR-21-5p.

Diabetic nephropathy (DN) is one of the most common and serious microvascular complications associated with diabetes. DN is the leading contributor to the majority of cases of end-stage renal disease (ESRD). Small extracellular vesicles (sEVs) can transport various genetic materials to recipient cells. The objective of this study was to explore how sEVs released from HK-2 cells when stimulated by high glucose levels influence renal tubular phenotypic transformation through miR-21-5p. Both human and cell studies were utilized to explore the crosstalk between proximal renal tubules in DN. sEVs from plasma and cells were isolated using ultracentrifugation, and the differential expression of miR-21-5p in plasma sEVs from DN patients versus healthy controls was quantified using Quantitative Real-time PCR (RT-qPCR). A DN model was constructed by stimulating HK-2 cells with glucose. The expression of epithelial-mesenchymal transition (EMT) proteins in each cell group was analyzed by Western Blot (WB), while miR-21-5p levels in both cells and their sEVs were quantified using RT-qPCR. A stable transfected HK-2 cell line was constructed. The CCK8 assay, scratch assay, and WB were employed to detect EMT proteins, aiming to explore how autocrine sEVs affect tubular phenotypic transformation in diabetic nephropathy (DN). The expression of miR-21-5p in plasma sEVs was significantly elevated in the DN group compared to the healthy control group. High glucose (HG) stimulation of HK-2 cells resulted in higher miR-21-5p expression in both cells and their sEVs, leading to enhanced proliferation, migration, and EMT capacities in these cells. Co-incubation of HK-2 cells with HG-sEVs significantly enhanced the proliferation, migration, and EMT capabilities of the recipient cells, but miR-21-5p knockdown reversed these effects. These results indicate that high glucose stimulates HK-2 cells to secrete sEVs, which promote DN proliferation, migration, and EMT through miR-21-5p, thereby offering new insights into the treatment of DN.

Zhang M ，Lu Y ，Wang L ，Mao Y ，Hu X ，Chen Z ... -  《-》

Exosome-transported circ_0001955 as a potent driver of breast cancer by regulating the miR-708-5p/PGK1 axis.

Increasing evidence shows that exosome-mediated delivery of circular RNA (circRNA) is implicated in breast cancer progression. This study aimed to elucidate the role of exosome-transported circ_0001955 in breast cancer. The expression of circ_0001955, miR-708-5p, and phosphoglycerate kinase 1 (PGK1) messenger RNA (mRNA) was detected by quantitative real-time polymerase chain reaction (qRT-PCR); the protein levels of PGK1 and hexokinase 2 (HK2) were detected by western blot (WB). 5'-Ethynyl-2'-deoxyuridine (EdU) and colony formation assay were used to determine cell proliferation. Glycolytic metabolism was analyzed by corresponding kits to detect the associated indicators. The role of circ_0001955 in vivo was studied by establishing animal models. The potential binding relationship between miR-708-5p and circ_0001955 or PGK1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Circ_0001955 was highly expressed in breast cancer tissues and cell lines, as well as in exosomes from breast cancer cell lines. The deficiency of circ_0001955 blocked proliferation, decreased the IC50 value of paclitaxel (PTX), and blocked glycolysis in MCF-7 and MDA-MB-231 cells. Circ_0001955 knockdown also inhibited tumor growth in vivo. Circ_0001955 directly combined with miR-708-5p, and the miR-708-5p inhibitor reversed the effects of sh-circ_0001955. PGK1 was a target of miR-708-5p, and circ_0001955 indirectly promoted PGK1 expression by binding to miR-708-5p. PGK1 overexpression abolished the function of miR-708-5p in breast cancer. Exosomal circ_0001955 excreted from breast cancer cells facilitated proliferation and glycolysis and enhanced the IC50 value of PTX in breast cancer cells by sponging miR-708-5p to upregulate PGK1.

Li W ，Jin G ，Zhou H ，Gao Y ，Ge Y ，Zhang H ... -  《-》

Circular RNA MTCL1 targets SMAD3 by sponging miR-145-5p for regulation of cell proliferation and migration in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a congenital disorder resulting from abnormal development of the enteric nervous system (ENS). Given the complexity of its pathogenesis, it is important to investigate the role of epigenetic inheritance in its development. As Circ-MTCL1 is abundant in brain tissue and colon tissue, whether it has a significant part in the development of ENS is worth exploring. This study clarifies its role in HSCR and identifies the specific molecular mechanisms involved. Diseased and dilated segment colon tissues diagnosed as HSCR were collected for the assessment of gene expression levels using RT-PCR. EdU and CCK-8 assays were adopted to evaluate cell proliferation, and Transwell assay was adopted to assess cell migration. The interaction between Circ-MTCL1, miR-145-5p and SMAD3 was confirmed by dual luciferase reporter gene analysis, RT-PCR and Western blotting. Circ-MTCL1 was down-regulated in the aganglionic colon tissues. The decreased expression of Circ-MTCL1 associated with a reduction in cell migration and proliferation. Bioinformatics analysis and cellular experiments confirmed its role might have been associated with the inhibition of miR-145-5p. MiR-145-5p was up-regulated in HSCR diseased segment colon tissues, exhibiting a negative correlation with Circ-MTCL1. Overexpression of miR-145-5p reversed the inhibition of cell migration and proliferation associated with Circ-MTCL1 down-regulation. The expression of SMAD3 was inhibited by miR-145-5p. The overexpression of SMAD3 eliminated the miR-145-5p-associated inhibition of cell migration and proliferation. Overexpression of miR-145-5p reversed the inhibitory effects of Circ-MTCL1 down-regulation-associated inhibition of cell migration and proliferation, while suppressing SMAD3 expression. Conversely, overexpression of SMAD3 counteracted the miR-145-5p-associated inhibition of cell migration and proliferation. Circ-MTCL1 may function as a miR-145-5p sponge, regulating the expression of SMAD3 and influencing cell migration and proliferation, thus participating in the development of HSCR.

Chen W ，Caiyun L ，Yang Y ，Xinwei H ，Nan L ，Jiaming Y ，Huirong Y ，Kai W ，Liucheng Y ... -  《-》